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Sample records for clostridium septicum alpha-toxin

  1. Clostridium septicum Empyema in an Immunocompetent Woman

    PubMed Central

    Granok, Alexander B.; Mahon, Patrick A.; Biesek, Genesio W.

    2010-01-01

    We report a case of a Clostridium septicum empyema in an immunocompetent woman following operation for an incarcerated internal hernia. The patient was successfully treated with pleural decortication and an extended course of postoperative antibiotics. This is the first report of such an infection in the medical literature. PMID:20490275

  2. Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin

    PubMed Central

    Oda, Masataka; Terao, Yutaka; Sakurai, Jun; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail. PMID:26633512

  3. Clostridium septicum brain abscesses in a premature neonate.

    PubMed

    Sadarangani, Sapna P; Batdorf, Rachel; Buchhalter, Lillian C; Mrelashvili, Anna; Banerjee, Ritu; Henry, Nancy K; Huskins, W Charles; Boyce, Thomas G

    2014-05-01

    Brain abscesses in neonates are typically caused by Gram-negative organisms. There are no previously described cases caused by Clostridium septicum. We present a case of a premature male infant who developed recurrent episodes of suspected necrotizing enterocolitis followed by brain abscesses, cerebritis and ventriculitis caused by C. septicum. PMID:24220230

  4. Acute aortic dissection caused by Clostridium septicum aortitis.

    PubMed

    Eplinius, Franziska; Hädrich, Carsten

    2014-11-01

    Clostridium septicum aortitis is a rare cause of aortic dissection. So far, only 28 cases have been described in literature before. Most of these cases occurred in elderly patients and an association to colonic neoplasms and/or atherosclerosis has been witnessed frequently. Here we report the case of a 32-year-old man with fatal aortic dissection due to aortic infection with C. septicum. Beside a case of a 22-year-old man who died of aortic dissection due to C. septicum aortitis this is the second case of C. septicum aortitis in a young individual with no signs of colonic neoplasms or atherosclerosis. PMID:25242573

  5. Clostridium septicum aortitis with associated sigmoid colon adenocarcinoma.

    PubMed

    Ge, Phillip S; de Virgilio, Christian

    2012-02-01

    We report an unusual case of Clostridium septicum aortitis with associated adenocarcinoma of the sigmoid colon. An 87-year-old man with multiple medical comorbidities presented with a 1-week history of severe abdominal pain in the left lower quadrant of his abdomen. Abdominal computed tomography showed, in addition to a mass in the sigmoid colon, a gas density within the wall of the abdominal aorta with extensive periaortic fat stranding and some additional gas densities in the proximal left common iliac artery. The patient refused surgery, and was treated with intravenous antibiotics. He died 5 weeks later. The development of Clostridiumsepticum aortitis, an extremely rare but life-threatening infection, is highly associated with an underlying colonic malignancy and demands immediate surgical intervention. PMID:22304871

  6. Complete Genome Sequence of Clostridium septicum Strain CSUR P1044, Isolated from the Human Gut Microbiota.

    PubMed

    Benamar, Samia; Cassir, Nadim; Caputo, Aurélia; Cadoret, Frédéric; La Scola, Bernard

    2016-01-01

    Clostridium septicum is one of the first pathogenic anaerobes to be identified. Here, we announce the genome draft sequence of C. septicum strain CSUR P1044 isolated from the gut of a healthy adult. Its chromosome genome consists of 3.2 Mbp with a plasmid of 32 Kbp. C. septicum strain CSUR P1044 has a G+C content of 27.5%, and is composed of 3,125 protein-coding genes together with 103 RNA genes, including 22 rRNA genes. PMID:27609912

  7. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity. PMID:18154441

  8. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

    PubMed Central

    2013-01-01

    Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells. PMID:23782465

  9. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

    PubMed

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  10. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

    PubMed Central

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  11. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases. PMID:27051186

  12. Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

    PubMed

    Saito, Hitoshi; Inoue, Masaharu; Tomiki, Masayoshi; Nemoto, Hiroshi; Komoriya, Tomoe; Kimata, Junko; Watanabe, Kunitomo; Kohno, Hideki

    2009-01-01

    Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin

  13. Fatal clostridium septicum myonecrosis in a captive canada lynx (Lynx canadensis).

    PubMed

    Izer, Jenelle M; Wilson, Ronald P; Cooper, Timothy K

    2014-09-01

    A 1-yr-old female Canada lynx (Lynx canadensis) presented for sudden onset of rapidly progressive bilateral pelvic limb paralysis. The lynx was chemically immobilized to perform a physical examination but expired shortly thereafter. On postmortem radiographs, there were myriad small irregular, round-to-spherical gas densities within the skeletal muscle of the right thigh and epaxial musculature. At gross necropsy, the muscles of the right thigh, right lateral abdominal wall, and epaxial region were emphysematous and necrohemorrhagic, with subcutaneous and muscular crepitant swelling. Multiple skin puncture wounds, consistent with bites, were present over the affected tissues. Clostridium septicum was isolated in pure anaerobic culture from the musculature of the right hind limb. Histopathologic examination confirmed the diagnosis of acute, severe necrohemorrhagic and gangrenous myositis and cellulitis. Gram stains demonstrated large gram-positive bacilli with subterminal spores. This is the first known documented case of C. septicum myonecrosis in a nondomestic felid. PMID:25314833

  14. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

    PubMed

    Sasaki, Yoshimasa; Kojima, Akemi; Aoki, Hiroshi; Ogikubo, Yasuaki; Takikawa, Noriyasu; Tamura, Yutaka

    2002-05-01

    The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia. PMID:11900959

  15. Clostridium septicum Gas Gangrene in Colon Cancer: Importance of Early Diagnosis

    PubMed Central

    Nanjappa, Sowmya; Shah, Sweta; Pabbathi, Smitha

    2015-01-01

    The Clostridia species are responsible for some of the deadliest diseases including gas gangrene, tetanus, and botulism. Clostridium septicum is a rare subgroup known to cause atraumatic myonecrosis and is associated with colonic malignancy or immunosuppression. It is a Gram-positive, anaerobic, spore-forming bacillus found in the gastrointestinal tract and can lead to direct, spontaneous infections of the bowel and peritoneal cavity. The anaerobic glycolysis of the tumor produces an acidic, hypoxic environment favoring germination of clostridial spores. Tumor-induced mucosal ulceration allows for translocation of sporulated bacteria from the bowel into the bloodstream, leading to fulminant sepsis. C. septicum bacteremia can have a variable presentation and is associated with greater than 60% mortality rate. The majority of deaths occur within the first 24 hours if diagnosis and appropriate treatment measures are not promptly started. We report a case of abdominal myonecrosis in a patient with newly diagnosed colon cancer. The aim of this study is to stress the importance of maintaining a high suspicion of C. septicum infection in patients with underlying colonic malignancy. PMID:26793397

  16. Spontaneous Occurrence of Gangrene Due to Clostridium septicum in a Patient With Advanced Endometrial Carcinoma

    PubMed Central

    Ghosh, Kris; Sutton, Gregory P.

    1994-01-01

    Background: We report the first known case of spontaneous, atraumatic Clostridium septicum gangrene occurring in a patient with recurrent endometrial adenocarcinoma. Case: A 63-year-old white female undergoing chemotherapy for recurrent endometrial adenocarcinoma presented with right “arthritis-like” shoulder pain. She denied fever, chills, or shoulder trauma. The patient was afebrile and her blood pressure was 100/50. Her right shoulder and upper extremity were remarkable for an area of dark blue discoloration with crepitus. The white blood cell (WBC) count was 8,200/μl with left shift. Serum creatinine, platelet count, and coagulation studies were normal. Computed tomography revealed gas in the right shoulder tissues. A Gram stain of fluid aspirated from the shoulder demonstrated gram-positive spore-forming rods. She declined surgical intervention and expired within hours of admission. Cultures of the right shoulder eventually grew Clostridium septicum. Conclusion: It is imperative to consider clostridial gangrene in the differential diagnosis for any patient with cancer and a fever of unknown origin. PMID:18475364

  17. Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes

    PubMed Central

    2014-01-01

    Background Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species. Results Strains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases. Conclusions Bovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro. PMID:24479821

  18. A microbiological hazard of rural living: Clostridium septicum brain abscess in a child with E coli 0157 associated haemolytic uraemic syndrome.

    PubMed

    Williams, Eleri J; Mitchell, Patrick; Mitra, Dipayan; Clark, Julia E

    2012-01-01

    Clostridium septicum infection in humans is rare and this is the second case report of focal C septicum brain abscesses associated with Escherichia coli 0157 haemolytic uraemic syndrome (HUS). The case presented in a child who lived on a rural farm. The abscesses initially progressed despite 7 months of appropriate antibiotic therapy and repeated image-guided aspiration. After definitive resection and prolonged antibiotic therapy, there was no recurrence. This case reminds us of this rare but important neurological complication of HUS and supports consideration of early definitive neurosurgical intervention in similar unusual cases. PMID:22736786

  19. Evaluation in broilers of the probiotic properties of Pichia pastoris and a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene.

    PubMed

    Gil de los Santos, João Rodrigo; Storch, Otávio Brod; Fernandes, Cristina Gevehr; Gil-Turnes, Carlos

    2012-05-01

    The probiotic properties of Pichia pastoris and of a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene were evaluated in broilers. One-day-old chicks randomly divided in four groups were fed with commercial feed devoid of antibacterials. The control group (1) received plain food, while the other groups were supplemented with either P. pastoris (2), the recombinant P. pastoris (3) or Bacillus cereus var. Toyoi (4). At day 49, live weights, feed efficiency and seroconversions were higher (P<0.05) in the supplemented groups than in the control groups. Group 3 showed the best results, while group 2 had lower weight gain than groups 3 and 4 although food conversion was better than in group 4. Seroconversions were not different (P>0.05) among the supplemented groups. Adverse reactions were not observed in histopathologic evaluation. We concluded that P. pastoris and the recombinant P. pastoris could be used as probiotics in broilers. PMID:22176763

  20. In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

    PubMed Central

    Uppalapati, Siva Ramakrishna; Kingston, Joseph Jeyabalaji; Qureshi, Insaf Ahmed; Murali, Harishchandra Sripathy; Batra, Harsh Vardhan

    2013-01-01

    Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture. PMID:24349173

  1. The Pore-Forming α-Toxin from Clostridium septicum Activates the MAPK Pathway in a Ras-c-Raf-Dependent and Independent Manner

    PubMed Central

    Chakravorty, Anjana; Awad, Milena M.; Cheung, Jackie K.; Hiscox, Thomas J.; Lyras, Dena; Rood, Julian I.

    2015-01-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host’s innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  2. The pore-forming α-toxin from clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner.

    PubMed

    Chakravorty, Anjana; Awad, Milena M; Cheung, Jackie K; Hiscox, Thomas J; Lyras, Dena; Rood, Julian I

    2015-02-01

    Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process. PMID:25675415

  3. Variable protection against experimental broiler necrotic enteritis after immunization with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant.

    PubMed

    Fernandes da Costa, Sérgio P; Mot, Dorien; Geeraerts, Sofie; Bokori-Brown, Monika; Van Immerseel, Filip; Titball, Richard W

    2016-06-01

    Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis, a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunization with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247-370) would provide protection against experimental necrotic enteritis. Immunized birds with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247-370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed-challenge. PMID:26743457

  4. Alpha-toxin of Staphylococcus aureus.

    PubMed Central

    Bhakdi, S; Tranum-Jensen, J

    1991-01-01

    Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occurs in both cases after membrane-bound toxin molecules collide via lateral diffusion to form ring-structured hexamers. The latter insert spontaneously into the lipid bilayer to form discrete transmembrane pores of effective diameter 1 to 2 nm. A hypothetical model is advanced in which the pore is lined by amphiphilic beta-sheets, one surface of which interacts with lipids whereas the other repels apolar membrane constitutents to force open an aqueous passage. The detrimental effects of alpha-toxin are due not only to the death of susceptible targets, but also to the presence of secondary cellular reactions that can be triggered via Ca2+ influx through the pores. Well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction. Such processes cause profound long-range disturbances such as development of pulmonary edema and promotion of blood coagulation.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1779933

  5. Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

    PubMed Central

    Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

    2014-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

  6. Staphylococcus aureus In Vitro Secretion of Alpha Toxin (hla) Correlates with the Affiliation to Clonal Complexes

    PubMed Central

    Monecke, Stefan; Müller, Elke; Büchler, Joseph; Stieber, Bettina; Ehricht, Ralf

    2014-01-01

    The alpha toxin of Staphylococcus aureus is a pore forming toxin that penetrates host cell membranes causing osmotic swelling, rupture, lysis and subsequently cell death. Haemolysin alpha is toxic to a wide range of different mammalian cells; i.e., neurotoxic, dermonecrotic, haemolytic, and it can cause lethality in a wide variety of animals. In this study, the in vitro alpha toxin production of 648 previously genotyped isolates of S. aureus was measured quantitatively using antibody microarrays. Isolates originated from medical and veterinary settings and were selected in order to represent diverse clonal complexes and defined clinical conditions. Generally, the production of alpha toxin in vitro is related to the clonal complex affiliation. For clonal complexes CC22, CC30, CC45, CC479, CC705 and others, invariably no alpha toxin production was noted under the given in vitro conditions, while others, such as CC1, CC5, CC8, CC15 or CC96 secreted variable or high levels of alpha toxin. There was no correlation between alpha toxin yield and clinical course of the disease, or between alpha toxin yield and host species. PMID:24940872

  7. Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model.

    PubMed Central

    Menzies, B E; Kernodle, D S

    1996-01-01

    A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies. PMID:8613399

  8. Foot infection by Clostridium sordellii: case report and review of 15 cases in France.

    PubMed

    Bouvet, Philippe; Sautereau, Jean; Le Coustumier, Alain; Mory, Francine; Bouchier, Christiane; Popoff, Michel-R

    2015-04-01

    We report a case of foot infection by Clostridium sordellii and review 15 human infections registered at a Reference Center in France during the period 1998 to 2011. All strains were found nontoxigenic, lacking the lethal toxin gene coding for TcsL. Like Clostridium septicum, several C. sordellii infections were associated with intestinal neoplasms. PMID:25609723

  9. Foot Infection by Clostridium sordellii: Case Report and Review of 15 Cases in France

    PubMed Central

    Sautereau, Jean; Le Coustumier, Alain; Mory, Francine; Bouchier, Christiane; Popoff, Michel-R.

    2015-01-01

    We report a case of foot infection by Clostridium sordellii and review 15 human infections registered at a Reference Center in France during the period 1998 to 2011. All strains were found nontoxigenic, lacking the lethal toxin gene coding for TcsL. Like Clostridium septicum, several C. sordellii infections were associated with intestinal neoplasms. PMID:25609723

  10. Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves.

    PubMed

    Valgaeren, Bonnie R; Pardon, Bart; Goossens, Evy; Verherstraeten, Stefanie; Roelandt, Sophie; Timbermont, Leen; Van Der Vekens, Nicky; Stuyvaert, Sabrina; Gille, Linde; Van Driessche, Laura; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip; Deprez, Piet

    2015-07-01

    Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin. PMID:26184311

  11. Veal Calves Produce Less Antibodies against C. Perfringens Alpha Toxin Compared to Beef Calves

    PubMed Central

    Valgaeren, Bonnie R.; Pardon, Bart; Goossens, Evy; Verherstraeten, Stefanie; Roelandt, Sophie; Timbermont, Leen; Van Der Vekens, Nicky; Stuyvaert, Sabrina; Gille, Linde; Van Driessche, Laura; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip; Deprez, Piet

    2015-01-01

    Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin. PMID:26184311

  12. Discrimination of clostridium species using a magnetic bead based hybridization assay

    NASA Astrophysics Data System (ADS)

    Pahlow, Susanne; Seise, Barbara; Pollok, Sibyll; Seyboldt, Christian; Weber, Karina; Popp, Jürgen

    2014-05-01

    Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5

  13. Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

    PubMed Central

    Bhakdi, S; Mannhardt, U; Muhly, M; Hugo, F; Ronneberger, H; Hungerer, K D

    1989-01-01

    Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin. PMID:2777380

  14. Method for Estimating the Presence of Clostridium perfringens in Food

    PubMed Central

    Harmon, S. M.; Kautter, D. A.

    1970-01-01

    The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 106C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed. Images PMID:4321712

  15. Identification and Validation of a Linear Protective Neutralizing Epitope in the β-Pore Domain of Alpha Toxin

    PubMed Central

    Oscherwitz, Jon; Cease, Kemp B.

    2015-01-01

    The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha

  16. Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

    PubMed

    Oscherwitz, Jon; Cease, Kemp B

    2015-01-01

    The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha

  17. Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor.

    PubMed

    Kreienkamp, H J; Sine, S M; Maeda, R K; Taylor, P

    1994-03-18

    Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr190, Cys192-Cys193 region of the ligand binding domain, substitution of Trp187 and Phe189 by non-aromatic residues and alteration of the proline sequence Pro194-X-X-Pro197. Glycosylation signals were inserted into the toxin-sensitive mouse alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/gamma contacts, respectively. PMID:7907588

  18. Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin

    PubMed Central

    Scherr, Tyler D.; Hanke, Mark L.; Huang, Ouwen; James, David B. A.; Horswill, Alexander R.; Bayles, Kenneth W.; Fey, Paul D.; Torres, Victor J.

    2015-01-01

    ABSTRACT The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. PMID:26307164

  19. Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis.

    PubMed

    Kulkarni, R R; Parreira, V R; Sharif, S; Prescott, J F

    2007-09-01

    Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens. PMID:17634510

  20. Optimization of the Production of Inactivated Clostridium novyi Type B Vaccine Using Computational Intelligence Techniques.

    PubMed

    Aquino, P L M; Fonseca, F S; Mozzer, O D; Giordano, R C; Sousa, R

    2016-07-01

    Clostridium novyi causes necrotic hepatitis in sheep and cattle, as well as gas gangrene. The microorganism is strictly anaerobic, fastidious, and difficult to cultivate in industrial scale. C. novyi type B produces alpha and beta toxins, with the alpha toxin being linked to the presence of specific bacteriophages. The main strategy to combat diseases caused by C. novyi is vaccination, employing vaccines produced with toxoids or with toxoids and bacterins. In order to identify culture medium components and concentrations that maximized cell density and alpha toxin production, a neuro-fuzzy algorithm was applied to predict the yields of the fermentation process for production of C. novyi type B, within a global search procedure using the simulated annealing technique. Maximizing cell density and toxin production is a multi-objective optimization problem and could be treated by a Pareto approach. Nevertheless, the approach chosen here was a step-by-step one. The optimum values obtained with this approach were validated in laboratory scale, and the results were used to reload the data matrix for re-parameterization of the neuro-fuzzy model, which was implemented for a final optimization step with regards to the alpha toxin productivity. With this methodology, a threefold increase of alpha toxin could be achieved. PMID:27003282

  1. Typing of Clostridium perfringens by in vitro amplification of toxin genes.

    PubMed

    Daube, G; China, B; Simon, P; Hvala, K; Mainil, J

    1994-12-01

    The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents. PMID:7822224

  2. In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

    PubMed Central

    Hong, Ka L.; Battistella, Luisa; Salva, Alysia D.; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved. PMID:25633102

  3. Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

    PubMed

    Kojima, A; Uchida, I; Sekizaki, T; Sasaki, Y; Ogikubo, Y; Tamura, Y

    2001-02-26

    We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens. PMID:11182502

  4. Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model

    PubMed Central

    Lemmens-den Toom, N. A.; Willemse, J.; Koning, R. A.; Demmers, J. A. A.; Dekkers, D. H. W.; Rijkers, E.; El Ghalbzouri, A.; Nibbering, P. H.; van Wamel, W.

    2016-01-01

    Background & Aim The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. Results All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Conclusion Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections. PMID:26741798

  5. A Multi-Omics Approach Identifies Key Hubs Associated with Cell Type-Specific Responses of Airway Epithelial Cells to Staphylococcal Alpha-Toxin

    PubMed Central

    Richter, Erik; Harms, Manuela; Ventz, Katharina; Gierok, Philipp; Chilukoti, Ravi Kumar; Hildebrandt, Jan-Peter; Mostertz, Jörg; Hochgräfe, Falko

    2015-01-01

    Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. PMID:25816343

  6. Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements.

    PubMed

    Legros, Christian; Céard, Brigitte; Vacher, Hélène; Marchot, Pascale; Bougis, Pierre E; Martin-Eauclaire, Marie-France

    2005-05-25

    The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard alpha-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Na(v)) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity. PMID:15725394

  7. Anti-Alpha-Toxin Monoclonal Antibody and Antibiotic Combination Therapy Improves Disease Outcome and Accelerates Healing in a Staphylococcus aureus Dermonecrosis Model

    PubMed Central

    Hilliard, Jamese J.; Datta, Vivekananda; Tkaczyk, Christine; Hamilton, Melissa; Sadowska, Agnieszka; Jones-Nelson, Omari; O'Day, Terrence; Weiss, William J.; Szarka, Szabolcs; Nguyen, Vien; Prokai, Laszlo; Suzich, JoAnn; Stover, C. Kendall

    2014-01-01

    Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections. PMID:25348518

  8. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

    PubMed Central

    Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

    2014-01-01

    Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

  9. Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR).

    PubMed

    Miserez, R; Frey, J; Buogo, C; Capaul, S; Tontis, A; Burnens, A; Nicolet, J

    1998-05-01

    Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the alpha-, beta- and epsilon-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the alpha- and epsilon-toxin genes but were devoid of the beta-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the alpha-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the alpha- and epsilon-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the epsilon-toxin gene, whereas the majority of the colonies were of type A with the alpha-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens. The beta-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation. PMID:9674169

  10. Dissecting the Contributions of Clostridium perfringens Type C Toxins to Lethality in the Mouse Intravenous Injection Model

    PubMed Central

    Fisher, Derek J.; Fernandez-Miyakawa, Mariano E.; Sayeed, Sameera; Poon, Rachael; Adams, Victoria; Rood, Julian I.; Uzal, Francisco A.; McClane, Bruce A.

    2006-01-01

    The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia. PMID:16926413

  11. NetB, a New Toxin That Is Associated with Avian Necrotic Enteritis Caused by Clostridium perfringens

    PubMed Central

    Keyburn, Anthony L; Boyce, John D; Vaz, Paola; Bannam, Trudi L; Ford, Mark E; Parker, Dane; Di Rubbo, Antonio; Rood, Julian I; Moore, Robert J

    2008-01-01

    For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential. PMID:18266469

  12. Clostridium perfringens

    PubMed Central

    Clifford, Walter J.; Anellis, Abe

    1971-01-01

    A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH4)2SO4 and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media. Images PMID:4332043

  13. Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893.

    PubMed

    Tabor, David E; Yu, Li; Mok, Hoyin; Tkaczyk, Christine; Sellman, Bret R; Wu, Yuling; Oganesyan, Vaheh; Slidel, Tim; Jafri, Hasan; McCarthy, Michael; Bradford, Patricia; Esser, Mark T

    2016-09-01

    Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development. PMID:27324766

  14. Targeting Alpha Toxin and ClfA with a Multimechanistic Monoclonal-Antibody-Based Approach for Prophylaxis of Serious Staphylococcus aureus Disease

    PubMed Central

    Tkaczyk, C.; Hamilton, M. M.; Sadowska, A.; Shi, Y.; Chang, C.S.; Chowdhury, P.; Buonapane, R.; Xiao, X.; Warrener, P.; Mediavilla, J.; Kreiswirth, B.; Suzich, J.; Stover, C. K.

    2016-01-01

    ABSTRACT Staphylococcus aureus produces numerous virulence factors, each contributing different mechanisms to bacterial pathogenesis in a spectrum of diseases. Alpha toxin (AT), a cytolytic pore-forming toxin, plays a key role in skin and soft tissue infections and pneumonia, and a human anti-AT monoclonal antibody (MAb), MEDI4893*, has been shown to reduce disease severity in dermonecrosis and pneumonia infection models. However, interstrain diversity and the complex pathogenesis of S. aureus bloodstream infections suggests that MEDI4893* alone may not provide adequate protection against S. aureus sepsis. Clumping factor A (ClfA), a fibrinogen binding protein, is an important virulence factor facilitating S. aureus bloodstream infections. Herein, we report on the identification of a high-affinity anti-ClfA MAb, 11H10, that inhibits ClfA binding to fibrinogen, prevents bacterial agglutination in human plasma, and promotes opsonophagocytic bacterial killing (OPK). 11H10 prophylaxis reduced disease severity in a mouse bacteremia model and was dependent on Fc effector function and OPK. Additionally, prophylaxis with 11H10 in combination with MEDI4893* provided enhanced strain coverage in this model and increased survival compared to that obtained with the individual MAbs. The MAb combination also reduced disease severity in murine dermonecrosis and pneumonia models, with activity similar to that of MEDI4893* alone. These results indicate that an MAb combination targeting multiple virulence factors provides benefit over a single MAb neutralizing one virulence mechanism by providing improved efficacy, broader strain coverage, and protection against multiple infection pathologies. PMID:27353753

  15. Use of alpha-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes.

    PubMed

    Cascante, M; Centelles, J J; Agius, L

    2000-12-15

    We investigated whether hepatocytes permeabilized with alpha-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2, 6-bisphosphate [Fru(2,6)P(2)], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P(2), rates of detritiation of [2-(3)H]glucose and [3-(3)H]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1, 6)P(2), decreased the rate of detritiation of [3-(3)H]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-(3)H]glucose or [3-(3)H]Glc6P as labelled substrate, there was dilution of metabolism of [3-(3)H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P(2). They provide an alternative explanation for the concept that there is more than one Glc6P pool. PMID:11104701

  16. Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

    PubMed Central

    Gierok, Philipp; Harms, Manuela; Richter, Erik; Hildebrandt, Jan-Peter; Lalk, Michael; Mostertz, Jörg; Hochgräfe, Falko

    2014-01-01

    Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o− under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o− cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage. PMID:24733556

  17. Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893

    PubMed Central

    Yu, Li; Mok, Hoyin; Tkaczyk, Christine; Sellman, Bret R.; Wu, Yuling; Oganesyan, Vaheh; Slidel, Tim; Jafri, Hasan; McCarthy, Michael; Bradford, Patricia; Esser, Mark T.

    2016-01-01

    Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development. PMID:27324766

  18. Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

    PubMed

    Albini, S; Brodard, I; Jaussi, A; Wollschlaeger, N; Frey, J; Miserez, R; Abril, C

    2008-02-01

    Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene. PMID:17855025

  19. Epsilon toxin: a fascinating pore-forming toxin.

    PubMed

    Popoff, Michel R

    2011-12-01

    Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons. PMID:21535407

  20. An unusual necrotic myositis by Clostridium perfringens in a German Shepherd dog: A clinical report, bacteriological and molecular identification

    PubMed Central

    Salari Sedigh, Hamideh; Rajabioun, Masoud; Razmyar, Jamshid; Kazemi Mehrjerdi, Hossein

    2015-01-01

    Clostridial myositis, considered to be rare in pet animals, is an acutely fatal toxaemic condition. Some species of clostridia are responsible for necrotic myositis. A 2-year-old male German shepherd dog was admitted with non-weight bearing lameness and massive swelling of the left hind limb. Clostridium perfringens type A with alpha toxin was diagnosed as a pathogenic agent. Based on the history, the bacteria were introduced inside the tissue via contaminated needle following intramuscular injection. Urgent medical therapy followed by surgical intervention was performed. The dog was discharged completely healthy after hospitalization for four weeks. The objective of this report was to describe necrotic myositis in a dog with an emphasis on clinical signs and treatment as well as bacteriological and molecular identification of the micro-organism. Because of the fatal entity of the disease, prompt diagnosis as well as proper and urgent treatment is very important for successful therapy. PMID:26973773

  1. An unusual necrotic myositis by Clostridium perfringens in a German Shepherd dog: A clinical report, bacteriological and molecular identification.

    PubMed

    Salari Sedigh, Hamideh; Rajabioun, Masoud; Razmyar, Jamshid; Kazemi Mehrjerdi, Hossein

    2015-01-01

    Clostridial myositis, considered to be rare in pet animals, is an acutely fatal toxaemic condition. Some species of clostridia are responsible for necrotic myositis. A 2-year-old male German shepherd dog was admitted with non-weight bearing lameness and massive swelling of the left hind limb. Clostridium perfringens type A with alpha toxin was diagnosed as a pathogenic agent. Based on the history, the bacteria were introduced inside the tissue via contaminated needle following intramuscular injection. Urgent medical therapy followed by surgical intervention was performed. The dog was discharged completely healthy after hospitalization for four weeks. The objective of this report was to describe necrotic myositis in a dog with an emphasis on clinical signs and treatment as well as bacteriological and molecular identification of the micro-organism. Because of the fatal entity of the disease, prompt diagnosis as well as proper and urgent treatment is very important for successful therapy. PMID:26973773

  2. Membrane vesicles of Clostridium perfringens Type A strains induce innate and adaptive immunity

    PubMed Central

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L.; Curtiss, Roy

    2014-01-01

    Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection. PMID:24631214

  3. Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia

    PubMed Central

    Hashiba, Masamitsu; Tomino, Atsutoshi; Takenaka, Nobuyoshi; Hattori, Tomonori; Kano, Hideki; Tsuda, Masanobu; Takeyama, Naoshi

    2016-01-01

    Patient: Male, 82 Final Diagnosis: Clostridium perfringens infection Symptoms: Anemia • fever • shock Medication: — Clinical Procedure: Antimicrobial chemotherapy Specialty: Infectious Diseases Objective: Rare disease Background: Clostridium perfringens (C. perfringens) can cause various infections, including gas gangrene, crepitant cellulitis, and fasciitis. While C. perfringens sepsis is uncommon, it is often rapidly fatal because the alpha toxin of this bacterium induces massive intravascular hemolysis by disrupting red blood cell membranes. Case Report: We present the case of a male patient with diabetes who developed a fatal liver abscess with massive intravascular hemolysis and septic shock caused by toxigenic C. perfringens. The peripheral blood smear showed loss of central pallor, with numerous spherocytes. Multiplex PCR only detected expression of the cpa gene, indicating that the pathogen was C. perfringens type A. Conclusions: C. perfringens infection should be considered in a febrile patient who has severe hemolytic anemia with a very low MCV, hemolyzed blood sample, and negative Coombs test. The characteristic peripheral blood smear findings may facilitate rapid diagnosis. PMID:27049736

  4. The NanI and NanJ Sialidases of Clostridium perfringens Are Not Essential for Virulence▿

    PubMed Central

    Chiarezza, Martina; Lyras, Dena; Pidot, Sacha J.; Flores-Díaz, Marietta; Awad, Milena M.; Kennedy, Catherine L.; Cordner, Leanne M.; Phumoonna, Tongted; Poon, Rachael; Hughes, Meredith L.; Emmins, John J.; Alape-Girón, Alberto; Rood, Julian I.

    2009-01-01

    The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence. PMID:19651873

  5. The Mycotoxin Deoxynivalenol Predisposes for the Development of Clostridium perfringens-Induced Necrotic Enteritis in Broiler Chickens

    PubMed Central

    Antonissen, Gunther; Ducatelle, Richard; Haesebrouck, Freddy; Timbermont, Leen; Verlinden, Marc; Janssens, Geert Paul Jules; Eeckhaut, Venessa; Eeckhout, Mia; De Saeger, Sarah; Hessenberger, Sabine; Martel, An; Croubels, Siska

    2014-01-01

    Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens. PMID:25268498

  6. Characterization of toxin plasmids in Clostridium perfringens type C isolates.

    PubMed

    Gurjar, Abhijit; Li, Jihong; McClane, Bruce A

    2010-11-01

    Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence. PMID:20823204

  7. Clostridium Difficile Infections

    MedlinePlus

    Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Nausea Abdominal pain or tenderness You might get C. difficile disease if you have an illness that ...

  8. Collagenase Clostridium Histolyticum Injection

    MedlinePlus

    ... disease (a thickening of tissue [plaque] inside the penis that causes the penis to curve). Collagenase Clostridium histolyticum injection is in ... the plaque of thickened tissue and allows the penis to be straightened.

  9. Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D.

    PubMed

    Uzal, Francisco A; Fisher, Derek J; Saputo, Juliann; Sayeed, Sameera; McClane, Bruce A; Songer, Glenn; Trinh, Hien T; Fernandez Miyakawa, Mariano E; Gard, Sharon

    2008-09-01

    Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa. PMID:18776108

  10. Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates

    PubMed Central

    Vidal, Jorge E.; Ohtani, Kaori; Shimizu, Tohru; McClane, Bruce A.

    2009-01-01

    Clostridium perfringens type C isolates cause necrotizing enteritis in humans and domestic animals. In vitro, type C isolates often produce beta toxin (CPB), beta2 toxin (CPB2), alpha toxin (CPA), perfringolysin O (PFO), and TpeL during (or after) late log-phase growth. In contrast, the current study found that many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. However, the luxS quorum sensing system is not required for the rapid upregulation of type C toxin production induced by contact with Caco-2 cells. These results provide the first indication of host cell:pathogen cross-talk affecting toxin production kinetics by any pathogenic Clostridium spp., identify in vivo vs. in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases. PMID:19438515

  11. Recent progress in understanding the pathogenesis of Clostridium perfringens type C infections

    PubMed Central

    McClane, B. A.

    2011-01-01

    Clostridium perfringens type C causes necrotizing enteritis in humans and several other animal species. Type C isolates must produce at least beta toxin (CPB) and alpha toxin (CPA) and most strains produce several other toxins including perfringolysin O (PFO) and TpeL. However, current evidence indicates that CPB is the main virulence factor for type C infections. Most of this evidence is based upon the loss of virulence shown by isogenic type C CPB knock out mutants on cells, and also in rabbit intestinal loops and in mouse models. This virulence is regained when these mutants are complemented with the wild-type cpb gene. Many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. PMID:21420802

  12. [Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica].

    PubMed

    Gamboa-Coronado, María del Mar; Mau-Inchaustegui, Silvia; Rodríguez-Cavallini, Evelyn

    2011-12-01

    Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments. PMID:22208067

  13. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

    PubMed Central

    Gohari, Iman Mehdizadeh; Arroyo, Luis; MacInnes, Janet I.; Timoney, John F.; Parreira, Valeria R.; Prescott, John F.

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  14. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis.

    PubMed

    Gohari, Iman Mehdizadeh; Arroyo, Luis; Macinnes, Janet I; Timoney, John F; Parreira, Valeria R; Prescott, John F

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  15. Bacteriophages of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  16. Clostridium tetani bacteraemia.

    PubMed

    Hallit, Rabih Riad; Afridi, Muhammad; Sison, Raymund; Salem, Elie; Boghossian, Jack; Slim, Jihad

    2013-01-01

    Tetanus is a neuromuscular disease in which Clostridium tetani exotoxin (tetanospasmin) produces muscle spasms, incapacitating its host. To our knowledge, C. tetani bacteraemia has never been reported in the literature. The ideal management of this entity remains unresolved given that there is no literature to guide the therapy. PMID:22977074

  17. Ca/sup 2 +/-stimulated catecholamine release from. cap alpha. -toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G protein

    SciTech Connect

    Ahnert-Hilger, G.; Braeutigam, M.; Gratzl, M.

    1987-12-01

    Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in ..cap alpha..-toxin-permeabilized cells with micromolar levels of free Ca/sup 2 +/. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca/sup 2 +/. Both types of experiments provide direct evidence for the operation of Ca/sup 2 +/-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca/sup 2 +/ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below ..mu..M free Ca/sup 2 +/ and plateaus at 1-5 ..mu..M free Ca/sup 2 +/, while a second occurs in the presence of larger amounts of free Ca/sup 2 +/ (10-100 ..mu..M). Ca/sup 2 +/-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg/sup 2 +//ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca/sup 2 +/-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.

  18. Clostridium difficile infection

    PubMed Central

    Viswanathan, VK; Mallozzi, MJ

    2010-01-01

    Clostridium difficile infection (CDI) is the primary cause of antibiotic-associated diarrhea and is a significant nosocomial disease. In the past ten years, variant toxin-producing strains of C. difficile have emerged, that have been associated with severe disease as well as outbreaks worldwide. This review summarizes current information on C. difficile pathogenesis and disease, and highlights interventions used to combat single and recurrent episodes of CDI. PMID:21327030

  19. Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model.

    PubMed

    Fernandez-Miyakawa, Mariano E; Jost, B Helen; Billington, Stephen J; Uzal, Francisco A

    2008-03-18

    Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats. PMID:17997054

  20. [Oncologic aspects of Clostridium difficile].

    PubMed

    Telekes, András

    2016-07-01

    Clostridium difficile infection is one of the most frequent among cancer patients. Its diagnosis is complicated by the fact that the symptoms of the infection and the side effects of the anticancer treatments could be similar. Chemotherapy itself might facilitate Clostridium difficile infection. Several risk factors are known but Clostridium difficile infection can develop in the absence of these. Neutreopenia is a risk factor for fatal Clostridium difficile infection and also the side effect of chemotherapy. Therefore, if symptoms of the potential infection develop (eg. diarrhoea more than three times a day, fever above 38.5 °C, colitis, rapid increase of serum creatinin) Clostridium difficile infection should be excluded. If the infection is confirmed it should be managed in the most efficient way. Orv. Hetil., 2016, 157(28), 1110-1116. PMID:27397423

  1. A live oral recombinant Salmonella enterica serovar typhimurium vaccine expressing Clostridium perfringens antigens confers protection against necrotic enteritis in broiler chickens.

    PubMed

    Kulkarni, R R; Parreira, V R; Jiang, Y-F; Prescott, J F

    2010-02-01

    Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium chi9352 vaccine vehicle. The expression of these Clostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle. PMID:20007363

  2. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov.

    PubMed

    Keis, S; Shaheen, R; Jones, D T

    2001-11-01

    On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species. In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp. represented by the strains NCP 262T and NRRL B643 are compared. In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing. These included 14 C. beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation. All of the C. beijerinckii strains were confirmed to have been classified correctly. The 'C. kaneboi' and 'C. butanologenum' strains require reclassification as C. acetobutylicum and C. beijerinckii, respectively. The commercial production strains were found to belong either to C. beijerinckii or to the unnamed Clostridium sp. For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species. These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease. On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species. The descriptions of C. acetobutylicum and C. beijerinckii have been emended. The names Clostridium saccharoperbutylacetonicum sp. nov. [type strain = N1-4 (HMT) = ATCC 27021T] and Clostridium saccharobutylicum sp. nov. (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species. PMID:11760952

  3. Enterotoxigenicity and Genetic Relatedness of Clostridium perfringens Isolates from Retail Foods in the United States

    PubMed Central

    Lin, Yuan-Tong; Labbe, Ronald

    2003-01-01

    Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45°C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa. None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe+ strains in retail foods and the genetic diversity among nonoutbreak strains. PMID:12620854

  4. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

    PubMed

    Collier, C T; Hofacre, C L; Payne, A M; Anderson, D B; Kaiser, P; Mackie, R I; Gaskins, H R

    2008-03-15

    This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE. PMID:18068809

  5. Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran

    PubMed Central

    Akhi, Mohammad Taghi; Bidar Asl, Saeid; Pirzadeh, Tahereh; Naghili, Behruz; Yeganeh, Fatemeh; Memar, Yousef; Mohammadzadeh, Yalda

    2015-01-01

    Background: Clostridium perfringens, a Gram-positive, anaerobic bacterium that produces at least 16 virulence factors including 12 toxins (α-ν), enterotoxin, hemolysin and neuraminidase, can create variable pathogenic condition, ranging from a food poisoning to life-threatening myonecrosis. Among C. perfringens strains, resistance to the drug choices such as penicillin as well as to alternatives of penicillin like metronidazole and clindamycin has also been observed. Objectives: The aim of this study was to determine the resistance of isolated toxigenic and non-toxigenic C. perfringens strains against common antimicrobial agents. Materials and Methods: In this descriptive study, a total of 136 stool specimens were collected. At first, cooked meat medium enrichment method was performed on samples at 45°C. Thereafter, a loopful of the enriched culture was transferred to blood agar and incubated anaerobically at 37°C for 24-72 hours. Colonies with double zone of hemolysis were identified by different biochemical tests such as phospholipase C (lecithinase) test, indole and urease production. The Minimum Inhibitory Concentration (MIC) for common antibiotics was determined by Etests (Epsilometer) and duplex Polymerase Chain Reaction (PCR) reaction was performed with specific primers for amplification of cpe (426 bp) and plc (283 bp) Genes. Results: Of 136 stool samples including diarrhea [48] and non-diarrhea [88] ones, 83 (61.02%) C. perfringens were cultured. Of these 83, 79 C. perfringens isolates showed the alpha-toxin (phospholipase C) production gene by PCR. Respectively, 3 (9.09%) and 2 (4.34%) cpe genes were present in diarrhea and non-diarrhea samples. Of 79 isolates of C. perfringens, 34 (43.03%) cases showed no resistance, 18 (22.78%) had one resistance and 27 (34.17%) isolates had multiple resistance to imipenem, metronidazole, ceftriaxone, clindamycin, chloramphenicol, and penicillin. Conclusions: Periodic evaluation of antimicrobial susceptibility for C

  6. Effect of tannins on the in vitro growth of Clostridium perfringens.

    PubMed

    Elizondo, Ana M; Mercado, Elsa C; Rabinovitz, Bettina C; Fernandez-Miyakawa, Mariano E

    2010-10-26

    Vegetable tannins are water-soluble polyphenolic compounds of varying molecular weights that occur abundantly in nature. The diet of many free-ranging wild animals contains significant amounts of tannins. Also, commercial tannins are used in animal industry as food additives to improve animal performance. In order to further determine the capacity of tannins to inhibit the development of intestinal diseases produced by Clostridium pefringens, we evaluated here the effect of tannins from quebracho, chestnut or combinations of both on C. perfringens and their toxins. The C. perfringens (types A, B, C, D and E) growth obtained from the intestine of healthy and diseased animals was reduced in a dose-dependent manner in the presence of quebracho tannins, chestnut tannins, combinations of both or a commercial formula based in these tannins. Although the minimal inhibitory concentration of both tannins varied between isolates, no statistically significant differences were observed between isolates from healthy or sick animals. Comparative analysis showed that the concentrations of quebracho tannin inhibiting the growth of C. perfringens were higher than chestnut tannin. In fact, antibacterial effect of quebracho tannin was increased up to 20 times with the addition of 25% of chestnut tannin and 85 times with 75% of chestnut tannin. Antibacterial activity of the commercial product was up to ~50 times higher than quebracho tannin alone. Quebracho tannin showed partial bactericidal activity, whereas chestnut tannin activity was stronger. Both tannins were able to reduce the alpha toxin lecithinase activity and epsilon toxin cytotoxicity in MDCK cells. These results suggest that tannin-supplemented diet could be useful to prevent some clostridial diseases. PMID:20471759

  7. Vaccines against Clostridium difficile

    PubMed Central

    Leuzzi, Rosanna; Adamo, Roberto; Scarselli, Maria

    2014-01-01

    Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens. PMID:24637887

  8. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Clostridium difficile Toxin B and Sensitive Detection in Human Fecal Matter

    PubMed Central

    Maher, Eamonn; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Toxin B is one of the major virulence factors of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity (Kd = 47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations. PMID:25734010

  9. Clostridium difficile infection.

    PubMed

    Smits, Wiep Klaas; Lyras, Dena; Lacy, D Borden; Wilcox, Mark H; Kuijper, Ed J

    2016-01-01

    Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota. PMID:27158839

  10. Genomics of Clostridium tetani.

    PubMed

    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. PMID:25638019

  11. [Toxins of Clostridium perfringens].

    PubMed

    Morris, W E; Fernández-Miyakawa, M E

    2009-01-01

    Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present. PMID:20085190

  12. Isolation of Clostridium thermocellum auxotrophs

    SciTech Connect

    Mendez, B.S.; Gomez, R.F.

    1982-02-01

    The conversion of biomass of fuels and chemical feedstocks by microbial fermentation offers the potential of solving two of today's important problems: waste accumulation and exhaustion of fossil fuels. Microorganisms with the capabilities of converting biomass components such as cellulos and hemicellulose to chemicals and fuels in a single step are of particular interest. One such microorganism is Clostridium thermocellum, a thermophilic anaerobe which degrades cellulose to ethanol and organic acids. For efficient industrial use, the cellulolytic capacity of this strain must be improved by genetic means. Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.

  13. Autism and Clostridium tetani.

    PubMed

    Bolte, E R

    1998-08-01

    Autism is a severe developmental disability believed to have multiple etiologies. This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals. A significant percentage of individuals with autism have a history of extensive antibiotic use. Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens. Clostridium tetani is an ubiquitous anaerobic bacillus that produces a potent neurotoxin. Intestinal colonization by C. tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells. The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS. This route bypasses TeNT's normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident. Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism. Lab animals injected in the brain with TeNT have exhibited many of these behaviors. Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia. When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis. A review of atypical tetanus cases, and strategies to test the validity of this paper's hypothesis, are included. PMID:9881820

  14. Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed.

    PubMed

    Kanakaraj, R; Harris, D L; Songer, J G; Bosworth, B

    1998-08-28

    A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes (cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2 x 10(4) colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens. PMID:9810619

  15. Genome sequencing and analysis of a type A Clostridium perfringens isolate from a case of bovine clostridial abomasitis.

    PubMed

    Nowell, Victoria J; Kropinski, Andrew M; Songer, J Glenn; MacInnes, Janet I; Parreira, Valeria R; Prescott, John F

    2012-01-01

    Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified. PMID:22412860

  16. Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants

    PubMed Central

    Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

    2016-01-01

    Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals. PMID:27004612

  17. CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Ma, Menglin; Sarker, Mahfuzur R.; McClane, Bruce A.

    2013-01-01

    ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. PMID:24105766

  18. Development and application of new mouse models to study the pathogenesis of Clostridium perfringens type C Enterotoxemias.

    PubMed

    Uzal, Francisco A; Saputo, Juliann; Sayeed, Sameera; Vidal, Jorge E; Fisher, Derek J; Poon, Rachael; Adams, Vicki; Fernandez-Miyakawa, Mariano E; Rood, Julian I; McClane, Bruce A

    2009-12-01

    Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality. PMID:19805537

  19. Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period

    PubMed Central

    Shindo, Yuji; Dobashi, Yoh; Sakai, Toshiyasu; Monma, Chie; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Background: Although Clostridium perfringens (C. perfringens) is well known as the causative agent of several forms of enteric disease, precise epidemiological and pathobiological aspects are still unknown. Methods: We retrospectively reviewed the culture results of samples collected in our hospital from 2001 through 2013. In addition, for the detection and toxinogenic typing of C. perfringens, polymerase-chain-reaction amplification (PCR)-based rapid analysis was performed in 6 cases using DNA extracted from paraffin-embedded tissues. Results: A total of 35 samples from 33 cases were positive for C. perfringens, representing an incidence of 0.017% (35/205, 114). Among 33 patients, 21 patients manifested sepsis and 7 patients had bacteremia. One of the septic cases was complicated by fatal intravascular hemolysis and thus, the prevalence was estimated at 3.0% among C. perfringens infections (1/33). The direct causative disease or state for C. perfringens infection was identified in 18 patients: surgery or intervention for cancers, 8 patients; chemotherapy for cancer, 2 patients; surgery or intervention for non-neoplastic disease, 6 patients; liver cirrhosis, 3 patients, etc. PCR-based toxinogenic typing of C. perfringens detected the alpha-toxin gene only in tissue from a patient who died of massive hemolysis; none of the toxin genes could be amplified in the other 5 cases examined. Conclusions: The prevalence of overt C. perfringens infection is low, but upon detection, infected patients should be carefully monitored for fatal acute hemolysis caused by type A C. perfringens. Furthermore, PCR-based rapid detection of C. perfringens and toxinogenic typing by archival pathological material is applicable as a diagnostic tool. PMID:25755747

  20. Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias▿

    PubMed Central

    Uzal, Francisco A.; Saputo, Juliann; Sayeed, Sameera; Vidal, Jorge E.; Fisher, Derek J.; Poon, Rachael; Adams, Vicki; Fernandez-Miyakawa, Mariano E.; Rood, Julian I.; McClane, Bruce A.

    2009-01-01

    Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality. PMID:19805537

  1. Electrotransformation of Clostridium thermocellum.

    PubMed

    Tyurin, Michael V; Desai, Sunil G; Lynd, Lee R

    2004-02-01

    Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign

  2. Clostridium perfringens in retail chicken.

    PubMed

    Nowell, Victoria J; Poppe, Cornelis; Parreira, Valeria R; Jiang, Yan-Fen; Reid-Smith, Richard; Prescott, John F

    2010-06-01

    Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% "atypical" genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken. PMID:19961943

  3. Clostridium difficile and the microbiota

    PubMed Central

    Seekatz, Anna M.; Young, Vincent B.

    2014-01-01

    Clostridium difficile infection (CDI) is the leading health care–associated illness. Both human and animal models have demonstrated the importance of the gut microbiota’s capability of providing colonization resistance against C. difficile. Risk factors for disease development include antibiotic use, which disrupts the gut microbiota, leading to the loss of colonization resistance and subsequent CDI. Identification of the specific microbes capable of restoring this function remains elusive. Future studies directed at how microbial communities influence the metabolic environment may help elucidate the role of the microbiota in disease development. These findings will improve current biotherapeutics for patients with CDI, particularly those with recurrent disease. PMID:25036699

  4. Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.

    PubMed

    Connan, Chloé; Denève, Cécile; Mazuet, Christelle; Popoff, Michel R

    2013-12-01

    Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. PMID:23769754

  5. Role of the Agr-like quorum-sensing system in regulating toxin production by Clostridium perfringens type B strains CN1793 and CN1795.

    PubMed

    Chen, Jianming; McClane, Bruce A

    2012-09-01

    Clostridium perfringens type B causes enteritis and enterotoxemia in domestic animals. By definition, these bacteria must produce alpha toxin (CPA), beta toxin (CPB) and epsilon toxin (ETX) although most type B strains also produce perfringolysin O (PFO) and beta2 toxin (CPB2). A recently identified Agr-like quorum-sensing (QS) system in C. perfringens controls all toxin production by surveyed type A, C, and D strains, but whether this QS is involved in regulating toxin production by type B strains has not been explored. Therefore, the current study introduced agrB null mutations into type B strains CN1795 and CN1793. Both type B agrB null mutants exhibited reduced levels of CPB, PFO, and CPA in their culture supernatants, and this effect was reversible by complementation. The reduced presence of CPB in culture supernatant involved decreased cpb transcription. In contrast, the agrB null mutants of both type B strains retained wild-type production levels of ETX and CPB2. In a Caco-2 cell model of enteritis, culture supernatants of the type B agrB null mutants were less cytotoxic than supernatants of their wild-type parents. However, in an MDCK cell in vitro model for enterotoxemic effects, supernatants from the agrB null mutants or wild-type parents were equally cytotoxic after trypsin activation. Coupling these and previous results, it is now evident that strain-dependent variations exist in Agr-like QS system regulation of C. perfringens toxin production. The cell culture results further support a role for trypsin in determining which toxins contribute to disease involving type B strains. PMID:22689820

  6. Role of the Agr-Like Quorum-Sensing System in Regulating Toxin Production by Clostridium perfringens Type B Strains CN1793 and CN1795

    PubMed Central

    Chen, Jianming

    2012-01-01

    Clostridium perfringens type B causes enteritis and enterotoxemia in domestic animals. By definition, these bacteria must produce alpha toxin (CPA), beta toxin (CPB) and epsilon toxin (ETX) although most type B strains also produce perfringolysin O (PFO) and beta2 toxin (CPB2). A recently identified Agr-like quorum-sensing (QS) system in C. perfringens controls all toxin production by surveyed type A, C, and D strains, but whether this QS is involved in regulating toxin production by type B strains has not been explored. Therefore, the current study introduced agrB null mutations into type B strains CN1795 and CN1793. Both type B agrB null mutants exhibited reduced levels of CPB, PFO, and CPA in their culture supernatants, and this effect was reversible by complementation. The reduced presence of CPB in culture supernatant involved decreased cpb transcription. In contrast, the agrB null mutants of both type B strains retained wild-type production levels of ETX and CPB2. In a Caco-2 cell model of enteritis, culture supernatants of the type B agrB null mutants were less cytotoxic than supernatants of their wild-type parents. However, in an MDCK cell in vitro model for enterotoxemic effects, supernatants from the agrB null mutants or wild-type parents were equally cytotoxic after trypsin activation. Coupling these and previous results, it is now evident that strain-dependent variations exist in Agr-like QS system regulation of C. perfringens toxin production. The cell culture results further support a role for trypsin in determining which toxins contribute to disease involving type B strains. PMID:22689820

  7. Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences

    PubMed Central

    Manich, Maria; Knapp, Oliver; Gibert, Maryse; Maier, Elke; Jolivet-Reynaud, Colette; Geny, Blandine; Benz, Roland; Popoff, Michel R.

    2008-01-01

    Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes GM2 as receptor and forms anion-selective channels. PMID:19018299

  8. Clostridium thermosaccharolyticum strain deficient in acetate production

    SciTech Connect

    Rothstein, D.M.

    1986-01-01

    A mutant of Clostridium thermosaccharolyticum that is blocked in acetate production was isolated after treatment with nitrosoguanidine and selection for fluoroacetate resistance. The mutant produced more ethanol than the parent strain did.

  9. Clostridium difficile and C. difficile Toxin Testing

    MedlinePlus

    ... C diff antigen; GDH Formal name: Clostridium difficile Culture; C. difficile Toxin, A and B; C. difficile Cytotoxin Assay; Glutamate Dehydrogenase Test Related tests: Stool Culture ; O&P At a Glance Test Sample The ...

  10. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations ...

  11. Clostridium difficile in paediatric populations

    PubMed Central

    Allen, Upton D

    2014-01-01

    An increase in Clostridium difficile infection incidence has been observed among hospitalized children in the United States. The present statement, targeted at clinicians caring for infants and children in community and institutional settings, summarizes the relevant information relating to the role of C difficile in childhood diarrhea and provides recommendations for diagnosis, prevention and treatment. Significant differences between adult and paediatric risk factors and disease are discussed, along with emerging therapies. The relationship between age and disease severity in children with a newly emergent and more fluoroqinolone-resistant strain of C difficile (North American Pulse-field type-1 [NAP1]) remains unknown. The importance of antimicrobial stewardship as a preventive strategy is highlighted. This statement replaces a previous Canadian Paediatric Society position statement on C difficile published in 2000. PMID:24627655

  12. Clostridium perfringens Type C Enterotoxemia.

    PubMed

    Niilo, L

    1988-08-01

    Forms of enteric disease caused by Clostridium perfringens type C are critically reviewed with emphasis on practical aspects and recent research findings. Available data indicate that more animal species may be fatally infected by type C of this organism than by any other type of C. perfringens. Fatal cases have been recorded in pigs, cattle, sheep, horses and humans. Newborn animals are typically the most susceptible, possibly related to aspects of bacterial colonization, intestinal digestive functions, and to some other, unexplained, factors. Both beta toxin and the bacterial cells are required to initiate pathogenesis at the tips of jejunal villi, and subsequent massive adherence of these cells to necrotic mucosa is a characteristic feature. Although major lesions occur in the intestine, death is due to toxemia. The disease can be effectively controlled by vaccination of the dam. Epizootiology of this disease is a possible area for further studies. PMID:17423103

  13. Phosphotransacetylase from Clostridium acidiurici1

    PubMed Central

    Robinson, James R.; Sagers, Richard D.

    1972-01-01

    The phosphotransacetylase from Clostridium acidiurici has two properties not observed for this enzyme in other bacteria: (i) it requires a divalent metal for activity, and (ii) it is not subject to uncoupling by arsenate. The enzyme has been obtained in highly purified form, with a specific activity 500-fold higher than crude extracts. Ferrous or manganous ions are required for maximal activity, with Mn2+ being 50 to 75% as effective as Fe2+. The acetyl group can be transferred from acetyl phosphate to coenzyme A in 20 mm arsenate without a net decrease in high-energy acyl linkages. Likewise, H32PO42− will exchange with acetyl-PO42− in the presence of arsenate without loss of acetyl phosphate. This suggests that the active site on the enzyme is capable of discriminating between phosphate and arsenate while permitting the reversible transfer of acyl groups between CoA and phosphate. Images PMID:16559158

  14. Clostridium difficile infection in patients with inflammatory bowel disease

    PubMed Central

    Biesiada, Grażyna; Perucki, William; Mach, Tomasz

    2014-01-01

    Clostridium difficile is a bacterium widely distributed in the human environment. In the last decade the incidence and severity of Clostridium difficile infection has grown, particularly in Europe and North America, making it one of the more common nosocomial infections. A group particularly susceptible to Clostridium difficile infection are patients with inflammatory bowel disease, especially those with involvement of the colon. This paper presents relevant data on Clostridium difficile infections in inflammatory bowel disease patients, including epidemiology, pathogenesis, diagnosis and treatment. PMID:25097707

  15. Switchgrass (Panicum virgatum) fermentation by sequential culture of Clostridium thermocellum and Clostridium beijerinckii: effect of particle size on gas production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fuel alcohols can be produced by fermenting cellulosic biomass. Clostridium beijerinckii produces both ethanol and butanol, but it is non-cellulolytic. Cellulose requires saccharification prior to fermentation by C. beijerinckii. In contrast, the thermophile, Clostridium thermocellum, is highly ce...

  16. The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation in Clostridium perfringens

    PubMed Central

    Varga, John; Stirewalt, Veronica L.; Melville, Stephen B.

    2004-01-01

    repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin). PMID:15292123

  17. Faecal carriage of Clostridium perfringens.

    PubMed Central

    Stringer, M. F.; Watson, G. N.; Gilbert, R. J.; Wallace, J. G.; Hassall, J. E.; Tanner, E. I.; Webber, P. P.

    1985-01-01

    The numbers and serotypes of Clostridium perfringens present in the faeces of three groups of hospital patients and young healthy laboratory workers were examined in studies lasting between 10 and 13 weeks. In one hospital some long-stay geriatric patients carried relatively high numbers of C. perfringens (greater than 10(7)/g) most of the time and it was not unusual in any one week for the majority of these patients to carry the same serotype(s). However, the numbers of C. perfringens in the faeces of young long-stay patients in the same hospital were in the range of 10(3)-10(4)/g and carriage of common serotypes was not observed. These results were similar to the findings with the young laboratory workers. This investigation indicates that two of the laboratory criteria often used in the investigation of C. perfringens food poisoning, i.e. faecal counts of greater than or equal to 10(5) C. perfringens/g and patients carrying the same serological type need to be interpreted with caution with suspected outbreaks involving some groups of geriatric long-stay hospital patients. PMID:2866214

  18. Clostridium difficile infections in China.

    PubMed

    Jin, Ke; Wang, Shixia; Huang, Zuhu; Lu, Shan

    2010-11-01

    Clostridium difficile (C. difficile) infection has become one of the major hospital-associated infections in Western countries in the last two decades. However, there is limited information on the status of C. difficile infection in Chinese healthcare settings. Given the large and increasing elderly population and the well-recognized problem of over-prescribing of broad spectrum antibiotics in China, it is critical to understand the epidemiology and potential risk factors that may contribute to C. difficile infection in China. A literature review of available published studies, including those in Chinese language-based journals, was conducted. A review of the currently available literature suggested the presence of C. difficile infections in China, but also suggested that these infections were not particularly endemic. This finding should lead to better designed and greatly expanded studies to provide a more reliable epidemiologically-based conclusion on the actual status of C. difficile infection in China, including the identification of any associated risk factors. Such information is ultimately valuable to develop appropriate strategies to prevent C. difficile infection and the vast negative impact of such infections in China and other developing countries. PMID:23554657

  19. Toxin plasmids of Clostridium perfringens.

    PubMed

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  20. Clostridium difficile infection in Thailand.

    PubMed

    Putsathit, Papanin; Kiratisin, Pattarachai; Ngamwongsatit, Puriya; Riley, Thomas V

    2015-01-01

    Clostridium difficile is the aetiological agent in ca. 20% of cases of antimicrobial-associated diarrhoea in hospitalised adults. Diseases caused by this organism range from mild diarrhoea to occasional fatal pseudomembranous colitis. The epidemiology of C. difficile infection (CDI) has changed notably in the past decade, following epidemics in the early 2000s of PCR ribotype (RT) 027 infection in North America and Europe, where there was an increase in disease severity and mortality. Another major event has been the emergence of RT 078, initially as the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Although there have been numerous investigations of the epidemiology of CDI in North America and Europe, limited studies have been undertaken elsewhere, particularly in Asia. Antimicrobial exposure remains the major risk factor for CDI. Given the high prevalence of indiscriminate and inappropriate use of antimicrobials in Asia, it is conceivable that CDI is relatively common among humans and animals. This review describes the level of knowledge in Thailand regarding C. difficile detection methods, prevalence and antimicrobial susceptibility profile, as well as the clinical features of, treatment options for and outcomes of the disease. In addition, antimicrobial usage in livestock in Thailand will be reviewed. A literature search yielded 18 studies mentioning C. difficile in Thailand, a greater number than from any other Asian country. It is possible that the situation in Thailand in relation to CDI may mirror the situation in other developing Asians countries. PMID:25537687

  1. Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

    PubMed Central

    Diekert, Gabriele B.; Thauer, Rudolf K.

    1978-01-01

    Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO2. Rates up to 0.4 μmol min−1 mg of wet cells−1 were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO2 reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min−1 mg of protein−1 (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO2 to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO2 do not possess a CO-oxidizing system. PMID:711675

  2. Alpha-toxin promotes Staphylococcus aureus mucosal biofilm formation.

    PubMed

    Anderson, Michele J; Lin, Ying-Chi; Gillman, Aaron N; Parks, Patrick J; Schlievert, Patrick M; Peterson, Marnie L

    2012-01-01

    Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla(+)) and low (hla(-)) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla(-)), MNPE (hla(+)), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1-5 × 10(8) CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla(-)), MN8 (hla(-)), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla(-)) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV). PMID:22919655

  3. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    PubMed Central

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  4. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    PubMed

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  5. Clostridium difficile recurrences in Stockholm.

    PubMed

    Sandell, Staffan; Rashid, Mamun-Ur; Jorup-Rönström, Christina; Ellström, Kristina; Nord, Carl Erik; Weintraub, Andrej

    2016-04-01

    Sixty-eight hospital-admitted patients with a first episode of Clostridium difficile infection (CDI) were included and followed up during 1 year. Faeces samples were collected at 1, 2, 6 and 12 months after inclusion and analyzed for the presence of C. difficile toxin B, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped and the MICs of the isolates were determined against eight antimicrobial agents. In 68 patients initially included, antibiotics, clinical signs and co-morbidities were analyzed and 56 were evaluable for recurrences. The mean number of different antibiotics given during 3 months prior to inclusion was 2.6 (range 0-6). Six patients had not received any antibiotics and three of them had diagnosed inflammatory bowel disease. Thirty-two patients (57%) had either a microbiological or clinical recurrence, 16 of whom had clinical recurrences that were confirmed microbiologically (13, 23%) or unconfirmed by culture (3, 5%). Twenty-nine patients were positive in at least one of the follow-up tests, 16 had the same ribotype in follow-up tests, i.e. relapse, and 13 a different ribotype, i.e., reinfection. Most common ribotypes were 078/126, 020, 023, 026, 014/077, 001 and 005. No strain of ribotype 027 was found. Strains ribotype 078/126 and 023 were positive for binary toxin and were the strains most prone to cause recurrence. All strains were sensitive to vancomycin and metronidazole. Patients with recurrences were significantly older (p = 0.02) and all patients had a high burden of comorbidities, which could explain the high fatality rate, 26 (38%) patients died during the 1-year follow-up. PMID:26802875

  6. Cellulolytic Activity of Clostridium acetobutylicum

    PubMed Central

    Lee, Song F.; Forsberg, Cecil W.; Gibbins, L. N.

    1985-01-01

    Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism. PMID:16346847

  7. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  8. Clostridium difficile phages: still difficult?

    PubMed Central

    Hargreaves, Katherine R.; Clokie, Martha R. J.

    2014-01-01

    Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

  9. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enteritis infection among chickens ...

  10. Coculture Production of Butanol by Clostridium Bacteria

    NASA Technical Reports Server (NTRS)

    Bergstrom, S. L.; Foutch, G. L.

    1985-01-01

    Production of butanol by anaerobic fermentation of sugars enhanced by use of two Clostridium species, one of which feeds on metabolic product of other. Renewed interest in fermentation process for making butanol stimulated by potential use of butanol as surfactant in enhanced oil recovery. Butanol also used as fuel or as chemical feedstock and currently produced synthetically from petroleum.

  11. Clostridium difficile in poultry and poultry meat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer t...

  12. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease and gas gangrene among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enterit...

  13. Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

    PubMed Central

    Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2013-01-01

    Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD+ and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2. PMID:23872566

  14. Epsilon-Toxin Production by Clostridium perfringens Type D Strain CN3718 Is Dependent upon the agr Operon but Not the VirS/VirR Two-Component Regulatory System

    PubMed Central

    Chen, Jianming; Rood, Julian I.; McClane, Bruce A.

    2011-01-01

    ABSTRACT Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. PMID:22167225

  15. Complete Genome Sequence of Clostridium clariflavum DSM 19732

    SciTech Connect

    Goodwin, Lynne A.; Davenport, Karen W.; Teshima, Hazuki; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Han, James; Pitluck, Sam; Nolan, Matt; Chen, Amy; Huntemann, Marcel; Mavromatis, K; Mikhailova, Natalia; Liolios, Konstantinos; Woyke, Tanja; Lynd, Lee R

    2012-01-01

    Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 proteincoding and 98 RNA genes, for the type strain DSM 19732.

  16. A case if infant botulism due to neurotoxigenic Clostridium butyricum type E associated with Clostridium difficile colitis.

    PubMed

    Fenicia, L; Da Dalt, L; Anniballi, F; Franciosa, G; Zanconato, S; Aureli, P

    2002-10-01

    Reported here is the sixth case of intestinal toxemia botulism caused by Clostridium butyricum type E in Italy since 1984. In this case, the patient was concomitantly affected with colitis due to Clostridium difficile toxin. A review of previously reported cases revealed that some of these patients may also have had intestinal toxemia botulism associated with Clostridium difficile colitis, based on the reported symptoms. Given that this association has been shown to exist not only in Italy but also in the USA, it is recommended that individuals with intestinal botulism and symptoms of colitis undergo testing for Clostridium difficile and its toxins in fecal samples. PMID:12479171

  17. A novel insecticidal serotype of Clostridium bifermentans.

    PubMed

    Seleena, P; Lee, H L; Lecadet, M M

    1997-12-01

    A novel Clostridium bifermentans strain toxic to mosquito larvae on ingestion was isolated from a soil sample collected from secondary forest floor. This strain was designated as serovar paraiba (C. b. paraiba) according to its specific H antigen. Clostridium bifermentans paraiba is most toxic to Anopheles maculatus Theobald larvae (LC50 = 0.038 mg/liter), whereas toxicity to Aedes aegypti (Linn.) (LC50 = 0.74 mg/liter) and Culex quinquefasciatus Say (LC50 = 0.11 mg/liter) larvae was 20 and 3 times lower, respectively. The toxicity to An. maculatus larvae is as high as that of Bacillus thuringiensis serovar israelensis. C. b. paraiba was also found to exhibit significant per os insecticidal activity toward adult Musca domestica (Linn.). PMID:9474569

  18. [Genetic modification systems for Clostridium acetobutylicum].

    PubMed

    Dong, Hongjun; Zhang, Yanping; Li, Yin

    2010-10-01

    Clostridium acetobutylicum, a biofuel-butanol producer, has attracted worldwide interests. Strain improvement is important for the process of biobutanol industrialization where efficient genetic modification systems are essential. In this review, the history of genetic modification systems of C. acetobutylicum was introduced, and the types and principles of these systems and their disadvantages are summarized and analysed. The development of updated genetic modification systems for C. acetobutylicum is also proposed. PMID:21218624

  19. Clostridium difficile Infection: A Worldwide Disease

    PubMed Central

    Burke, Kristin E.

    2014-01-01

    Clostridium difficile, an anaerobic toxigenic bacterium, causes a severe infectious colitis that leads to significant morbidity and mortality worldwide. Both enhanced bacterial toxins and diminished host immune response contribute to symptomatic disease. C. difficile has been a well-established pathogen in North America and Europe for decades, but is just emerging in Asia. This article reviews the epidemiology, microbiology, pathophysiology, and clinical management of C. difficile. Prompt recognition of C. difficile is necessary to implement appropriate infection control practices. PMID:24516694

  20. Persistent and Recurrent Clostridium difficile Colitis

    PubMed Central

    Cole, Shola A.; Stahl, Thomas J.

    2015-01-01

    Clostridium difficile infection (CDI) is the most frequent cause of nosocomial diarrhea. It has become a significant dilemma in the treatment of patients, and causes increasing morbidity that, in extreme cases, may result in death. Persistent and recurrent disease hamper attempts at eradication of this infection. Escalating levels of treatment and novel therapeutics are being utilized and developed to treat CDI. Further trials are warranted to definitively determine what protocols can be used to treat persistent and recurrent disease. PMID:26034401

  1. Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome.

    PubMed Central

    Wilkinson, S R; Young, M

    1995-01-01

    A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin. PMID:7814334

  2. Draft Genome Sequence of Clostridium butyricum Strain NOR 33234, Isolated from an Elderly Patient with Diarrhea

    PubMed Central

    Kwok, Jamie S. L.; Ip, Margaret; Chan, Ting-Fung; Lam, Wai-Yip

    2014-01-01

    Clostridium butyricum is one of the species frequently present in patients’ stool samples. However, the identification of this species is sometimes difficult. Here, we present the draft genome of Clostridium butyricum NOR 33234, which was isolated from a patient with suspected Clostridium difficile infection-associated diarrhea and resembles Clostridium clostridioforme in biochemical tests. PMID:25540356

  3. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia.

    PubMed

    Sting, Reinhard

    2009-01-01

    The production of Clostridium (C.) perfringens toxins in the intestine is an important cause of enteritis and enterotoxaemia in livestock. In the present study, the alpha toxin and the genes encoding beta2 and epsilon toxin could be frequently detected by means of phenotypical and PCR examinations in these bacteria. The C. perfringens isolates originated from 1213 field samples taken from diseased or perished livestock located in the north-eastern administrative districts of Baden-Württemberg (Germany) from 2005 to 2008. The beta2 toxin gene of C perfringens was detected in all animal species examined, comprising pigs, the small ruminants sheep and goats, cattle, horses, rabbits, alpacas and lamas, and fallow deer. Among all the animal species included in this study, pigs attracted attention by a high quota of 74.2% (610 of 822) cpb2-positive C. perfringens isolates in comparison to the other animal species tested, revealing a quota of 20.8% (72 of 346). Beta2 toxigenic isolates could be predominantly cultivated from the faeces of young piglets. The beta toxin gene was detected in isolates from piglets and small ruminants only, amounting to 82.5% (33 of 40) in piglets in combination with the cpb2 gene. In this context, cpb2/cpb-positive C. perfringens isolates of piglets could be clearly detected more often in the intestine of perished animals (18 of 158) than in faeces (15 of 629). Furthermore, cpb2-bearing C. perfringens isolates were detected in cattle, horses, rabbits, alpacas and lamas, and fallow deer to a notable degree. The detection of C. perfringens isolates carrying the epsilon toxin gene was restricted to sheep and goats. Of a total of 242 small ruminants that succumbed to sudden death, 71 (29.3%) harboured epsilon toxin-positive C. perfringens isolates in their intestines. These cases clustered seasonally in the second quarter (April, May, and June) of the year. Neither the isolates bearing the beta2 nor beta toxin gene nor those carrying the epsilon

  4. Physical and genetic map of the Clostridium saccharobutylicum (formerly Clostridium acetobutylicum) NCP 262 chromosome.

    PubMed

    Keis, S; Sullivan, J T; Jones, D T

    2001-07-01

    A physical and genetic map of the Clostridium saccharobutylicum NCP 262 chromosome was constructed. The order of macrorestriction fragments was determined by analysing fragments generated after single and double digestion with the restriction enzymes BssHII, I-CeuI, Sse8387I, RsrII and SfiI and separation by PFGE. The I-CeuI backbone of C. saccharobutylicum was constructed by indirect end-labelling with rrs- and 3' rrl-specific probes located on either side of the I-CeuI site in the rrn operon, and reciprocal separation of BssHII and I-CeuI digestion products by two-dimensional PFGE. The positions of BssHII fragments on the physical map were determined using a library of linking clones containing BssHII cleavage sites. The size of the circular genome was estimated to be 5.3 Mb with a mean resolution of approximately 140 kb. The chromosome of C. saccharobutylicum contains 12 rrn operons, located on 46% of the chromosome, which are transcribed divergently from the deduced origin of replication. The genetic map was constructed by determining the location of 28 genes involved in house-keeping, heat-shock response, sporulation, electron transfer and acid- and solvent-formation. Comparison of the C. saccharobutylicum genetic map with those of the spore-forming bacteria Bacillus subtilis, Clostridium acetobutylicum, Clostridium perfringens and Clostridium beijerinckii indicated C. saccharobutylicum to be most similar to the latter two Clostridium species, with the order of the genes within the gyrAB and recA loci being conserved. PMID:11429467

  5. Blastocystis sp. Infection Mimicking Clostridium Difficile Colitis

    PubMed Central

    Gil, Gaby S.; Chaudhari, Shobhana; Shady, Ahmed; Caballes, Ana; Hong, Joe

    2016-01-01

    We report an unusual case of severe diarrhea related to Blastocystis sp. infection in a patient with end stage renal disease on hemodialysis. The patient was admitted due to profuse diarrhea associated with fever and leukocytosis. Pertinent stool work-up such as leukocytes in stool, stool culture, clostridium difficile toxin B PCR, and serology for hepatitis A, hepatitis B, and hepatitis C and cytomegalovirus screening were all negative. Ova and parasite stool examination revealed Blastocystis sp. The patient was given intravenous metronidazole with clinical improvement by day three and total resolution of symptoms by day ten. PMID:27247810

  6. An Update on Clostridium difficile Toxinotyping

    PubMed Central

    Janezic, Sandra

    2015-01-01

    Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature. PMID:26511734

  7. Alternative medium for Clostridium perfringens sporulation.

    PubMed Central

    Tórtora, J C

    1984-01-01

    A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains. Comparatively this medium gave a greater percentage of sporulation than five other media described previously. PMID:6331307

  8. Clostridium difficile colitis: pathogenesis and host defence.

    PubMed

    Abt, Michael C; McKenney, Peter T; Pamer, Eric G

    2016-10-01

    Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile. This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis. PMID:27573580

  9. Inducing and Quantifying Clostridium difficile Spore Formation.

    PubMed

    Shen, Aimee; Fimlaid, Kelly A; Pishdadian, Keyan

    2016-01-01

    The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation. PMID:27507338

  10. Clostridium difficile: from obscurity to superbug.

    PubMed

    Brazier, J S

    2008-01-01

    According to the UK media and popular press, Clostridium difficile is now a fully fledged member of that notorious but ill-defined group of microorganisms portrayed to the general public as superbugs. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous superbug responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This review tracks the rise in scientific knowledge and public awareness of this organism. PMID:18476496

  11. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

  12. Annotation of the Clostridium Acetobutylicum Genome

    SciTech Connect

    Daly, M. J.

    2004-06-09

    The genome sequence of the solvent producing bacterium Clostridium acetobutylicum ATCC824, has been determined by the shotgun approach. The genome consists of a 3.94 Mb chromosome and a 192 kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases, closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria.

  13. Novel Risk Factors for Recurrent Clostridium difficile Infection in Children

    PubMed Central

    Nicholson, Maribeth R.; Thomsen, Isaac P.; Slaughter, James C.; Creech, C. Buddy; Edwards, Kathryn M.

    2014-01-01

    Objectives Clostridium difficile, a common cause of antibiotic-associated diarrhea, has been reported to recur in high rates in adults. The rates and risk factors for recurrent Clostridium difficile infection (rCDI) in children have not been well established. Methods We conducted a retrospective cohort study of 186 pediatric patients seen at a tertiary care referral center over a 5-year period diagnosed with a primary infection with Clostridium difficile. Children with recurrent disease, defined as return of symptoms of Clostridium difficile infection and positive testing ≤60 days after the completion of therapy, were compared to children who did not experience an episode of recurrence. Results Of the 186 pediatric patients included in this study, 41 (22%) experienced recurrent Clostridium difficile infection. On univariable analysis, factors significantly associated with recurrent Clostridium difficile infection included malignancy, recent hospitalization, recent surgery, antibiotic use, number of antibiotic exposures by class, acid blocker use, immunosuppressant use, and hospital acquired disease. On multivariable analysis, malignancy (OR=3.39, 95% CI=1.52–7.85), recent surgery (OR=2.40, 95% CI=1.05–5.52), and the number of antibiotic exposures by class (OR=1.33, 95% CI=1.01–1.75) were significantly associated with recurrent disease in children. Conclusions The rate of recurrent Clostridium difficile infection in children was 22%. Recurrence was significantly associated with the risk factors of malignancy, recent surgery, and the number of antibiotic exposures by class. PMID:25199038

  14. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma.

    PubMed

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-04-14

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2(nd) day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2(nd) TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  15. Clostridium novyi, sordellii, and tetani: mechanisms of disease.

    PubMed

    Aronoff, David M

    2013-12-01

    Clostridia represent a diverse group of spore-forming gram positive anaerobes that include several pathogenic species. In general, diseases caused by clostridia are a result of intoxication of the infected host. Thus, clostridial toxins have been targeted for diagnostic, therapeutic, and preventive strategies against infection. Studying the mechanisms of action of clostridial toxins has not only shed light on the pathogenesis of infection but has provided important new insights into cell biology and immunology. A primary purpose of this manuscript is to provide a succinct review on the mechanisms of disease caused by intoxication by the pathogens Clostridium tetani, Clostridium novyi, and Clostridium sordellii. PMID:24036420

  16. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma

    PubMed Central

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-01-01

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2nd day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2nd TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  17. Clostridium to treat cancer: dream or reality?

    PubMed Central

    Lambin, Philippe

    2015-01-01

    In their paper “Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses”, Roberts et al. describe the induction of antitumor responses following local spore administration of an attenuated C. novyi strain (C. novyi-NT). Stereotactic intratumoral spore injection led to significant survival advantages in a murine orthotopic brain model and local bacterial treatment produced robust responses in a set of spontaneous canine soft tissue carcinomas. Their preclinical findings in both models, provided the basis for a phase 1 investigational clinical study in patients with solid tumors that were either refractory to standard treatment or without an available standard treatment available (NCT01924689). The results of the first patient enrolled in this trial, a 53-year-old female with a retroperitoneal leiomyosarcoma, are described. Next to the non-armed C. novyi-NT described in this paper, very potent genetically modified Clostridium expressing anti-cancer therapeutic genes are also being developed. Are treatments with these non-pathogenic clostridia a viable alternative cancer treatment? PMID:26046067

  18. Secretion of clostridium cellulase by E. coli

    DOEpatents

    Yu, Ida Kuo

    1998-01-01

    A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

  19. First Report of Clostridium lavalense Isolated in Human Blood Cultures

    PubMed Central

    Bourque, Christine; Thibault, Louise; Côté, Jean-Charles; Domingo, Marc-Christian

    2016-01-01

    An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors. PMID:27478446

  20. Flooding and Clostridium difficile infection: a case-crossover analysis

    EPA Science Inventory

    Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospttalized and/or receiving antibiotics; however, community­ associated infections affecting otherwise healthy individuals have become more ...

  1. Methylenetetrahydrofolate dehydrogenase from Clostridium formicoaceticum and methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase (combined) from Clostridium thermoaceticum

    SciTech Connect

    Ljungdahl, L.G.; O'Brien, W.E.; Moore, M.R.; Liu, M.T.

    1980-01-01

    Methylenetetrahydrofolate dehydrogenase is widely distributed and has been found in every cell type investigated. The NAD-specific enzyme has been purified to homogeneity from Clostridium formicoaceticum and the NADP-specific enzyme has been obtained from Clostridium thermoaceticum. Other sources of the NADP-specific enzyme are Streptococcus species, Escherichia coli, Clostridium cylindrosporum, Salmonella typhimurium, yeast, liver from various animals, calf thymus, and plants. The NAD-specific enzyme has been demonstrated in Acetobacterium woodii, some methane bacteria, and in Ehrlich ascites tumor cells. Of considerable interest are the observations that in porcine and ovine livers, as well as in yeast, methylenetetrahydrofolate dehydrogenase purified to homogeneity also contains methylenetetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities. Now it appears that the purified methylenetetrahydrofolate dehydrogenase from C. thermoaceticum also has cyclohydrolase but not synthetase activity. Methylenetetrahydrofolate dehydrogenase has been discussed previously in this series, as has methenyltetrahydrofolate cyclohydrolase. In C. formicoaceticum and C. thermoaceticum these tetrahydrofolate-dependent enzymes participate in a sequence of metabolic reactions by which carbon dioxide is reduced to the methyl group of 5-methyltetrahydrofolate which in turn is utilized for the synthesis of acetate. This pathway provides the mechanism for disposing of reducing equivalents generated in glycolysis.

  2. Small RNAs in the Genus Clostridium

    PubMed Central

    Chen, Yili; Indurthi, Dinesh C.; Jones, Shawn W.; Papoutsakis, Eleftherios T.

    2011-01-01

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. PMID:21264064

  3. Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4 ▿

    PubMed Central

    Nakayama, Shunichi; Kiyoshi, Keiji; Kadokura, Toshimori; Nakazato, Atsumi

    2011-01-01

    We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system. PMID:21764954

  4. Improved Medium for Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  5. Improved medium for enumeration of Clostridium perfringens.

    PubMed

    Harmon, S M; Kautter, D A; Peeler, J T

    1971-10-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 mug of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  6. Isolating and Purifying Clostridium difficile Spores.

    PubMed

    Edwards, Adrianne N; McBride, Shonna M

    2016-01-01

    The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications. PMID:27507337

  7. Quantitation of Clostridium perfringens in foods.

    PubMed

    ANGELOTTI, R; HALL, H E; FOTER, M J; LEWIS, K H

    1962-05-01

    A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks. PMID:13861594

  8. Carbohydrate-based Clostridium difficile vaccines.

    PubMed

    Monteiro, Mario A; Ma, Zuchao; Bertolo, Lisa; Jiao, Yuening; Arroyo, Luis; Hodgins, Douglas; Mallozzi, Michael; Vedantam, Gayatri; Sagermann, Martin; Sundsmo, John; Chow, Herbert

    2013-04-01

    Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface. PMID:23560922

  9. [New aspects on Clostridium difficile infection].

    PubMed

    von Müller, Lutz

    2016-08-01

    Clostridium difficile infection (CDI) is a frequent and complex disease which is influenced by the repertoire of bacterial virulence factors, by host immunity and by the intestinal microbiome. These complex interaction opens a number of options which may be used for treatment in the future. One example for new treatment options is fecal microbiota transplantation (FMT). Driven by C. difficile related research activities the knowledge of protective microorganism is increasing and it may be assumed that bacteriotherapy by next-generation probiotics may be used very soon also for other diseases. Very often, CDI reflects to the clinician that antibiotic therapy is associated with side effects. Therefore, C. difficile is the guilty conscience which helps to implement targeted and restrictive antibiotic use in the daily practice. PMID:27509341

  10. Biotechnological potential of Clostridium butyricum bacteria

    PubMed Central

    Szymanowska-Powałowska, Daria; Orczyk, Dorota; Leja, Katarzyna

    2014-01-01

    In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties. PMID:25477923

  11. Current State of Clostridium difficile Treatment Options

    PubMed Central

    Venugopal, Anilrudh A.; Johnson, Stuart

    2012-01-01

    Recent reports of reduced response to standard therapies for Clostridium difficile infection (CDI) and the risk for recurrent CDI that is common with all currently available treatment agents have posed a significant challenge to clinicians. Current recommendations include metronidazole for treatment of mild to moderate CDI and vancomycin for severe CDI. Results from small clinical trials suggest that nitazoxanide and teicoplanin may be alternative options to standard therapies, whereas rifaximin has demonstrated success in uncontrolled trials for the management of multiple recurrences. Anecdotal reports have also suggested that tigecycline might be useful as an adjunctive agent for the treatment of severe complicated CDI. Reports of resistance will likely limit the clinical use of fusidic acid and bacitracin and, possibly, rifaximin if resistance to this agent becomes widespread. Treatment of patients with multiple CDI recurrences and those with severe complicated CDI is based on limited clinical evidence, and new treatments or strategies are needed. PMID:22752868

  12. Clostridium difficile: clinical disease and diagnosis.

    PubMed Central

    Knoop, F C; Owens, M; Crocker, I C

    1993-01-01

    Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions. PMID:8358706

  13. Purification and characterization of Clostridium difficile toxin.

    PubMed Central

    Rolfe, R D; Finegold, S M

    1979-01-01

    Recent evidence indicates that toxigenic Clostridium difficile strains are a major cause of antimicrobial-associated ileocecitis in laboratory animals and pseudomembranous colitis in humans. C. difficile ATCC 9689 was cultivated in a synthetic medium to which 3% ultrafiltrated proteose peptone was added. Purification of the toxin from broth filtrate was accomplished through ultrafiltration (100,000 nominal-molecular-weight-limit membrane), precipitation with 75% (NH4)2SO4, and chromatographic separation using Bio-Gel A 5m followed by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-25 column. The purified toxin displayed only one band on polyacrylamide gel electrophoresis, and approximately 170 pg was cytopathic for human amnion cells. The isolated toxin was neutralized by Clostridium sordelli antitoxin, heat labile (56 degrees C for 30 min), and inactivated at pH 4 and 9; it had an isoelectric point of 5.0, increased vascular permeability in rabbits, and caused ileocecitis in hamsters when injected intracecally. Treatment of the toxin with trypsin, chymotrypsin, pronase, amylase, or ethylmercurithiosalicylate caused inactivation, whereas lipase had no effect. By gel filtration, its molecular weight was estimated as 530,000. Upon reduction and denaturation, the toxin dissociated into 185,000- and 50,000-molecular-weight components, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive dissociation yielded only the 50,000-molecular-weight component. The toxin appears to be protoplasmic and is released into the surrounding environment upon autolysis of the cells. Attempts to correlate specific enzymatic activity with the toxin have been unsuccessful. These studies will help delineate the role of C. difficile toxin in antimicrobial-associated colitis and diarrhea. Images PMID:478634

  14. An unusual case of pneumocephalus.

    PubMed

    Bhogal, P; Bhatnagar, G; Manieson, J; Booth, T; Prendergast, C

    2011-01-01

    The authors present an unusual cause of pneumocephalus in a previously fit and well female octogenarian who presented with acute onset altered level of consciousness and generalised weakness. Radiological imaging demonstrated widespread cerebritis with pneumocephalus and gas within the superior sagittal sinus. Blood culture grew Clostridium septicum, a virulent but rare organism that can infect normal tissues. The close association between C septicum and both haematological and bowel malignancies must be considered if this organism is cultured. PMID:22673713

  15. Two simultaneous botulism outbreaks in Barcelona: Clostridium baratii and Clostridium botulinum.

    PubMed

    Lafuente, S; Nolla, J; Valdezate, S; Tortajada, C; Vargas-Leguas, H; Parron, I; Sáez-Nieto, J A; Portaña, S; Carrasco, G; Moguel, E; Sabate, S; Argelich, R; Caylà, J A

    2013-09-01

    Botulism is a severe neuroparalytic disorder that can be potentially life-threatening. In Barcelona, Spain, no outbreaks had been reported in the past 25 years. However, in September 2011, two outbreaks occurred involving two different families. A rare case of Clostridium baratii which produced a neurotoxin F outbreak was detected in five family members who had shared lunch, and several days before that another family was affected by C. botulinum toxin A which was probably present in homemade pâté. PMID:23158693

  16. Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline.

    PubMed

    Shin, Yeseul; Kang, Seok-Seong; Paek, Jayoung; Jin, Tae Eun; Song, Hong Seok; Kim, Hongik; Park, Hee-Moon; Chang, Young-Hyo

    2016-06-01

    Two bacterial strains, YHK0403(T) and YHK0508, isolated from soil under a corroded gas pipe line, were revealed as Gram-negative, obligately anaerobic, spore-forming and mesophilic bacteria. The cells were rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were members of the genus Clostridium and were the most closely related to Clostridium scatologenes KCTC 5588(T) (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177(T) (95.8%), Clostridium drakei KCTC 5440(T) (95.7%) and Clostridium tyrobutyricum KCTC 5387(T) (94.9%). The G + C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1γ type with meso-diaminopimelic acid. The major polar lipid was diphosphatidylglycerol (DPG), and other minor lipids were revealed as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unknown glycolipids (GL1 and GL2), an unknown aminoglycolipid (NGL), two unknown aminophospholipids (PN1 and PN2) and four unknown phospholipids (PL1 to PL4). Predominant fatty acids were C16:0 and C16:1cis9 DMA. The major end products from glucose fermentation were identified as butyrate (12.2 mmol) and acetate (9.8 mmol). Collectively, the results from a wide range of phenotypic tests, chemotaxonomic tests, and phylogenetic analysis indicated that the two isolates represent novel species of the genus Clostridium, for which the name Clostridium kogasensis sp. nov. (type strain, YHK0403(T) = KCTC 15258(T) = JCM 18719(T)) is proposed. PMID:26899448

  17. Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum sp. nov

    NASA Technical Reports Server (NTRS)

    Paster, B. J.; Russell, J. B.; Yang, C. M.; Chow, J. M.; Woese, C. R.; Tanner, R.

    1993-01-01

    In previous studies, gram-positive bacteria which grew rapidly with peptides or an amino acid as the sole energy source were isolated from bovine rumina. Three isolates, strains C, FT (T = type strain), and SR, were considered to be ecologically important since they produced up to 20-fold more ammonia than other ammonia-producing ruminal bacteria. On the basis of phenotypic criteria, the taxonomic position of these new isolates was uncertain. In this study, the 16S rRNA sequences of these isolates and related bacteria were determined to establish the phylogenetic positions of the organisms. The sequences of strains C, FT, and SR and reference strains of Peptostreptococcus anaerobius, Clostridium sticklandii, Clostridium coccoides, Clostridium aminovalericum, Acetomaculum ruminis, Clostridium leptum, Clostridium lituseburense, Clostridium acidiurici, and Clostridium barkeri were determined by using a modified Sanger dideoxy chain termination method. Strain C, a large coccus purported to belong to the genus Peptostreptococcus, was closely related to P. anaerobius, with a level of sequence similarity of 99.6%. Strain SR, a heat-resistant, short, rod-shaped organism, was closely related to C. sticklandii, with a level of sequence similarity of 99.9%. However, strain FT, a heat-resistant, pleomorphic, rod-shaped organism, was only distantly related to some clostridial species and P. anaerobius. On the basis of the sequence data, it was clear that strain FT warranted designation as a separate species. The closest known relative of strain FT was C. coccoides (level of similarity, only 90.6%). Additional strains that are phenotypically similar to strain FT were isolated in this study.(ABSTRACT TRUNCATED AT 250 WORDS).

  18. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

    PubMed

    Li, Qi; Chen, Jun; Minton, Nigel P; Zhang, Ying; Wen, Zhiqiang; Liu, Jinle; Yang, Haifeng; Zeng, Zhe; Ren, Xiaodan; Yang, Junjie; Gu, Yang; Jiang, Weihong; Jiang, Yu; Yang, Sheng

    2016-07-01

    Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia. PMID:27213844

  19. Group II Intron-Anchored Gene Deletion in Clostridium

    PubMed Central

    Jia, Kaizhi; Zhu, Yan; Zhang, Yanping; Li, Yin

    2011-01-01

    Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established. PMID:21304965

  20. Promoters and proteins from Clostridium thermocellum and uses thereof

    DOEpatents

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  1. A prediction model for Clostridium difficile recurrence

    PubMed Central

    LaBarbera, Francis D.; Nikiforov, Ivan; Parvathenani, Arvin; Pramil, Varsha; Gorrepati, Subhash

    2015-01-01

    Background Clostridium difficile infection (CDI) is a growing problem in the community and hospital setting. Its incidence has been on the rise over the past two decades, and it is quickly becoming a major concern for the health care system. High rate of recurrence is one of the major hurdles in the successful treatment of C. difficile infection. There have been few studies that have looked at patterns of recurrence. The studies currently available have shown a number of risk factors associated with C. difficile recurrence (CDR); however, there is little consensus on the impact of most of the identified risk factors. Methods Our study was a retrospective chart review of 198 patients diagnosed with CDI via Polymerase Chain Reaction (PCR) from January 2009 to Jun 2013. In our study, we decided to use a machine learning algorithm called the Random Forest (RF) to analyze all of the factors proposed to be associated with CDR. This model is capable of making predictions based on a large number of variables, and has outperformed numerous other models and statistical methods. Results We came up with a model that was able to accurately predict the CDR with a sensitivity of 83.3%, specificity of 63.1%, and area under curve of 82.6%. Like other similar studies that have used the RF model, we also had very impressive results. Conclusions We hope that in the future, machine learning algorithms, such as the RF, will see a wider application. PMID:25656667

  2. The Changing Epidemiology of Clostridium difficile Infections

    PubMed Central

    Freeman, J.; Bauer, M. P.; Baines, S. D.; Corver, J.; Fawley, W. N.; Goorhuis, B.; Kuijper, E. J.; Wilcox, M. H.

    2010-01-01

    Summary: The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents. PMID:20610822

  3. Clostridium perfringens in Meat and Meat Products

    PubMed Central

    Hall, Herbert E.; Angelotti, Robert

    1965-01-01

    A total of 262 specimens of meat and meat dishes were examined for the presence of Clostridium perfringens. Of this total, 161 were raw, unprocessed beef, veal, lamb, pork, or chicken; 101 were processed meats and meat dishes. C. perfringens was isolated from 113 (43.1%) of these specimens. The highest percentage of contamination (82%) was found in veal cuts, and the lowest (4.7%) in sliced sandwich meats and spreads. Only 2 of the 113 isolates were shown to produce heat-resistant spores, which indicates a very low incidence (0.8%) of contamination. These findings indicate that outbreaks of C. perfringens food-borne disease in the Cincinnati area are caused principally by the contamination of the food with vegetative cells or spores of the organism after cooking. Studies of the effects of various holding temperatures on the growth of C. perfringens indicated that, in the range of 5 to 15 C, no multiplication would occur, but that viable cells would still be present at the end of a 5-day holding period. Extremely rapid growth occurred at temperatures around 45 C, and complete inhibition of growth was accomplished between 49 and 52 C. PMID:14325274

  4. Action of nitroheterocyclic drugs against Clostridium difficile

    PubMed Central

    Kumar, Manish; Adhikari, Sudip; Hurdle, Julian G.

    2014-01-01

    The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile. PMID:25129314

  5. Secretome analysis of Clostridium difficile strains.

    PubMed

    Boetzkes, Alexander; Felkel, Katharina Wiebke; Zeiser, Johannes; Jochim, Nelli; Just, Ingo; Pich, Andreas

    2012-08-01

    Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified. PMID:22398929

  6. Characterization of Functional Prophages in Clostridium difficile.

    PubMed

    Sekulović, Ognjen; Fortier, Louis-Charles

    2016-01-01

    Bacteriophages (phages) are present in almost, if not all ecosystems. Some of these bacterial viruses are present as latent "prophages," either integrated within the chromosome of their host, or as episomal DNAs. Since prophages are ubiquitous throughout the bacterial world, there has been a sustained interest in trying to understand their contribution to the biology of their host. Clostridium difficile is no exception to that rule and with the recent release of hundreds of bacterial genome sequences, there has been a growing interest in trying to identify and classify these prophages. Besides their identification in bacterial genomes, there is also growing interest in determining the functionality of C. difficile prophages, i.e., their capacity to escape their host and reinfect a different strain, thereby promoting genomic evolution and horizontal transfer of genes through transduction, for example of antibiotic resistance genes. There is also some interest in using therapeutic phages to fight C. difficile infections.The objective of this chapter is to share with the broader C. difficile research community the expertise we developed in the study of C. difficile temperate phages. In this chapter, we describe a general "pipeline" comprising a series of experiments that we use in our lab to identify, induce, isolate, propagate, and characterize prophages. Our aim is to provide readers with the necessary basic tools to start studying C. difficile phages. PMID:27507339

  7. Clostridium botulinum in cattle and dairy products.

    PubMed

    Lindström, Miia; Myllykoski, Jan; Sivelä, Seppo; Korkeala, Hannu

    2010-04-01

    The use of plastic-wrapped and nonacidified silage as cattle feed has led to an increasing number of botulism outbreaks due to Clostridium botulinum Groups I-III in dairy cattle. The involvement of Groups I and II organisms in cattle botulism has raised concern of human botulism risk associated with the consumption of dairy products. Multiplication of C. botulinum in silage and in the gastrointestinal tract of cattle with botulism has been reported, thus contamination of the farm environment and raw milk, and further transmission through the dairy chain, are possible. The standard milk pasteurization treatment does not eliminate spores, and the intrinsic factors of many dairy products allow botulinal growth and toxin production. Although rare, several large botulism outbreaks due to both commercial and home-prepared dairy products have been reported. Factors explaining these outbreaks include most importantly temperature abuse, but also unsafe formulation, inadequate fermentation, insufficient thermal processing, post-process contamination, and lack of adequate quality control for adjunct ingredients were involved. The small number of outbreaks is probably explained by a low incidence of spores in milk, the presence of competitive bacteria in pasteurized milk and other dairy products, and growth-inhibitory combinations of intrinsic and extrinsic factors in cultured and processed dairy products. PMID:20301016

  8. Elimination of formate production in Clostridium thermocellum

    DOE PAGESBeta

    Rydzak, Thomas; Lynd, Lee R.; Guss, Adam M.

    2015-07-11

    We study the ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield are far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, pyruvate:formate lyase (pfl) and respective PFL-activating enzyme were deleted. Formate production in the resulting Δpfl strain was eliminated and acetate production decreased by 50% on both complex and defined medium. Growth ratemore » of Δpfl decreased by 2.9-fold on defined medium and diauxic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δpfl growth rate to 80% of the parent strain. Finally, we discuss the role of pfl in metabolic engineering strategies and C1 metabolism.« less

  9. Laboratory Diagnosis of Clostridium difficile Infection

    PubMed Central

    Tenover, Fred C.; Baron, Ellen Jo; Peterson, Lance R.; Persing, David H.

    2011-01-01

    The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy. PMID:21854871

  10. Biosynthesis of amino acids in Clostridium pasteurianum

    PubMed Central

    Dainty, R. H.; Peel, J. L.

    1970-01-01

    1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) 14C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus 14C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of 14C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate → threonine → glycine ⇌ serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine. PMID:5419750

  11. Crystal structure of Clostridium difficile toxin A.

    PubMed

    Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen; Farrow, Melissa A; Lisher, John P; Farquhar, Erik; Giedroc, David P; Spiller, Benjamin W; Melnyk, Roman A; Lacy, D Borden

    2016-01-01

    Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon(1,2). The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host(3,4). The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics. PMID:27571750

  12. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  13. Crystal structure of Clostridium difficile toxin A

    PubMed Central

    Chumbler, Nicole M.; Rutherford, Stacey A.; Zhang, Zhifen; Farrow, Melissa A.; Lisher, John P.; Farquhar, Erik; Giedroc, David P.; Spiller, Benjamin W.; Melnyk, Roman A.; Lacy, D. Borden

    2016-01-01

    Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics. PMID:27512603

  14. ClosTron-mediated engineering of Clostridium

    PubMed Central

    Kuehne, Sarah A.; Minton, Nigel P.

    2012-01-01

    Members of the genus Clostridium are of both medical and industrial importance. The molecular tools necessary to study and exploit their wide ranging physiological diversity through directed mutational analysis have until recently been lacking. The situation was transformed in the mid-2000s with the specific adaptation of intron re-targeting technology to the genus, through the development of the ClosTron. By making a handful of nucleotide changes to the group II intron encoding region, the intron can be directed to insert into almost any region within the genome. Through the use of a retrotransposition-activated marker (RAM), based on the ermB gene, successful insertion is selected on the basis of acquisition of resistance to erythromycin. The re-targeted region is designed using an online re-targeting algorithm (www.clostron.com), and then an order is placed with DNA2.0 for both the synthesis of the re-targeted region and its custom cloning into the ClosTron vector. Re-targeted ClosTrons are delivered ready for use in 10–14 days, allowing mutants to be isolated 5–7 days after receipt. Its availability has revolutionized clostridial molecular biology. PMID:22750794

  15. Cellulosomics of the cellulolytic thermophile Clostridium clariflavum

    PubMed Central

    2014-01-01

    Background Clostridium clariflavum is an anaerobic, thermophilic, Gram-positive bacterium, capable of growth on crystalline cellulose as a single carbon source. The genome of C. clariflavum has been sequenced to completion, and numerous cellulosomal genes were identified, including putative scaffoldin and enzyme subunits. Results Bioinformatic analysis of the C. clariflavum genome revealed 49 cohesin modules distributed on 13 different scaffoldins and 79 dockerin-containing proteins, suggesting an abundance of putative cellulosome assemblies. The 13-scaffoldin system of C. clariflavum is highly reminiscent of the proposed cellulosome system of Acetivibrio cellulolyticus. Analysis of the C. clariflavum type I dockerin sequences indicated a very high level of conservation, wherein the putative recognition residues are remarkably similar to those of A. cellulolyticus. The numerous interactions among the cellulosomal components were elucidated using a standardized affinity ELISA-based fusion-protein system. The results revealed a rather simplistic recognition pattern of cohesin-dockerin interaction, whereby the type I and type II cohesins generally recognized the dockerins of the same type. The anticipated exception to this rule was the type I dockerin of the ScaB adaptor scaffoldin which bound selectively to the type I cohesins of ScaC and ScaJ. Conclusions The findings reveal an intricate picture of predicted cellulosome assemblies in C. clariflavum. The network of cohesin-dockerin pairs provides a thermophilic alternative to those of C. thermocellum and a basis for subsequent utilization of the C. clariflavum cellulosomal system for biotechnological application. PMID:26413154

  16. Current status of Clostridium difficile infection epidemiology.

    PubMed

    Lessa, Fernanda C; Gould, Carolyn V; McDonald, L Clifford

    2012-08-01

    The dramatic changes in the epidemiology of Clostridium difficile infection (CDI) during recent years, with increases in incidence and severity of disease in several countries, have made CDI a global public health challenge. Increases in CDI incidence have been largely attributed to the emergence of a previously rare and more virulent strain, BI/NAP1/027. Increased toxin production and high-level resistance to fluoroquinolones have made this strain a very successful pathogen in healthcare settings. In addition, populations previously thought to be at low risk are now being identified as having severe CDI. Recent genetic analysis suggests that C. difficile has a highly fluid genome with multiple mechanisms to modify its content and functionality, which can make C. difficile adaptable to environmental changes and potentially lead to the emergence of more virulent strains. In the face of these changes in the epidemiology and microbiology of CDI, surveillance systems are necessary to monitor trends and inform public health actions. PMID:22752867

  17. Phosphorylation of proteins in Clostridium thermohydrosulfuricum

    SciTech Connect

    Londesborough, J.

    1986-02-01

    Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (..gamma..-/sup 32/P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10..mu..M fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 ..mu..M brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by /sub 32/P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.

  18. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  19. Clostridium difficile Is an Autotrophic Bacterial Pathogen

    PubMed Central

    Köpke, Michael; Straub, Melanie; Dürre, Peter

    2013-01-01

    During the last decade, Clostridium difficile infection showed a dramatic increase in incidence and virulence in the Northern hemisphere. This incessantly challenging disease is the leading cause of antibiotic-associated and nosocomial infectious diarrhea and became life-threatening especially among elderly people. It is generally assumed that all human bacterial pathogens are heterotrophic organisms, being either saccharolytic or proteolytic. So far, this has not been questioned as colonization of the human gut gives access to an environment, rich in organic nutrients. Here, we present data that C. difficile (both clinical and rumen isolates) is also able to grow on CO2+H2 as sole carbon and energy source, thus representing the first identified autotrophic bacterial pathogen. Comparison of several different strains revealed high conservation of genes for autotrophic growth and showed that the ability to use gas mixtures for growth decreases or is lost upon prolonged culturing under heterotrophic conditions. The metabolic flexibility of C. difficile (heterotrophic growth on various substrates as well as autotrophy) could allow the organism in the gut to avoid competition by niche differentiation and contribute to its survival when stressed or in unfavorable conditions that cause death to other bacteria. This may be an important trait for the pathogenicity of C. difficile. PMID:23626782

  20. [Epidemiology of Clostridium difficile infection in Spain].

    PubMed

    Asensio, Angel; Monge, Diana

    2012-06-01

    There has been increasing interest in Clostridium difficile infection (CDI) due its association with healthcare and its impact on morbidity and mortality in the elderly. During the last few years there has been a growing increase in the number of published studies on the incidence, changes on the clinical presentation and on the epidemiology, with the description of new risk factors. The frequency of CDI in Spain is not sufficiently characterised. The available data indicates that incidence is within the range of that of surrounding countries but increasing. Furthermore, the high and growing use of broad spectrum antibiotics, both in our hospitals and in the community setting, are factors that favour the increase of the disease. The hyper-virulent ribotype 027 has not spread in our hospitals. We need to know with enhanced validity and accuracy the incidence of CDI, both community and healthcare-associated, the information on outbreaks, the incidence on certain population groups, the characterisation of circulating ribotypes and the impact of the disease in terms of mortality and health costs. We need to implement programs for the improvement of antibiotic therapy in the hospital, as well as in the community. Furthermore, the knowledge and the performance of standard precautions need to be improved, particularly hand hygiene, and the specific measures to limit the transmission of C. difficile among the healthcare institutions. PMID:22136747

  1. Parameters Affecting Solvent Production by Clostridium pasteurianum

    PubMed Central

    Dabrock, Birgit; Bahl, Hubert; Gottschalk, Gerhard

    1992-01-01

    The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol. PMID:16348691

  2. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

    Verherstraeten, Stefanie; Goossens, Evy; Valgaeren, Bonnie; Pardon, Bart; Timbermont, Leen; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet; Wade, Kristin R.; Tweten, Rodney; Van Immerseel, Filip

    2015-01-01

    The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. PMID:26008232

  3. Effects of butanol on Clostridium acetobutylicum.

    PubMed Central

    Bowles, L K; Ellefson, W L

    1985-01-01

    The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism. Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained. Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control. Butanol, one of the end products of the fermentation, had numerous harmful effects on C. acetobutylicum. At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake. Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH. Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent. The effects of butanol on C. acetobutylicum are complex, inhibiting several interrelated membrane processes. PMID:2868690

  4. ANALYSES OF WOUND EXUDATES FOR CLOSTRIDIAL TOXINS.

    PubMed

    NOYES, H E; PRITCHARD, W L; BRINKLEY, F B; MENDELSON, J A

    1964-03-01

    Noyes, Howard E. (Walter Reed Army Institute of Research, Washington, D.C.), William L. Pritchard, Floyd B. Brinkley, and Janice A. Mendelson. Analyses of wound exudates for clostridial toxins. J. Bacteriol. 87:623-629. 1964.-Earlier studies indicated that death of goats with traumatic wounds of the hindquarter could be related to the number of clostridia in the wounds, and that toxicity of wound exudates for mice and guinea pigs could be partially neutralized by commercial trivalent gas gangrene antitoxin. This report describes in vitro and in vivo analyses of wound exudates for known clostridial toxins. Wounds were produced by detonation of high-explosive pellets. Wound exudates were obtained by cold saline extraction of both necrotic tissues and gauze sponges used to cover the wounds. Exudates were sterilized by Seitz filtration in the cold. In vitro tests were used to measure alpha-, theta-, and mu-toxins of Clostridium perfringens and the epsilon-toxin of C. novyi. Mouse protection tests, employing commercial typing antisera, were used to analyze exudates for other clostridial toxins. Lethality of wound exudates for mice could be related to (i) the numbers of clostridia present in the wound, (ii) survival time of the goats, and (iii) positive lecithovitellin (LV) tests of the exudates. However, the LV tests could not be neutralized by antitoxin specific for C. perfringens alpha-toxin. Mice were not protected by typing antisera specific for types A, C, or D C. perfringens or C. septicum but were protected by antisera specific for type B C. perfringens and types A and B C. novyi. PMID:14127581

  5. Clostridium infection (jisizheng) in yaks in Qinghai, China.

    PubMed

    Changqing, Q; Xueli, Y

    2001-10-01

    Since the mid-1980s, outbreaks of a disease characterized by a sudden onset, acute deaths and extensive haemorrhages in the viscera and digestive tract of yaks have been prevalent in Qilian, Qinghai, China. The disease is known as jisiheng by local people. Virulent Clostridium perfringens type A and Clostridium haemolytica were isolated from yaks that had died of jisizheng. In 1996 and 1997, yaks were immunized with a polyvalent inactivated vaccine against C. perfringens and with an inactivated vaccine against C. haemolyticum, and this prevented the occurrence of jisizheng. PMID:11583378

  6. Adherence of Clostridium thermocellum to cellulose.

    PubMed Central

    Bayer, E A; Kenig, R; Lamed, R

    1983-01-01

    The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species. Images PMID:6630152

  7. The emergence of 'hypervirulence' in Clostridium difficile.

    PubMed

    Cartman, Stephen T; Heap, John T; Kuehne, Sarah A; Cockayne, Alan; Minton, Nigel P

    2010-08-01

    The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the 'laboratory flask' (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD. PMID:20547099

  8. Form and Function of Clostridium thermocellum Biofilms

    PubMed Central

    Dumitrache, Alexandru; Allen, Grant; Liss, Steven N.; Lynd, Lee R.

    2013-01-01

    The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h−1) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion. PMID:23087042

  9. Colonic Immunopathogenesis of Clostridium difficile Infections

    PubMed Central

    Turnwald, Bradley P.; Koo, Hoonmo L.; Garey, Kevin W.; Jiang, Zhi-Dong; Aitken, Samuel L.; DuPont, Herbert L.

    2014-01-01

    There are major gaps in our understanding of the immunopathogenesis of Clostridium difficile infections (CDIs). In this study, 36 different biomarkers were examined in the stools of CDI and non-CDI patients using the Proteome Profiler human cytokine array assay and quantitative enzyme-linked immunosorbent assay. Diarrheal stools from patients with CDI (CDI-positive diarrheal stools) showed higher relative amounts of the following inflammatory markers than the diarrheal stools from CDI-negative patients (CDI-negative diarrheal stools): C5a, CD40L, granulocyte colony-stimulating factor, I-309, interleukin-13 (IL-13), IL-16, IL-27, monocyte chemoattractant protein 1, tumor necrosis factor alpha, and IL-8. IL-8 and IL-23 were present in a larger number of CDI-positive diarrheal stools than CDI-negative diarrheal stools. Th1 and Th2 cytokines were not significantly different between the CDI-positive and CDI-negative diarrheal stools. Lactoferrin and calprotectin concentrations were also higher in the CDI-positive diarrheal stools. Our results demonstrate that CDI elicits a proinflammatory host response, and we report for the first time that IL-23 is a major marker in CDI-positive diarrheal stools. IL-23 may explain the lack of a robust immunological response exhibited by a proportion of CDI patients and may relate to recurrence; the IL-23 levels induced during CDI in these patients may be inadequate to sustain the cellular immunity conferred by this cytokine in promoting the induction and proliferation of effector memory T cells. PMID:24477852

  10. Prospects for a vaccine for Clostridium difficile.

    PubMed

    Kyne, L; Kelly, C P

    1998-09-01

    Clostridium difficile diarrhoea and colitis is a new disease that is attributable to broad spectrum antibiotic therapy. During the past 2 decades C. difficile has become one of the most common nosocomial pathogens in the developed world. As changing demographics create an increasingly elderly population and the use of broad spectrum antimicrobials continues to expand, C. difficile is likely to become increasingly problematic. Disease caused by this organism is caused by the inflammatory actions of its 2 toxins, A and B, on the intestinal mucosa. Human antibody responses to these toxins are common in the general population and in patients with C. difficile-associated disease. There is substantial, albeit inconclusive, evidence to indicate that antitoxin antibodies provide protection against severe, prolonged or recurrent C. difficile diarrhoea. Immunity induced by oral or parenteral passive administration of antibody is protective in animal models of C. difficile infection. In humans, intravenous passive immunisation with pooled human immunoglobulin has been successful in the treatment of recurrent and severe C. difficile colitis. Human trials of oral passive immunotherapy with bovine immunoglobulin therapy are in progress. Formalin-inactivated culture filtrate from toxigenic C. difficile, as well as purified and inactivated toxins, have been used to successfully immunise animals. Similar preparations are under investigation as possible human vaccines. Antibiotic therapy is effective in treating most individual patients with C. difficile diarrhoea, but has proven ineffective in reducing the overall incidence of nosocomial infection. Active immunisation is probably the most promising approach to long term control of this difficult iatrogenic disease. PMID:18020593

  11. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    PubMed Central

    Genth, Harald; Schelle, Ilona; Just, Ingo

    2016-01-01

    Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA)—is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+) in mammalian target cells. PMID:27089365

  12. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B.

    PubMed

    Genth, Harald; Schelle, Ilona; Just, Ingo

    2016-01-01

    Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the "Large clostridial glycosylating toxins." These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB-together with Toxin A (TcdA)-is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn(2+) > Co(2+) > Mg(2+) > Ca(2+), Cu(2+), Zn(2+). TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn(2+) and 180 µM for Mg(2+). TcsL and TcdB further require co-stimulation by monovalent K⁺ (not by Na⁺). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K⁺ and Mg(2+) (rather than Mn(2+)) in mammalian target cells. PMID:27089365

  13. Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs.

    PubMed

    Marks, Stanley L; Kather, Elizabeth J

    2003-06-24

    Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution. PMID:12742714

  14. Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

    PubMed Central

    Kanamori, K; Weiss, R L; Roberts, J D

    1989-01-01

    Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways. PMID:2564848

  15. Clostridium difficile in mixed populations of animals and humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: Since 2003, there has been an emergence of BI/NAP1 strain of Clostridium difficile (Cd) in North American hospitals. The origins of this epidemic strain have yet to be determined. However, PFGE analysis has shown ~80% similarity between this strain and some swine isolates. The objecti...

  16. Clostridium difficile Ribotype 027, Toxinotype III, the Netherlands

    PubMed Central

    van den Berg, Renate J.; Debast, Sylvia; Visser, Caroline E.; Veenendaal, Dick; Troelstra, Annet; van der Kooi, Tjallie; van den Hof, Susan; Notermans, Daan W.

    2006-01-01

    Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A–negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control. PMID:16704846

  17. Clostridium novyi causing necrotising fasciitis in an injecting drug user

    PubMed Central

    Noone, M; Tabaqchali, M; Spillane, J B

    2002-01-01

    Necrotising fasciitis with pronounced local oedema is described in an injecting drug user. Clostridium novyi was an unexpected single pathogen isolated from infected tissue. The patient was among a cluster of cases, all injecting drug users, presenting with toxaemia and soft tissue infection. The causal role and pathogenicity of C novyi is discussed. PMID:11865011

  18. Clostridium glycolicum Isolated from a Patient with Otogenic Brain Abscesses▿

    PubMed Central

    Van Leer, C.; Wensing, A. M. J.; van Leeuwen, J. P.; Zandbergen, E. G. J.; Swanink, C. M. A.

    2009-01-01

    We describe a case of brain abscesses with gas formation following otitis media, for which the patient treated himself by placing clay in his ear. Several microorganisms, including Clostridium glycolicum, were cultured from material obtained from the patient. This is the first report of an infection in an immunocompetent patient associated with this microorganism. PMID:19109475

  19. Clostridium difficile strains from community-associated infections.

    PubMed

    Limbago, Brandi M; Long, Cherie M; Thompson, Angela D; Killgore, George E; Hannett, George E; Havill, Nancy L; Mickelson, Stephanie; Lathrop, Sarah; Jones, Timothy F; Park, Mahin M; Harriman, Kathleen H; Gould, L Hannah; McDonald, L Clifford; Angulo, Frederick J

    2009-09-01

    Clostridium difficile isolates from presumed community-associated infections (n = 92) were characterized by toxinotyping, pulsed-field gel electrophoresis, tcdC and cdtB PCR, and antimicrobial susceptibility. Nine toxinotypes (TOX) and 31 PFGE patterns were identified. TOX 0 (48, 52%), TOX III (18, 20%), and TOX V (9, 10%) were the most common; three isolates were nontoxigenic. PMID:19571021

  20. Clostridium difficile in retail meat and processing plants in Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence and severity of disease associated with toxigenic Clostridium difficile (Cd) have increased in hospitals in North America from the emergence of newer, more virulent strains of Cd. Toxigenic Cd has been isolated from food animals and retail meat with potential implications of transfer ...

  1. Genome of a chronic osteitis-causing Clostridium tetani.

    PubMed

    Fournier, P-E; Levy, P-Y; Million, M; Croce, O; Blanc-Tailleur, C; Brouqui, P; Raoult, D

    2014-01-01

    We sequenced the genome of a Clostridium tetani strain that caused chronic tibial osteitis without any clinical sign of tetanus in a 26-year-old man previously vaccinated against this disease. The genome contained a plasmid that harboured the tetX-tetR tetanospasmin operon, and was highly similar to that of a tetanus-causing strain. PMID:25356334

  2. Severe Clostridium difficile-associated disease in children.

    PubMed

    Pokorn, Marko; Radsel, Anja; Cizman, Milan; Jereb, Matjaz; Karner, Primoz; Kalan, Gorazd; Grosek, Stefan; Andlovic, Alenka; Rupnik, Maja

    2008-10-01

    Three cases of Clostridium difficile-associated disease in children were detected within a short time interval. Intensive therapy was required in 2 cases with colectomy in one of them. One of the severe cases was community-acquired. Two patients had underlying diseases (Hirschprung disease, Down syndrome) and also tested positive for enteric viruses (rotavirus, calicivirus). PMID:18756189

  3. Genome Sequence of the Autotrophic Acetogen Clostridium magnum DSM 2767

    PubMed Central

    Uhlig, Ronny; Fischer, Ralf-Jörg; Daniel, Rolf

    2016-01-01

    Here we report the draft genome sequence (6.6 Mbp) of the type strain Clostridium magnum, an acetogen with two operons coding for two separate Rnf complexes. C. magnum grows on a broad range of organic substrates and converts CO2 and H2 to acetate using the Wood-Ljungdahl pathway. PMID:27284147

  4. PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AN INTEGRATED SWINE OPERATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the prevalence of Clostridium difficile among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Isolation of C. difficile was performed u...

  5. Genome of a chronic osteitis-causing Clostridium tetani

    PubMed Central

    Fournier, P-E; Levy, P-Y; Million, M; Croce, O; Blanc-Tailleur, C; Brouqui, P; Raoult, D

    2014-01-01

    We sequenced the genome of a Clostridium tetani strain that caused chronic tibial osteitis without any clinical sign of tetanus in a 26-year-old man previously vaccinated against this disease. The genome contained a plasmid that harboured the tetX-tetR tetanospasmin operon, and was highly similar to that of a tetanus-causing strain. PMID:25356334

  6. Small Molecules Take A Big Step Against Clostridium difficile.

    PubMed

    Beilhartz, Greg L; Tam, John; Melnyk, Roman A

    2015-12-01

    Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile. PMID:26547239

  7. Clostridium difficile from healthy food animals: Optimized isolation and prevalence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate (TCCFB) followed by alcohol shock and isolation on tryp...

  8. Clostridium difficile prevalence in an integrated swine operation in Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently there has been an epidemic of human disease in North America caused by the bacterium Clostridium difficile (Cd). It appears to be a new strain that is more virulent than previous strains, produces more toxins, and causes more severe disease (McDonald et al., 2005). The origin of the new s...

  9. 3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: 3-Methylindole (3-MI) is a degradation product of L-tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions which influence 3-MI production in Clostridium scatologenes ATCC 25775 were investigated. Methods and Results: Cells cultured in anaerobic ...

  10. Detection of Clostridium perfringens epsilon toxin by ELISA.

    PubMed

    Naylor, R D; Martin, P K; Sharpe, R T

    1987-03-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test. PMID:2884704

  11. Isolation of Clostridium difficile from healthy food animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Clostridium difficile-associated disease is increasingly reported and studies indicate that food animals may be sources of human infections. Methods: The presence of C. difficile in 345 swine fecal, 1,325 dairy cattle fecal, and 371 dairy environmental samples were examined. Two isolati...

  12. BUTANOL PRODUCTION FROM WHEAT STRAW HYDROLYSATE USING CLOSTRIDIUM BEIJERINCKII

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation, 48.9 gL**-1 glucose was used to produce 20.1 gL**-1 ABE with a productivity and yield of 0.28 gL**-1h**-1 and 0....

  13. ID Learning Unit: Understanding and Interpreting Testing for Clostridium difficile

    PubMed Central

    Solomon, Daniel A.; Milner, Danny A.

    2014-01-01

    Understanding and interpreting the molecular tests for Clostridium difficile is challenging because there are several different types of assays and most laboratories combine multiple tests in order to assess for presence of disease. This learning unit demonstrates the basic principles of each test along with its strengths and weaknesses, and illustrates how the tests are used in clinical practice. PMID:25734081

  14. Varied prevalence of Clostridium difficile in an integrated swine operation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the prevalence of Clostridium difficile among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Preliminary results are based on 131 C. d...

  15. Cultures of "Clostridium acetobutylicum" from various collections comprise Clostridium acetobutylicum, Clostridium beijerinckii, and two other distinct types based on DNA-DNA reassociation.

    PubMed

    Johnson, J L; Toth, J; Santiwatanakul, S; Chen, J S

    1997-04-01

    The best-known acetone-butanol (solvent)-producing bacterium is the Weizmann organism, Clostridium acetobutylicum, which was used for starch-based industrial fermentation. In the past two decades, cultures of "C. acetobutylicum" from various culture collections have included organisms that were isolated for sugar (molasses)-based industrial solvent production. Recent biochemical and genetic studies have revealed significant differences among some of these "C. acetobutylicum" strains. We used DNA-DNA reassociation to analyze 39 cultures of "C. acetobutylicum" and phenotypically similar organisms from major collections. The results of this study clearly identified four groups intergroup reassociation values of less than 30%. All of the intragroup values except the value for one strain were 68% or more, which supported species status for each group. The C. acetobutylicum group (with ATCC 824 as the type strain) consisted of 17 cultures and had average reassociation values of 10% with the other three groups. All strains of C. acetobutylicum produced riboflavin in milk, and the cultures were bright yellow, which is useful for differentiating this species from the other three groups. The Clostridium beijerinckii group (with VPI 5481 [= ATCC 25752] as the type strain) consisted of 16 cultures and included strains NCIMB 8052 and NCP 270. Strains NCP 262 and NRRL B643 constituted the third group, whereas strain N1-4 ("Clostridium saccharoperbutylacetonicum") and its derivative, strain N1-4081, formed the fourth group. At present, the last two groups are each represented by only one independent strain; definitive descriptions of these two groups as two new or revived species will require further phenotypic characterization, as well as identification of additional strains. C. beijerinckii NCP 270, Clostridium sp. strain NRRL B643, and "C. saccharoperbutylacetonicum" were used in industrial solvent production from molasses, which confirms that the new organisms used for the

  16. Development of a triplex real-time PCR assay for the simultaneous detection of Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum in milk.

    PubMed

    Morandi, Stefano; Cremonesi, Paola; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2015-08-01

    Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species. PMID:25870135

  17. The ClosTron: Mutagenesis in Clostridium refined and streamlined.

    PubMed

    Heap, John T; Kuehne, Sarah A; Ehsaan, Muhammad; Cartman, Stephen T; Cooksley, Clare M; Scott, Jamie C; Minton, Nigel P

    2010-01-01

    The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. PMID:19891996

  18. Switchgrass (Panicum virgatum) fermentation by Clostridium thermocellum and Clostridium saccharoperbutylacetonicum sequential culture in a continuous flow reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study was conducted to evaluate fermentation by Clostridium thermocellum and C. saccharoperbutylacetonicum in a continuous-flow, high-solids reactor. Liquid medium was continuously flowed through switchgrass (2 mm particle size) at one of three flow rates: 83.33 mL h-1 (2 L d-1), 41.66 mL h-1(1 ...

  19. Clostridium difficile associated infection, diarrhea and colitis

    PubMed Central

    Hookman, Perry; Barkin, Jamie S

    2009-01-01

    A new, hypervirulent strain of Clostridium difficile, called NAP1/BI/027, has been implicated in C. difficile outbreaks associated with increased morbidity and mortality since the early 2000s. The epidemic strain is resistant to fluoroquinolones in vitro, which was infrequent prior to 2001. The name of this strain reflects its characteristics, demonstrated by different typing methods: pulsed-field gel electrophoresis (NAP1), restriction endonuclease analysis (BI) and polymerase chain reaction (027). In 2004 and 2005, the US Centers for Disease Control and Prevention (CDC) emphasized that the risk of C. difficile-associated diarrhea (CDAD) is increased, not only by the usual factors, including antibiotic exposure, but also gastrointestinal surgery/manipulation, prolonged length of stay in a healthcare setting, serious underlying illness, immune-compromising conditions, and aging. Patients on proton pump inhibitors (PPIs) have an elevated risk, as do peripartum women and heart transplant recipients. Before 2002, toxic megacolon in C. difficile-associated colitis (CDAC), was rare, but its incidence has increased dramatically. Up to two-thirds of hospitalized patients may be infected with C. difficile. Asymptomatic carriers admitted to healthcare facilities can transmit the organism to other susceptible patients, thereby becoming vectors. Fulminant colitis is reported more frequently during outbreaks of C. difficile infection in patients with inflammatory bowel disease (IBD). C. difficile infection with IBD carries a higher mortality than without underlying IBD. This article reviews the latest information on C. difficile infection, including presentation, vulnerable hosts and choice of antibiotics, alternative therapies, and probiotics and immunotherapy. We review contact precautions for patients with known or suspected C. difficile-associated disease. Healthcare institutions require accurate and rapid diagnosis for early detection of possible outbreaks, to initiate

  20. FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

    NASA Astrophysics Data System (ADS)

    Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

    2013-07-01

    Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

  1. Selective medium for isolation of Clostridium butyricum from human feces.

    PubMed Central

    Popoff, M R

    1984-01-01

    A selective medium, Clostridium butyricum isolation medium (BIM), is described for the isolation of C. butyricum from human feces. The BIM is a synthetic minimal medium and contains trimethoprim (16 micrograms/ml), D-cycloserine (10 micrograms/ml), and polymyxin B sulfate (20 micrograms/ml) as selective inhibitory agents. Qualitative tests indicated that C. butyricum and other butyric acid-producing clostridia grew on BIM, Clostridium sphenoides and Bacillus cereus produced small colonies, and other clostridia and other obligate anaerobic or facultatively anerobic bacteria were inhibited. Quantitative recovery of C. butyricum from cultures or seeded fecal samples was comparable with BIM and with complex medium, but the quantitative recovery of the other butyric acid-producing clostridia tested (C. beijerinckii, C. acetobutylicum) was lower with BIM than with complex medium. The BIM should aid the rapid isolation of C. butyricum from fecal samples and should be useful for bacteriological investigation of neonatal necrotizing enterocolitis. PMID:6490827

  2. Models for the study of Clostridium difficile infection

    PubMed Central

    Best, Emma L.; Freeman, Jane; Wilcox, Mark H.

    2012-01-01

    Models of Clostridium difficile infection (C. difficile) have been used extensively for Clostridium difficile (C. difficile) research. The hamster model of C. difficile infection has been most extensively employed for the study of C. difficile and this has been used in many different areas of research, including the induction of C. difficile, the testing of new treatments, population dynamics and characterization of virulence. Investigations using in vitro models for C. difficile introduced the concept of colonization resistance, evaluated the role of antibiotics in C. difficile development, explored population dynamics and have been useful in the evaluation of C. difficile treatments. Experiments using models have major advantages over clinical studies and have been indispensible in furthering C. difficile research. It is important for future study programs to carefully consider the approach to use and therefore be better placed to inform the design and interpretation of clinical studies. PMID:22555466

  3. Beneficial and harmful roles of bacteria from the Clostridium genus.

    PubMed

    Samul, Dorota; Worsztynowicz, Paulina; Leja, Katarzyna; Grajek, Włodzimierz

    2013-01-01

    Bacteria of the Clostridium genus are often described only as a biological threat and a foe of mankind. However, many of them have positive properties and thanks to them they may be used in many industry branches (e.g., in solvents and alcohol production, in medicine, and also in esthetic cosmetology). During the last 10 years interest in application of C. botulinum and C. tetani in medicine significantly increased. Currently, the structure and biochemical properties of neurotoxins produced by these bacterial species, as well as possibilities of application of such toxins as botulinum as a therapeutic factor in humans, are being intensely researched. The main aim of this article is to demonstrate that bacteria from Clostridium spp. are not only pathogens and the enemy of humanity but they also have many important beneficial properties which make them usable among many chemical, medical, and cosmetic applications. PMID:24432307

  4. Clostridium difficile spore biology: sporulation, germination, and spore structural proteins

    PubMed Central

    Paredes-Sabja, Daniel; Shen, Aimee; Sorg, Joseph A.

    2014-01-01

    Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of world-wide concern. Due to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. While important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and B. subtilis at the level of sporulation, germination and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. PMID:24814671

  5. Clostridium perfringens in animal disease: a review of current knowledge.

    PubMed

    Niilo, L

    1980-05-01

    The diseases caused by various types of Clostridium perfringens are critically reviewed in the light of current knowledge. Particular emphasis is placed on information concerning these diseases in Canadian livestock. There are two etiologically clearly-defined acute C. perfringens diseases recognized in Canada: hemorrhagic enteritis of the new born calf, caused by C. perfringens type C, and enterotoxemia of sheep, caused by type D. Clostridium perfringens type A may play a role as a secondary pathological agent in various disease conditions, such as necrotic enteritis of chickens. It may also cause wound infections and may provide a source for human food poisoning outbreaks. There appears to be a considerable lack of knowledge regarding the distribution of C. perfringens types, their pathogenesis, diagnosis and the incidence of diseases caused by this organism. PMID:6253040

  6. Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service.

    PubMed

    Ferroni, A; Merckx, J; Ancelle, T; Pron, B; Abachin, E; Barbut, F; Larzul, J; Rigault, P; Berche, P; Gaillard, J L

    1997-12-01

    An outbreak of nosocomial diarrhea that occurred in a pediatric orthopedic service between 1 December 1993 and 15 April 1994 is reported. A total of 37 patients (mean age, 9.6 years; range, 2 months-19.3 years) were involved in the outbreak, including six patients with bacteriologically documented Clostridium difficile infection. A multivariate analysis identified lincomycin treatment for at least three days as the only significant risk factor. Stool samples from four asymptomatic patients were also positive for Clostridium difficile and its cytotoxins. Isolates from all patients belonged to serogroup C, were highly resistant to lincomycin, and exhibited the same restriction pattern by pulsed-field gel electrophoresis. The outbreak ended after treatment with lincomycin was discontinued and hygiene control measures were implemented. PMID:9495676

  7. The utilization of a commercial soil nucleic acid extraction kit and PCR for the detection of Clostridium tetanus and Clostridium chauvoei on farms after flooding in Taiwan.

    PubMed

    Huang, Shr-Wei; Chan, Jacky Peng-Wen; Shia, Wei-Yau; Shyu, Chin-Lin; Tung, Kwon-Chung; Wang, Chi-Young

    2013-05-01

    Clostridial diseases are zoonoses and are classified as soil-borne diseases. Clostridium chauvoei and Clostridium tetani cause blackleg disease and tetanus, respectively. Since bacteria and spores are re-distributed by floods and then, subsequently, contaminate soils, pastures and water; the case numbers associated with clostridial diseases usually increase after floods. Because Taiwan is often affected by flood damage during the typhoon season, possible threats from these diseases are present. Thus, this study's aim is to apply a combination of a commercial nucleic acid extraction kit and PCR to assess the prevalence of Clostridia spp. in soil and to compare the positivity rates for farms before and after floods. The minimum amounts of Clostridium tetanus and Clostridium chauvoei that could be extracted from soils and detected by PCR were 10 and 50 colony forming units (cfu), respectively. In total, 76 samples were collected from the central and southern regions of Taiwan, which are the areas that are most frequently damaged by typhoons. Noteworthy, the positive rates for Clostridium tetanus and Clostridium chauvoei in Pingtung county after the severe floods caused by a typhoon increased significantly from 13.73 and 7.84% to 53.85 and 50.00%, respectively. This study for the first time provides the evidence from surveillance data that there are changes in the environmental distribution of Clostridium spp. after floods. This study indicates that screening for soil-related zoonotic pathogens is a potential strategy that may help to control these diseases. PMID:23208321

  8. Closed Genome Sequence of Clostridium pasteurianum ATCC 6013

    PubMed Central

    Rotta, Carlo; Poehlein, Anja; Schwarz, Katrin; McClure, Peter; Daniel, Rolf

    2015-01-01

    We report here the closed genome of Clostridium pasteurianum ATCC 6013, a saccharolytic, nitrogen-fixing, and spore-forming Gram-positive obligate anaerobe. The organism is of biotechnological interest due to the production of solvents (butanol and 1,3-propanediol) but can be associated with food spoilage. The genome comprises a total of 4,351,223 bp. PMID:25700419

  9. Clostridium difficile in Children: To Treat or Not to Treat?

    PubMed Central

    2014-01-01

    Clostridium difficile infection has been increasing since 2000 in children and in adults. Frequent antibiotics use, comorbidity, and the development of hypervirulent strains have increased the risk of infection. Despite the high carriage rates of C. difficile, infants rarely develop clinical infection. Discontinuing antibiotics and supportive management usually leads to resolution of disease. Antibiotics use should be stratified depending on the patient's age and severity of the disease. PMID:25061582

  10. Clostridium chauvoei-associated meningoencephalitis in a calf.

    PubMed

    2016-01-16

    ·Meningoencephalitis in a calf associated with Clostridium chauvoei infection. ·Bovine papular stomatitis in calves. ·Otitis media due to Mycoplasma bovis in calves. ·Sporadic porcine abortion due to Nocardia species. ·Spotty liver disease in hens. These are among matters discussed in the disease surveillance report for September 2015 from SAC Consulting: Veterinary Services (SAC C VS). PMID:26769810

  11. Clostridium difficile Cell Attachment Is Modified by Environmental Factors

    PubMed Central

    Waligora, Anne-Judith; Barc, Marie-Claude; Bourlioux, Pierre; Collignon, Anne; Karjalainen, Tuomo

    1999-01-01

    Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa. PMID:10473442

  12. Historical and current perspectives on Clostridium botulinum diversity.

    PubMed

    Smith, Theresa J; Hill, Karen K; Raphael, Brian H

    2015-05-01

    For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current Clostridium botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity. PMID:25312020

  13. [Massive intravascular hemolysis secondary to sepsis due to Clostridium perfringens].

    PubMed

    Pita Zapata, E; Sarmiento Penide, A; Bautista Guillén, A; González Cabano, M; Agulla Budiño, J A; Camba Rodríguez, M A

    2010-05-01

    Massive hemolysis secondary to sepsis caused by Clostridium perfringens is a rare entity but appears fairly often in the literature. In nearly all published reports, the clinical course is rapid and fatal. We describe the case of a 75-year-old woman with diabetes who was admitted with symptoms consistent with acute cholecystitis. Deteriorating hemodynamics and laboratory findings were consistent with intravascular hemolysis, coagulation disorder, and renal failure. Gram-positive bacilli of the Clostridium species were detected in blood along with worsening indicators of hemolysis. In spite of antibiotic and surgical treatment, hemodynamic support and infusion of blood products, the patient continued to decline and died in the postoperative recovery unit 14 hours after admission. Mortality ranges from 70% to 100% in sepsis due to Clostridium perfringens, and risk of death is greater if massive hemolysis is present, as in the case we report. Only a high degree of clinical suspicion leading to early diagnosis and treatment can improve the prognosis. This bacterium should therefore be considered whenever severe sepsis and hemolysis coincide. PMID:20527348

  14. [Toxins of Clostridium perfringens as a natural and bioterroristic threats].

    PubMed

    Omernik, Andrzej; Płusa, Tadeusz

    2015-09-01

    Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens. PMID:26449576

  15. Clostridium tertium Bacteremia in a Patient with Glyphosate Ingestion

    PubMed Central

    You, Myung-Jo; Shin, Gee-Wook; Lee, Chang-Seop

    2015-01-01

    Patient: Female, 44 Final Diagnosis: Clostridium tertium bacteremia Symptoms: Fever Medication: Ertapenem • Metronidazole Clinical Procedure: — Specialty: Infectious Disease Objective: Unknown etiology Background: Clostridium tertium is distributed in the soil and in animal and human gastrointestinal tracts. C. tertium has been isolated from patients with blood diseases, immune disorders, and abdominal surgeries. Glyphosate is toxic, causing cause eye and skin irritation, gastrointestinal pain, and vomiting. Ingestion of herbicides modifies the gastrointestinal environment, which stresses the living organisms. However, there has been little attention to cases of bacteremia in patients recovering from suicide attempt by ingesting herbicide. Case Report: Clostridium tertium was identified in a 44-year-old female who attempted suicide by glyphosate (a herbicide) ingestion. The 16S rRNA sequences from all colonies were 99% identical with that of C. tertium (AB618789) found on a BLAST search of the NCBI database. The bacterium was cultured on TSA under aerobic and anaerobic conditions. Antimicrobial susceptibility tests performed under both aerobic and anaerobic conditions showed that the bacterium was susceptible to penicillin, a combination of β-lactamase inhibitor and piperacillin or amoxicillin, and first- and second- generation cephalosporins. However, it was resistant to third- and fourth-generation cephalosporins. Conclusions: Glyphosate herbicide might be a predisposing factor responsible for the pathogenesis of C. tertium. The results highlight the need for careful diagnosis and selection of antibiotics in the treatment of this organism. PMID:25577783

  16. Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

    PubMed Central

    Tompkin, R. B.; Christiansen, L. N.; Shaparis, A. B.; Bolin, H.

    1974-01-01

    Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. PMID:4368631

  17. Purification and characterization of konjac glucomannan degrading enzyme from anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group.

    PubMed

    Nakajima, N; Matsuura, Y

    1997-10-01

    Konjac glucomannan degrading enzyme was purified to homogeneity from the culture broth of an anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group. The enzyme was composed of a single polypeptide chain with a molecular weight of 50,000-53,000. The enzyme was an endo-beta-mannanase that acted specifically on the polysaccharides such as konjac glucomannan and coffee mannan, producing exclusively their smaller oligosaccharides and the monosaccharides. The optimal pH of the enzyme for the hydrolysis of konjac glucomannan was around 7-8 and the enzyme was stable in rather alkaline pH range of 8-10. The enzyme reaction was activated by the addition of CaCl2 and dithiothreitol. It was suggested that the enzyme might contribute to the decomposition of konjac glucomannan in human digestive tract. PMID:9362121

  18. Complete Genome Sequence of Clostridium sp. Strain DL-VIII, a Novel Solventogenic Clostridium Species Isolated from Anaerobic Sludge

    PubMed Central

    Taghavi, Safiyh; Izquierdo, Javier A.

    2013-01-01

    We report the genome sequence of Clostridium sp. strain DL-VIII, a novel Gram-positive, endospore-forming, solventogenic bacterium isolated from activated anaerobic sludge of a wastewater treatment plant. Aside from a complete sol operon, the 6,477,357-bp genome of DL-VIII reveals genes for several unique enzymes with applications in lignocellulose degradation, including two phenolic acid decarboxylases. PMID:23929491

  19. Draft Genome Sequence of Clostridium sporogenes Strain UC9000 Isolated from Raw Milk

    PubMed Central

    La Torre, Angela; Zotta, Teresa; Orrù, Luigi; Lamontanara, Antonella; Cocconcelli, Pier Sandro

    2016-01-01

    Clostridium sporogenes is a causative agent of food spoilage and is often used as the nontoxigenic surrogate for Clostridium botulinum. Here, we described the draft genome sequence and annotation of C. sporogenes strain UC9000 isolated from raw milk. PMID:27081128

  20. Two Serious Cases of Infection with Clostridium celatum after 40 Years in Hiding?

    PubMed Central

    Hoegh, Silje Vermedal; Holt, Hanne Marie; Justesen, Ulrik Stenz

    2015-01-01

    Clostridium celatum [ce.la'tum. L. adj. celatum hidden] has been known since 1974, when it was isolated from human feces. In 40 years, no association with human infection has been reported. In this work, we present two serious cases of infection with the anaerobic Gram-positive rod Clostridium celatum. PMID:26560535

  1. Antimicrobial susceptibility of Clostridium difficile isolated from food animals on farms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. Clostridium difficile can also inhabit healthy food animals and transmi...

  2. Mathematical modeling and growth kinetics of Clostridium sporogenes in cooked beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium sporogenes PA 3679 is a common surrogate for proteolytic Clostridium botulinum for thermal process development and validation. However, little information is available concerning the growth kinetics of C. sporogenes in food. Therefore, the objective of this study was to investigate the...

  3. Draft Genome Sequence of Clostridium sporogenes Strain UC9000 Isolated from Raw Milk.

    PubMed

    La Torre, Angela; Bassi, Daniela; Zotta, Teresa; Orrù, Luigi; Lamontanara, Antonella; Cocconcelli, Pier Sandro

    2016-01-01

    Clostridium sporogenesis a causative agent of food spoilage and is often used as the nontoxigenic surrogate forClostridium botulinum Here, we described the draft genome sequence and annotation ofC. sporogenesstrain UC9000 isolated from raw milk. PMID:27081128

  4. Genome sequence of Clostridium tunisiense TJ, isolated from drain sediment from a pesticide factory.

    PubMed

    Sun, Lili; Wang, Yu; Yu, Chunyan; Zhao, Yongqin; Gan, Yinbo

    2012-12-01

    Clostridium tunisiense is a Gram-positive, obligate anaerobe that was first isolated in an anaerobic environment under eutrophication. Here we report the first genome sequence of the Clostridium tunisiense TJ isolated from drain sediment of a pesticide factory in Tianjin, China. The genome is of great importance for both basic and application research. PMID:23209212

  5. Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4.

    PubMed

    Mori, Y

    1990-01-01

    Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10), it required CO(2) and Na(2)CO(3) for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism. PMID:16348106

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

    PubMed

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

    2014-12-01

    Clostridium perfringens has been suspected as an enteropathogen in dogs. However, its exact role in gastrointestinal (GI) disorders in dogs remains unknown. Recent studies suggest the importance of an altered intestinal microbiota in the activation of virulence factors of enteropathogens. The aim of this study was to evaluate the relationship between diarrhea, dysbiosis, and the presence of C. perfringens and its enterotoxin (CPE). Fecal samples were collected prospectively from 95 healthy control dogs and 104 dogs with GI disease and assessed for bacterial abundances and the presence of CPE using quantitative PCR and ELISA, respectively. C. perfringens was detected in all dogs. Potentially enterotoxigenic C. perfringens were detected in 33.7% (32/95) of healthy control dogs and 48.1% (50/104) diseased dogs, respectively. CPE was detected by ELISA in 1.0% (1/95) of control dogs and 16.3% (17/104) of diseased dogs. Abundances of Fusobacteria, Ruminococcaceae, Blautia, and Faecalibacterium were significantly decreased in diseased dogs, while abundances of Bifidobacterium, Lactobacillus, and Escherichia coli were significantly increased compared to control dogs. The microbial dysbiosis was independent of the presence of the enterotoxigenic C. perfringens or CPE. In conclusion, the presence of CPE as well as fecal dysbiosis was associated with GI disease. However, the presence of C. perfringens was not indicative of GI disease in all cases of diarrhea, and the observed increased abundance of enterotoxigenic C. perfringens may be part of intestinal dysbiosis occurring in GI disease. The significance of an intestinal dysbiosis in dogs with GI disease deserves further attention. PMID:25458422

  7. Intravascular Hemolysis and Septicemia due to Clostridium perfringens Emphysematous Cholecystitis and Hepatic Abscesses

    PubMed Central

    Cochrane, Justin; Bland, Lacie; Noble, Mary

    2015-01-01

    Context. Clostridium perfringens septicemia is often associated with translocation from the gastrointestinal or gastrourinary tract and occurs in patients who have malignancy or are immunocompromised. Clostridium perfringens septicemia is usually fatal without early identification, source control, and antibiotics. Case. We present a case of a 65-year-old female with Clostridium perfringens septicemia secondary to emphysematous cholecystitis, with progression to hepatic abscesses. Conclusion. Septicemia secondary to Clostridium perfringens is generally fatal if not detected early. Source control with surgery or percutaneous drainage and early antibiotic therapy is imperative. Hyperbaric oxygen therapy may reduce mortality. Clinicians caring for patients with sepsis and intravascular hemolysis must have Clostridium perfringens septicemia on their differential diagnosis with a low threshold for starting antibiotics and pursuing source of infection. PMID:26229537

  8. Electrochemical detoxification of phenolic compounds in lignocellulosic hydrolysate for Clostridium fermentation.

    PubMed

    Lee, Kyung Min; Min, Kyoungseon; Choi, Okkyoung; Kim, Ki-Yeon; Woo, Han Min; Kim, Yunje; Han, Sung Ok; Um, Youngsoon

    2015-01-01

    Lignocellulosic biomass is being preferred as a feedstock in the biorefinery, but lignocellulosic hydrolysate usually contains inhibitors against microbial fermentation. Among these inhibitors, phenolics are highly toxic to butyric acid-producing and butanol-producing Clostridium even at a low concentration. Herein, we developed an electrochemical polymerization method to detoxify phenolic compounds in lignocellulosic hydrolysate for efficient Clostridium fermentation. After the electrochemical detoxification for 10h, 78%, 77%, 82%, and 94% of p-coumaric acid, ferulic acid, vanillin, and syringaldehyde were removed, respectively. Furthermore, 71% of total phenolics in rice straw hydrolysate were removed without any sugar-loss. Whereas the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were completely inhibited in un-detoxified hydrolysate, those in detoxifying rice straw hydrolysate were recovered to 70-100% of the control cultures. The electrochemical detoxification method described herein provides an efficient strategy for producing butanol and butyric acid through Clostridium fermentation with lignocellulosic hydrolysate. PMID:25863199

  9. Clostridium difficile Enterocolitis and Reactive Arthritis: A Case Report and Review of the Literature

    PubMed Central

    Cappella, Michela; Pugliese, Fabrizio; Zucchini, Andrea; Marchetti, Federico

    2016-01-01

    Reactive arthritis is a rare complication of Clostridium difficile enterocolitis, especially in children. We review the 6 pediatric cases published in the English and non-English literature and discuss their clinical presentation, outcome, treatment, and pathophysiology. We also report the seventh case of Clostridium difficile reactive arthritis in a 6-year-old boy who was treated with amoxicillin-clavulanate for 10 days because of an upper respiratory infection. After the antibiotic course, the child developed at the same time diarrhea with positive stool culture for Clostridium difficile and an asymmetric polyarthritis. Nonsteroidal anti-inflammatory drugs and metronidazole completely resolved the pain, joint swelling, and diarrhea. After twelve months of follow-up there has been no recurrence. This report confirms the self-limiting course of Clostridium difficile reactive arthritis. Clostridium difficile testing in children with gastrointestinal symptoms and acute onset of joint pain should be always considered. PMID:27190666

  10. Clostridium difficile Enterocolitis and Reactive Arthritis: A Case Report and Review of the Literature.

    PubMed

    Cappella, Michela; Pugliese, Fabrizio; Zucchini, Andrea; Marchetti, Federico

    2016-01-01

    Reactive arthritis is a rare complication of Clostridium difficile enterocolitis, especially in children. We review the 6 pediatric cases published in the English and non-English literature and discuss their clinical presentation, outcome, treatment, and pathophysiology. We also report the seventh case of Clostridium difficile reactive arthritis in a 6-year-old boy who was treated with amoxicillin-clavulanate for 10 days because of an upper respiratory infection. After the antibiotic course, the child developed at the same time diarrhea with positive stool culture for Clostridium difficile and an asymmetric polyarthritis. Nonsteroidal anti-inflammatory drugs and metronidazole completely resolved the pain, joint swelling, and diarrhea. After twelve months of follow-up there has been no recurrence. This report confirms the self-limiting course of Clostridium difficile reactive arthritis. Clostridium difficile testing in children with gastrointestinal symptoms and acute onset of joint pain should be always considered. PMID:27190666

  11. Fusion of a thermophilic phage endolysin to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  12. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  13. First Complete Genome Sequence of Clostridium sporogenes DSM 795T, a Nontoxigenic Surrogate for Clostridium botulinum, Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Terabayashi, Yasunobu; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2015-01-01

    The first complete genome sequence of Clostridium sporogenes DSM 795T, a nontoxigenic surrogate for Clostridium botulinum, was determined in a single contig using the PacBio single-molecule real-time technology. The genome (4,142,990 bp; G+C content, 27.98%) included 86 sets of >1,000-bp identical sequence pairs and 380 tandem repeats. PMID:26227598

  14. Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of Clostridium species isolated from Saudi Arabia.

    PubMed

    AlMogbel, Mohammed Suliman

    2016-01-01

    The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha'il region of Saudi Arabia in September 2014 using MALDI-TOF-MS. Clostridium spp. were identified by Bruker MALDI-TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI-TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI-TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI-TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms. PMID:26991272

  15. Traits of selected Clostridium strains for syngas fermentation to ethanol.

    PubMed

    Martin, Michael E; Richter, Hanno; Saha, Surya; Angenent, Largus T

    2016-03-01

    Syngas fermentation is an anaerobic bioprocess that could become industrially relevant as a biorefinery platform for sustainable production of fuels and chemicals. An important prerequisite for commercialization is adequate performance of the biocatalyst (i.e., sufficiently high production rate, titer, selectivity, yield, and stability of the fermentation). Here, we compared the performance of three potential candidate Clostridium strains in syngas-to-ethanol conversion: Clostridium ljungdahlii PETC, C. ljungdahlii ERI-2, and Clostridium autoethanogenum JA1-1. Experiments were conducted in a two-stage, continuously fed syngas-fermentation system that had been optimized for stable ethanol production. The two C. ljungdahlii strains performed similar to each other but different from C. autoethanogenum. When the pH value was lowered from 5.5 to 4.5 to induce solventogenesis, the cell-specific carbon monoxide and hydrogen consumption (similar rate for all strains at pH 5.5), severely decreased in JA1-1, but hardly in PETC and ERI-2. Ethanol production in strains PETC and ERI-2 remained relatively stable while the rate of acetate production decreased, resulting in a high ethanol/acetate ratio, but lower overall productivities. With JA1-1, lowering the pH severely lowered rates of both ethanol and acetate production; and as a consequence, no pronounced shift to solventogenesis was observed. The highest overall ethanol production rate of 0.301 g · L(-1)  · h(-1) was achieved with PETC at pH 4.5 with a corresponding 19 g/L (1.9% w/v) ethanol concentration and a 5.5:1 ethanol/acetate molar ratio. A comparison of the genes relevant for ethanol metabolism revealed differences between C. ljungdahlii and C. autoethanogenum that, however, did not conclusively explain the different phenotypes. PMID:26331212

  16. Controversies Surrounding Clostridium difficile Infection in Infants and Young Children

    PubMed Central

    Nicholson, Maribeth R.; Thomsen, Isaac P.; Edwards, Kathryn M.

    2014-01-01

    Clostridium difficile is a frequent cause of antibiotic-associated diarrhea in adults and older children. However, as many as 80% of infants can be asymptomatically colonized. The reasons for this have not been well established but are believed to be due to differences in toxin receptors or toxin internalization. Determining which children who test positive for C. difficile warrant treatment is exceedingly difficult, especially in the setting of increased rates of detection and the rising risk of disease in children lacking classic risk factors for C. difficile.

  17. On the difficulties of isolating Clostridium difficile from hospital environments.

    PubMed

    Malik, D J; Patel, K V; Clokie, M R J; Shama, G

    2013-06-01

    Spores of Clostridium difficile were deposited on to a stainless steel surface and subsequently exposed to a chlorine-releasing disinfectant (dichloroisocyanurate). Recovery of the spores was carried out using RODAC plates containing a variety of selective and non-selective agars. The non-selective agar media yielded higher recoveries of both control and chlorine-stressed spores. Our results show that the antibiotics used in selective media imposed an additional stress on both disinfectant-treated and untreated spores resulting in considerably reduced recoveries. This could lead to a serious underestimate of the extent of environmental contamination by this organism. PMID:23643391

  18. Infectious Diarrhea: Norovirus and Clostridium difficile in Older Adults.

    PubMed

    White, Mary B; Rajagopalan, Shobita; Yoshikawa, Thomas T

    2016-08-01

    Norovirus infection usually results in acute gastroenteritis, often with incapacitating nausea, vomiting, and diarrhea. It is highly contagious and resistant to eradication with alcohol-based hand sanitizer. Appropriate preventative and infection control measures can mitigate the morbidity and mortality associated with norovirus infection. Clostridium difficile infection is the leading cause of health care-associated diarrhea in the United States. Antibiotic use is by far the most common risk factor for C difficile colonization and infection. Appropriate preventive measures and judicious use of antibiotics can help mitigate the morbidity and mortality associated with C difficile infection. PMID:27394020

  19. Evaluation and Modifications of Media for Enumeration of Clostridium perfringens

    PubMed Central

    Hauschild, A. H. W.; Hilsheimer, R.

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  20. Evaluation and modifications of media for enumeration of Clostridium perfringens.

    PubMed

    Hauschild, A H; Hilsheimer, R

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  1. Rapid Technique for the Enumeration of Clostridium perfringens

    PubMed Central

    Marshall, Robert S.; Steenbergen, J. Frank; McClung, L. S.

    1965-01-01

    A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar. PMID:14339262

  2. Clostridium perfringens and other anaerobes isolated from bile.

    PubMed Central

    Sakaguchi, Y; Murata, K; Kimura, M

    1983-01-01

    Clostridium perfringens was isolated from bile in 13 cases of 150 patients examined. The serotypes of C perfringens strains isolated from bile and faeces were investigated using antisera to Hobbs' type 1-17. Two or more serological types were often found in a single specimen, but in the same patient the serotypes of C perfringens strains isolated from the bile were identical with those from the faeces. Beta-glucuronidase production in these C perfringens serotypes was tested with the API-Strep system. Strains agglutinated with Hobbs' antisera produced beta-glucuronidase, but non-agglutinated strains did not. PMID:6298284

  3. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  4. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

    PubMed Central

    Silva, R. O. S.; Salvarani, F.M.; Assis, R.A.; Martins, N.R.S.; Pires, P.S.; Lobato, F.C.F.

    2009-01-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  5. [Characteristics of Clostridium tetani and laboratory diagnosis of tetanus].

    PubMed

    Smietańska, Karolina; Rokosz-Chudziak, Natalia; Rastawicki, Waldemar

    2013-01-01

    The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans. PMID:24730217

  6. Pneumatosis intestinalis in a patient with recurrent Clostridium difficile infection

    PubMed Central

    Ha, Duc; Tsai, Chung-Jyi

    2012-01-01

    A 65-year-old man with long-standing diarrhoea, recurrent Clostridium difficile infection (CDI) in the previous 5 months presented to the gastroenterology clinic with recurrent diarrhoea and abdominal cramping. Physical examination was negative for signs of acute abdomen. Stool C difficile PCR was positive. Abdominal imaging demonstrated an extensive pneumatosis intestinalis involving the small bowel and a dilated small bowel loop. He was treated conservatively with oral vancomycin for recurrent CDI with resolution of diarrhoea and abdominal cramping on 1-month follow-up visit. PMID:23112256

  7. Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation

    DOE PAGESBeta

    Sander, Kyle B.; Wilson, Charlotte M.; M. Rodriquez, Jr.; Klingeman, Dawn Marie; Davison, Brian H.; Brown, Steven D.; Rydzak, T.

    2015-12-12

    Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.

  8. Challenges for standardization of Clostridium difficile typing methods.

    PubMed

    Huber, Charlotte A; Foster, Niki F; Riley, Thomas V; Paterson, David L

    2013-09-01

    Typing of Clostridium difficile facilitates understanding of the epidemiology of the infection. Some evaluations have shown that certain strain types (for example, ribotype 027) are more virulent than others and are associated with worse clinical outcomes. Although restriction endonuclease analysis (REA) and pulsed-field gel electrophoresis have been widely used in the past, PCR ribotyping is the current method of choice for typing of C. difficile. However, global standardization of ribotyping results is urgently needed. Whole-genome sequencing of C. difficile has the potential to provide even greater epidemiologic information than ribotyping. PMID:23784128

  9. Community-Acquired Clostridium Difficile Infection: Awareness and Clinical Implications

    PubMed Central

    Juneau, Cheryl; Mendias, Elnora (Nonie) P.; Wagal, Nihas; Loeffelholz, Michael; Savidge, Tor; Croisant, Sharon; Dann, Sara

    2013-01-01

    The epidemiology of Clostridium difficile infection (CDI) is changing. CDI, usually depicted as a nosocomial infection in the elderly, is now occurring in community-dwelling persons who are younger and otherwise dissimilar. A more virulent isolate (North American Pulsed Field type 1 (NAP1) associated with increased morbidity and mortality, has been identified. In 2005, similar strains were associated with severe disease in community-dwelling patients at a rate of 7.6/100,000. Screening patients with potential CDI symptoms and implementing preventative measures, including judicious use of antibiotics, can reduce disease burden. PMID:23814528

  10. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens.

    PubMed

    Silva, R O S; Salvarani, F M; Assis, R A; Martins, N R S; Pires, P S; Lobato, F C F

    2009-04-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  11. New advances in the treatment of Clostridium difficile infection (CDI)

    PubMed Central

    Hedge, Dennis D; Strain, Joe D; Heins, Jodi R; Farver, Debra K

    2008-01-01

    Clostridium difficile infections (CDI) have increased in frequency throughout the world. In addition to an increase in frequency, recent CDI epidemics have been linked to a hypervirulent C. difficile strain resulting in greater severity of disease. Although most mild to moderate cases of CDI continue to respond to metronidazole or vancomycin, refractory and recurrent cases of CDI may require alternative therapies. This review provides a brief overview of CDI and summarizes studies involving alternative antibiotics, toxin binders, probiotics, and immunological therapies that can be considered for treatment of acute and recurrent CDI in severe and refractory situations. PMID:19209277

  12. Clostridium difficile Infection in Patients with Inflammatory Bowel Disease.

    PubMed

    Fu, Nancy; Wong, Titus

    2016-06-01

    Clostridium difficile infection (CDI) is now the leading cause of nosocomial infection. There has been an upsurge of CDI in patients with inflammatory bowel disease (IBD). IBD patients with CDI have increased morbidity and mortality. The establishment, proliferation, and recurrence of CDI in IBD patients form a complex interplay of microbial, environmental, and host-susceptibility factors. Different risk factors have been found predisposing IBD patients to CDI. Vancomycin performs better than metronidazole in treating IBD patients with CDI. Fecal microbiota transplantation continues to be a very effective therapy. New therapeutic modalities such as vaccinations and bile salts are currently being investigated. PMID:27137789

  13. Characteristic strategy of assimilation of various saccharides by Clostridium cellulovorans.

    PubMed

    Inamori, Takako; Aburaya, Shunsuke; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-12-01

    Clostridium cellulovorans can effectively assimilate not only cellulose but also hemicellulose by producing cellulosomal and non-cellulosomal enzymes. However, little is known about how C. cellulovorans assimilates various saccharides in media containing polysaccharides and oligosaccharides. In this research, we investigated the property of saccharide incorporation and assimilation by C. cellulovorans. Faster growth in media containing xylan and cellulose was achieved by switching polysaccharides, in which xylan was first assimilated, followed by cellulose. Furthermore, the presence of polysaccharides that can be easily degraded might increase the assimilation rate of lignocellulose by promoting growth. These properties of C. cellulovorans could be suitable for the effective utilization of lignocellulosic biomass. PMID:27586595

  14. Clostridium difficile: the anaerobe that made the grade.

    PubMed

    Brazier, Jon S

    2012-04-01

    Unlike other anaerobic bacteria of clinical importance, Clostridium difficile has managed to enter into the realm of public awareness. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous "superbug" responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This report picks out key moments, particularly in the UK, which tracked the rise in both the public and political awareness of this organism. PMID:22293217

  15. Structure, Function and Regulation of the Clostridium cellulovorans Cellulosome

    SciTech Connect

    Doi, Roy H

    2008-06-01

    Our major goal for this project (2004-2008) was to obtain an understanding ofthe structure, function, and regulation of the Clostridium cellulovorans cellulosomes. Our specific goals were to select genes for cellulosomal and non-cellulosomal enzymes and characterize their products, to study the synergistic action between cellulosomal and non-cellulosomal enzymes, to study the composition of cellulosomes when cells were grown with different carbon sources, continue our studies on the scaffolding protein and examine heterologous expression of cellulosomal genes in Bacillus subtilis. We fulfilled the specific goals of our proposal.

  16. Probiotics and Antibiotic-Associated Diarrhea and Clostridium difficile Infection

    NASA Astrophysics Data System (ADS)

    Surawicz, Christina M.

    Diarrhea is a common side effect of antibiotics. Antibiotics can cause diarrhea in 5-25% of individuals who take them but its occurrence is unpredictable. Diarrhea due to antibiotics is called antibiotic-associated diarrhea (AAD). Diarrhea may be mild and resolve when antibiotics are discontinued, or it may be more severe. The most severe form of AAD is caused by overgrowth of Clostridium difficile which can cause severe diarrhea, colitis, pseudomembranous colitis, or even fatal toxic megacolon. Rates of diarrhea vary with the specific antibiotic as well as with the individual susceptibility.

  17. Role of chemotaxis in solvent production by Clostridium acetobutylicum

    SciTech Connect

    Gutierrez, N.A.; Maddox, I.S.

    1987-08-01

    The motility of Clostridium acetobutylicum has been investigated during a typical batch fermentation process for solvent production. The motility is characterized by runs during the early phase of sugar utilization and acid production, but this changes to tumbles during the onset of solventogenesis. Sugars and undissociated acetic and butyric acids have been shown to be attractants for the bacterium, while acetone, butanol, ethanol, and dissociated acetate and butyrate are repellents. It is suggested that chemotactic responses explain why highly motile cells are strongly solventogenic.

  18. Citrate, a specific substrate for the isolation of Clostridium sphenoides.

    PubMed Central

    Walther, R; Hippe, H; Gottschalk, G

    1977-01-01

    With a medium containing citrate as the carbon and energy source, 10 clostridial strains were isolated from various mud samples. Characterization of these strains revealed that they all belonged to the same species, Clostridium sphenoides. Strains of this organism obtained from culture collections were also able to grow citrate, whereas 15 other clostridial species tested were not. Citrate was fermented by C. sphenoides to acetate, ethanol, carbon dioxide, and hydrogen. Experiments with stereospecifically 14C-labeled citrate indicated that citrate lyase was involved in citrate degradation. Images PMID:869540

  19. A fatal postpartum Clostridium sordellii associated toxic shock syndrome.

    PubMed Central

    Bitti, A; Mastrantonio, P; Spigaglia, P; Urru, G; Spano, A I; Moretti, G; Cherchi, G B

    1997-01-01

    Clostridium sordellii is an infrequent human pathogen. It has been demonstrated to be occasionally responsible for myonecrosis or gas gangrene. Interestingly, in the obstetric literature, some cases of postpartum maternal deaths have been associated with C sordellii infection causing a rapidly lethal toxin mediated syndrome. This is the first reported case of postpartum death in a 29 year old woman, in which a toxigenic C sordellii was isolated from the patient's blood antemortem during the fatal toxic shock, strongly indicating its role in this rare syndrome. PMID:9155682

  20. First Australian isolation of epidemic Clostridium difficile PCR ribotype 027.

    PubMed

    Riley, Thomas V; Thean, Sarah; Hool, Graham; Golledge, Clayton L

    2009-06-15

    We report the first isolation in Australia of a hypervirulent epidemic strain of Clostridium difficile, PCR ribotype 027. It was isolated from a 43-year-old woman with a permanent ileostomy, who appears to have been infected while travelling in the United States. The isolate was positive for toxin A, toxin B and binary toxin, and resistant to fluoroquinolone antimicrobials, and had characteristic deletions in the tcdC gene. All diagnostic laboratories and health care facilities in Australia should now be on high alert for this organism. PMID:19527210

  1. Clostridial disease of the gut.

    PubMed

    Borriello, S P

    1995-06-01

    Clostridia are an important cause of morbidity and mortality in humans and animals. Some of the most common clostridial infections are those of the gut. The primary infections in humans are Clostridium perfringens food poisoning and Clostridium difficile-mediated antibiotic-associated diarrhea and colitis. Less common but important infections include non-food poisoning C. perfringens nosocomial diarrhea and C. perfringens type C necrotizing jejunitis (pig-bel). C. perfringens is also the dominant cause of gastrointestinal infections in animals, although Clostridium septicum causing braxy in sheep, Clostridium colinum causing ulcerative enteritis is avian species, and Clostridium spiroforme causing enterotoxemia in rabbits are important exceptions. PMID:7548565

  2. Alpha-Toxin and Gamma-Toxin Jointly Promote Staphylococcus aureus Virulence in Murine Septic Arthritis

    PubMed Central

    Nilsson, Ing-Marie; Hartford, Orla; Foster, Timothy; Tarkowski, Andrzej

    1999-01-01

    Septic arthritis is a common and feared complication of staphylococcal infections. Staphylococcus aureus produces a number of potential virulence factors including certain adhesins and enterotoxins. In this study we have assessed the roles of cytolytic toxins in the development of septic arthritis by inoculating mice with S. aureus wild-type strain 8325-4 or isogenic mutants differing in the expression of alpha-, beta-, and gamma-toxin production patterns. Mice inoculated with either an alpha- or beta-toxin mutant showed degrees of inflammation, joint damage, and weight decrease similar to wild-type-inoculated mice. In contrast, mice inoculated with either double (alpha- and gamma-toxin-deficient)- or triple (alpha-, beta-, and gamma-toxin-deficient)-mutant S. aureus strains showed lower frequency and severity of arthritis, measured both clinically and histologically, than mice inoculated with the wild-type strain. We conclude that simultaneous production of alpha- and gamma-toxin is a virulence factor in S. aureus arthritis. PMID:10024541

  3. Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O'Toole 1935) Prévot 1938.

    PubMed

    Lawson, Paul A; Citron, Diane M; Tyrrell, Kerin L; Finegold, Sydney M

    2016-08-01

    The recent proposal by Lawson and Rainey (2015) to restrict the genus Clostridium to Clostridium butyricum and related species has ramifications for the members of the genera that fall outside this clade that should not be considered as Clostridium sensu stricto. One such organism of profound medical importance is Clostridioides difficile that is a major cause of hospital-acquired diarrhea and mortality in individuals. Based on 16S rRNA gene sequence analysis, the closest relative of Clostridium difficile is Clostridium mangenotii with a 94.7% similarity value and both are located within the family Peptostreptococcaceae that is phylogenetically far removed from C. butyricum and other members of Clostridium sensu stricto. Clostridium difficile is Clostridium mangenotii each produce abundant H2 gas when grown in PYG broth and also produce a range of straight and branched chain saturated and unsaturated fatty acids with C16:0 as a major product. The cell wall peptidoglycan contains meso-DAP as the diagnostic diamino acid. Based on phenotypic, chemotaxonomic and phylogenetic analyses, novel genus Clostridioides gen. nov. is proposed for Clostridium difficile as Clostridioides difficile gen. nov. comb. nov. and that Clostridium mangenotii be transferred to this genus as Clostridioides mangenotii comb. nov. The type species of Clostridioides is Clostridioides difficile. PMID:27370902

  4. Type IV pili promote early biofilm formation by Clostridium difficile.

    PubMed

    Maldarelli, Grace A; Piepenbrink, Kurt H; Scott, Alison J; Freiberg, Jeffrey A; Song, Yang; Achermann, Yvonne; Ernst, Robert K; Shirtliff, Mark E; Sundberg, Eric J; Donnenberg, Michael S; von Rosenvinge, Erik C

    2016-08-01

    Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI. PMID:27369898

  5. A case of reactive arthritis due to Clostridium difficile colitis

    PubMed Central

    Essenmacher, Alex C.; Khurram, Nazish; Bismack, Gregory T.

    2016-01-01

    Reactive arthritis is an acute, aseptic, inflammatory arthropathy following an infectious process but removed from the site of primary infection. It is often attributed to genitourinary and enteric pathogens, such as Chlamydia, Salmonella, Shigella, Campylobacter, and Yersinia, in susceptible individuals. An uncommon and less recognized cause of this disease is preceding colonic infection with Clostridium difficile, an organism associated with pseudomembranous colitis and diarrhea in hospitalized patients and those recently exposed to antibiotics. Recognition of this association may be complicated by non-specific presentation of diarrhea, the interval between gastrointestinal and arthritic symptoms, and the wide differential in mono- and oligoarthritis. We present the case of a 61-year-old, hospitalized patient recently treated for C. difficile colitis who developed sudden, non-traumatic, right knee pain and swelling. Physical examination and radiographs disclosed joint effusion, and sterile aspiration produced cloudy fluid with predominant neutrophils and no growth on cultures. Diagnostic accuracy is enhanced by contemporaneous laboratory investigations excluding other entities such as gout and rheumatoid arthritis and other infections that typically precede reactive arthritis. Contribution of Clostridium infection to reactive arthritis is an obscure association frequently difficult to prove, but this organism is warranted inclusion in the differential of reactive arthritis. PMID:26908381

  6. Tequila vinasses acidogenesis in a UASB reactor with Clostridium predominance.

    PubMed

    Marino-Marmolejo, E N; Corbalá-Robles, L; Cortez-Aguilar, R C; Contreras-Ramos, S M; Bolaños-Rosales, R E; Davila-Vazquez, G

    2015-01-01

    Tequila vinasses represent an acidic, highly concentrated pollutant effluent generated during the distillation step of Tequila production. Although acidogenesis of Tequila vinasses has been reported for some reactor configurations, a characterization of the bacteria present during this metabolic process is lacking in the literature. Hydraulic retention times (HRT) between 36 and 6 h and organic loading rates (OLR) from 5 to 30 g COD L(-1) d(-1) were assessed in a UASB reactor fed with Tequila vinasses. Results showed that OLR excerted a stronger effect (p ≤ 0.0001) on parameters such as gas production rate, pH, and acidity than HRT. While it was clear that shorter HRT were related to higher volatile fatty acid production levels. Figures above 2 Lgas Lreactor (-1) d(-1) (where "gas" could be a mixture of methane and hydrogen) were attained only with an OLR as high as 30 g COD L(-1) d(-1). Bacterial identification of a sludge sample at the end of the experiment revealed that acid-tolerant microorganisms that remained in the reactor were exclusively affiliated to the Clostridium genera, being the first report of organisms identification for Tequila vinasses acidogenesis. These findings are relevant to the field of biotechnology since acidogenesis of Tequila vinasses using identified and studied microorganism abilities (i.e. Clostridium strains) presents the opportunity of optimizing processes intended for different metabolites production (butanol, volatile fatty acids, hydrogen, solvents). PMID:26301166

  7. Identification and Characterization of Clostridium sordellii Toxin Gene Regulator

    PubMed Central

    Sirigi Reddy, Apoorva Reddy; Girinathan, Brintha Parasumanna; Zapotocny, Ryan

    2013-01-01

    Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable. PMID:23873908

  8. Effective Sequestration of Clostridium difficile Protein Toxins by Calcium Aluminosilicate

    PubMed Central

    Pokusaeva, Karina; Carpenter, Robert

    2015-01-01

    Clostridium difficile is a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible for C. difficile infection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors for Clostridium difficile pathogenesis. Given the toxin-centric mechanism by which C. difficile pathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treat C. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdB in vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic- or chemotherapy-induced C. difficile infection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB. PMID:26149988

  9. A case of reactive arthritis due to Clostridium difficile colitis.

    PubMed

    Essenmacher, Alex C; Khurram, Nazish; Bismack, Gregory T

    2016-01-01

    Reactive arthritis is an acute, aseptic, inflammatory arthropathy following an infectious process but removed from the site of primary infection. It is often attributed to genitourinary and enteric pathogens, such as Chlamydia, Salmonella, Shigella, Campylobacter, and Yersinia, in susceptible individuals. An uncommon and less recognized cause of this disease is preceding colonic infection with Clostridium difficile, an organism associated with pseudomembranous colitis and diarrhea in hospitalized patients and those recently exposed to antibiotics. Recognition of this association may be complicated by non-specific presentation of diarrhea, the interval between gastrointestinal and arthritic symptoms, and the wide differential in mono- and oligoarthritis. We present the case of a 61-year-old, hospitalized patient recently treated for C. difficile colitis who developed sudden, non-traumatic, right knee pain and swelling. Physical examination and radiographs disclosed joint effusion, and sterile aspiration produced cloudy fluid with predominant neutrophils and no growth on cultures. Diagnostic accuracy is enhanced by contemporaneous laboratory investigations excluding other entities such as gout and rheumatoid arthritis and other infections that typically precede reactive arthritis. Contribution of Clostridium infection to reactive arthritis is an obscure association frequently difficult to prove, but this organism is warranted inclusion in the differential of reactive arthritis. PMID:26908381

  10. Role of collagenase clostridium histolyticum in Peyronie’s disease

    PubMed Central

    Peak, Taylor C; Mitchell, Gregory C; Yafi, Faysal A; Hellstrom, Wayne J

    2015-01-01

    Peyronie’s disease is a localized connective tissue disease characterized by an active, inflammatory phase and a stable, quiescent phase, with the eventual development of collagenous plaques within the tunica albuginea of the penis. Risk factors primarily associated with Peyronie’s disease include Dupuytren’s contracture, penile trauma, and family history. A variety of treatment strategies have been utilized, including oral and topical agents, electromotive drug administration, intralesional injections, extracorporeal shockwave therapy, penile traction, and surgery. However, most of these strategies are ineffective, with surgery being the only definitive treatment. Collagenase clostridium histolyticum is a newly US Food and Drug Administration-approved agent for intralesional injection. It is thought to downregulate many of the disease-related genes, cytokines, and growth factors and degrade collagen fibers. It also suppresses cell attachment, spreading, and proliferation. Collagenase clostridium histolyticum has been clinically proven to be a safe and effective therapeutic option, demonstrating decreases in penile curvature and plaque consistency, as well as increases in patient satisfaction. During clinical evaluation, the Peyronie’s Disease Questionnaire was validated as an effective tool for assessing treatment outcomes. PMID:26491251

  11. Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentans

    SciTech Connect

    Shin, C.Y.; Crawford, D.L.

    1995-12-31

    A Clostridium capable of degrading 2,4,6-trinitrotoluene (TNT) cometabolically was isolated from a mixed culture obtained from a bioreactor fed TNT. This bacterium, identified as a strain of Clostridium bifermentans, and designated strain CYS-1, was able to degrade TNT via 4-amino-2,6-dinitrotoluene (4-ADNT) and 2,4-diamino-6-nitrotoluene (2,4-DANT) to aliphatic polar products which are now being identified and are assumed to be organic acids. CYS 1 cells are tolerant of TNT and capable of degrading it at starting concentrations of up to {ge}100 mg/L TNT. The number of cells inoculated and the availability of cosubstrate nutrients are significant factors influencing TNT degradation, as are TNT tolerance and survival of the cells at high TNT concentrations. In liquid media, at high TNT concentrations, TNT toxicity could be overcome by increasing the amount of inoculum and supplementing the culture with appropriate rich organic cosubstrates. Under these conditions, the reduction of 4-ADNT to 2,4-DANT occurred very fast, whereas the further degradation of 2,4-DANT proceeded more slowly.

  12. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

    SciTech Connect

    SWAMINATHAN,S.; ESWARAMOORTHY,S.

    2001-11-19

    The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxins are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].

  13. Immunopathology and Cytokine Responses in Commercial Broiler Chickens with Gangrenous Dermatitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gangrene dermatitis (GD) is an emerging disease of increasing economic importance in poultry that results from infection by Clostridium septicum and C. perfringens (CP) type A. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. To gain better u...

  14. GENOME-WIDE DIFFERENTIAL GENE EXPRESSION PROFILES IN BROILER CHICKENS WITH GANGRENOUS DERMATITIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gangrenous dermatitis (GD) is a disease of poultry associated with the infection of Clostridium septicum (CS) and/or C. perfringens (CP) type A. While GD causes significant morbidity, mortality, and economic loss to the poultry industry, the fundamental mechanisms underlying this host-pathogen inte...

  15. Simultaneous and enhanced production of thermostable amylases and ethanol from starch by cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

    SciTech Connect

    Hyun, H.H.; Zeikus, J.G.

    1985-05-01

    Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of ..beta..-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60/sup 0/C and produced 9 U of ..beta..-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and > 120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.

  16. Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems.

    PubMed

    Brunt, Jason; Plowman, June; Gaskin, Duncan J H; Itchner, Manoa; Carter, Andrew T; Peck, Michael W

    2014-09-01

    Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed. PMID:25210747

  17. Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum.

    PubMed Central

    Das, A; Hugenholtz, J; Van Halbeek, H; Ljungdahl, L G

    1989-01-01

    Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain. PMID:2808299

  18. Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas.

    PubMed

    Ramió-Pujol, Sara; Ganigué, Ramon; Bañeras, Lluís; Colprim, Jesús

    2014-12-01

    The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity. PMID:26421736

  19. Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

    PubMed Central

    Hyun, H. H.; Zeikus, J. G.

    1985-01-01

    Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed. PMID:16346791

  20. Comparative analysis of the ability of Clostridium clariflavum strains and Clostridium thermocellumto utilize hemicellulose and unpretreated plant material

    SciTech Connect

    Izquierdo, Javier A.; Pattathil, Sivakumar; Guseva, Anna; Hahn, Michael G.; Lynd, Lee R.

    2014-11-18

    Among themophilic consolidated bioprocessing (CBP) candidate organisms, environmental isolates of Clostridium clariflavum have demonstrated the ability to grow on xylan, and the genome of C. clariflavum DSM 19732 has revealed a number of mechanisms that foster solubilization of hemicellulose that are distinctive relative to the model cellulolytic thermophile Clostridium thermocellum. Growth experiments on xylan, xylooligosaccharides, and xylose reveal that C. clariflavum strains are able to completely break down xylan to xylose and that the environmental strain C. clariflavum sp. 4-2a is able to grow on monomeric xylose. C. clariflavum strains were able to utilize a larger proportion of unpretreated switchgrass, and solubilize a higher proportion of glucan, xylan, and arabinan, with strain 4-2a reaching the highest extent of solubilization of these components (64.7 to 69.4%) compared to C. thermocellum (29.5 to 42.5%). In addition, glycome immunoanalyses of residual plant biomass reveal differences in the extent of degradation of easily accessible xylans, with C. clariflavum strains having increased solubilization of this fraction of xylans relative to C. thermocellum. In conclusion, C. clariflavum strains exhibit higher activity than C. thermocellum in the breakdown of hemicellulose and are capable of degrading xylan to xylooligomers and xylose. This capability seems to also play a role in the higher levels of utilization of unpretreated plant material.

  1. Comparative analysis of the ability of Clostridium clariflavum strains and Clostridium thermocellumto utilize hemicellulose and unpretreated plant material

    DOE PAGESBeta

    Izquierdo, Javier A.; Pattathil, Sivakumar; Guseva, Anna; Hahn, Michael G.; Lynd, Lee R.

    2014-11-18

    Among themophilic consolidated bioprocessing (CBP) candidate organisms, environmental isolates of Clostridium clariflavum have demonstrated the ability to grow on xylan, and the genome of C. clariflavum DSM 19732 has revealed a number of mechanisms that foster solubilization of hemicellulose that are distinctive relative to the model cellulolytic thermophile Clostridium thermocellum. Growth experiments on xylan, xylooligosaccharides, and xylose reveal that C. clariflavum strains are able to completely break down xylan to xylose and that the environmental strain C. clariflavum sp. 4-2a is able to grow on monomeric xylose. C. clariflavum strains were able to utilize a larger proportion of unpretreated switchgrass,more » and solubilize a higher proportion of glucan, xylan, and arabinan, with strain 4-2a reaching the highest extent of solubilization of these components (64.7 to 69.4%) compared to C. thermocellum (29.5 to 42.5%). In addition, glycome immunoanalyses of residual plant biomass reveal differences in the extent of degradation of easily accessible xylans, with C. clariflavum strains having increased solubilization of this fraction of xylans relative to C. thermocellum. In conclusion, C. clariflavum strains exhibit higher activity than C. thermocellum in the breakdown of hemicellulose and are capable of degrading xylan to xylooligomers and xylose. This capability seems to also play a role in the higher levels of utilization of unpretreated plant material.« less

  2. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    PubMed

    Janganan, Thamarai K; Mullin, Nic; Tzokov, Svetomir B; Stringer, Sandra; Fagan, Robert P; Hobbs, Jamie K; Moir, Anne; Bullough, Per A

    2016-10-01

    Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores. PMID:27375261

  3. Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef.

    PubMed

    Emswiler, B S; Pierson, C J; Kotula, A W

    1977-03-01

    The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef. PMID:16345236

  4. Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef

    PubMed Central

    Emswiler, B. S.; Pierson, C. J.; Kotula, A. W.

    1977-01-01

    The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef. PMID:16345236

  5. Effect of Lysozyme on Ionic Forms of Spores of Clostridium perfringens Type A

    PubMed Central

    Ando, Yoshiaki

    1975-01-01

    H spores of Clostridium perfringens type A (two strains) were more sensitive to germination by lysozyme than native spores. Resistance to lysozyme of H spores was restored by calcium loading. PMID:236284

  6. Draft Genome Sequence of Clostridium pasteurianum NRRL B-598, a Potential Butanol or Hydrogen Producer.

    PubMed

    Kolek, Jan; Sedlár, Karel; Provazník, Ivo; Patáková, Petra

    2014-01-01

    We present a draft genome sequence of Clostridium pasteurianum NRRL B-598. This strain ferments saccharides by two-stage acetone-butanol (AB) fermentation, is oxygen tolerant, and has high hydrogen yields. PMID:24652980

  7. Non-Clostridium perfringens infectious agents producing necrotic enteritis-like lesions in poultry.

    PubMed

    Uzal, F A; Sentíes-Cué, C G; Rimoldi, G; Shivaprasad, H L

    2016-06-01

    Necrotic enteritis (NE) produced by Clostridium perfringens is amongst the most prevalent enteric diseases of chickens and turkeys. However, several other bacterial, parasitic and viral agents can cause clinical signs, gross and microscopic lesions in poultry very similar to those of NE and the diseases produced by those agents need to be differentiated from NE. The main differential diagnoses for C. perfringens NE include bacterial (Clostridium colinum, Clostridium sordellii, Clostridium difficile, Pasteurella multocida, Brachyspira spp.), parasitic (Eimeria spp., Histomonas meleagridis) and viral (Duck Herpesvirus type 1, Avian Paramyxovirus type 1) diseases. Confirmation of the diagnosis of these diseases requires identification of the aetiological agents by morphological, cultural and/or molecular methods. PMID:27009483

  8. 'Clostridium massiliodielmoense', a new species isolated from the human gut microbiota.

    PubMed

    Tidjani Alou, Maryam; Sokhna, Cheikh; Bittar, Fadi; Khelaifia, Saber; Fournier, Pierre-Edouard; Raoult, Didier

    2016-09-01

    Herein, we report the main characteristics of 'Clostridium massiliodielmoense' strain mt26 (= CSUR P2255), representative of a new species isolated from the gastrointestinal tract of a healthy 28-month-old Senegalese boy. PMID:27366325

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

    PubMed Central

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

    2014-01-01

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

  10. Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

    PubMed

    el Idrissi, A H; Ward, G E

    1992-06-15

    Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias. PMID:1496812

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

    PubMed

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

    2014-09-01

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

  12. Metabolic control of Clostridium thermocellum via selective inhibition and compensatory product formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium thermocellum is a thermophilic, anaerobic bacterium that catabolizes recalcitrant plant fibers such as cellulose. Cellulose is depolymerized by an extracellular, membrane-associated enzyme system, and the sugars are then transported across the cell membrane for fermentation. C. thermoc...

  13. Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains

    PubMed Central

    Le Maréchal, Caroline; Souillard, Rozenn; Bayon-Auboyer, Marie-Hélène; Mermoud, Isabelle; Desoutter, Denise; Fach, Patrick

    2015-01-01

    Animal botulism is mainly associated with Clostridium botulinum group III strains producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of fourteen strains of Clostridium botulinum producing type C/D and two strains producing type D/C isolated in France, and one strain producing type D/C that originated from New Caledonia. PMID:26430029

  14. Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains.

    PubMed

    Woudstra, Cédric; Le Maréchal, Caroline; Souillard, Rozenn; Bayon-Auboyer, Marie-Hélène; Mermoud, Isabelle; Desoutter, Denise; Fach, Patrick

    2015-01-01

    Animal botulism is mainly associated with Clostridium botulinum group III strains producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of fourteen strains of Clostridium botulinum producing type C/D and two strains producing type D/C isolated in France, and one strain producing type D/C that originated from New Caledonia. PMID:26430029

  15. A cluster of three cases of botulism due to Clostridium baratii type F, France, August 2015.

    PubMed

    Tréhard, Hélène; Poujol, Isabelle; Mazuet, Christelle; Blanc, Quentin; Gillet, Yves; Rossignol, Frédérique; Popoff, Michel-Robert; Jourdan Da Silva, Nathalie

    2016-01-01

    A cluster of three cases of food-borne botulism due to Clostridium baratii type F occurred in France in August 2015. All cases required respiratory assistance. Consumption of a Bolognese sauce at the same restaurant was the likely source of contamination. Clostridium baratii was isolated both from stool specimens from the three patients and ground meat used to prepare the sauce. This is the second episode reported in France caused by this rare pathogen. PMID:26848055

  16. Clostridium perfringens in Long Island Sound sediments: An urban sedimentary record

    USGS Publications Warehouse

    Buchholtz ten Brink, M. R.; Mecray, E.L.; Galvin, E.L.

    2000-01-01

    Clostridium perfringens is a conservative tracer and an indicator of sewage-derived pollution in the marine environment. The distribution of Clostridium perfringens spores was measured in sediments from Long Island Sound, USA, as part of a regional study designed to: (1) map the distribution of contaminated sediments; (2) determine transport and dispersal paths; (3) identify the locations of sediment and contaminant focusing; and (4) constrain predictive models. In 1996, sediment cores were collected at 58 stations, and surface sediments were collected at 219 locations throughout the Sound. Elevated concentrations of Clostridium perfringens in the sediments indicate that sewage pollution is present throughout Long Island Sound and has persisted for more than a century. Concentrations range from undetectable amounts to 15,000 spores/g dry sediment and are above background levels in the upper 30 cm at nearly all core locations. Sediment focusing strongly impacts the accumulation of Clostridium perfringens spores. Inventories in the cores range from 28 to 70,000 spores/cm2, and elevated concentrations can extend to depths of 50 cm. The steep gradients in Clostridium perfringens profiles in muddier cores contrast with concentrations that are generally constant with depth in sandier cores. Clostridium perfringens concentrations rarely decrease in the uppermost sediment, unlike those reported for metal contaminants. Concentrations in surface sediments are highest in the western end of the Sound, very low in the eastern region, and intermediate in the central part. This pattern reflects winnowing and focusing of Clostridium perfringens spores and fine-grained sediment by the hydrodynamic regime; however, the proximity of sewage sources to the westernmost Sound locally enhances the Clostridium perfringens signals.

  17. The potential economic value of screening hospital admissions for Clostridium difficile.

    PubMed

    Bartsch, S M; Curry, S R; Harrison, L H; Lee, B Y

    2012-11-01

    Asymptomatic Clostridium difficile carriage has a prevalence reported as high as 51-85 %; with up to 84 % of incident hospital-acquired infections linked to carriers. Accurately identifying carriers may limit the spread of Clostridium difficile. Since new technology adoption depends heavily on its economic value, we developed an analytic simulation model to determine the cost-effectiveness screening hospital admissions for Clostridium difficile from the hospital and third party payer perspectives. Isolation precautions were applied to patients testing positive, preventing transmission. Sensitivity analyses varied Clostridium difficile colonization rate, infection probability among secondary cases, contact isolation compliance, and screening cost. Screening was cost-effective (i.e., incremental cost-effectiveness ratio [ICER] ≤ $50,000/QALY) for every scenario tested; all ICER values were ≤ $256/QALY. Screening was economically dominant (i.e., saved costs and provided health benefits) with a ≥10.3 % colonization rate and ≥5.88 % infection probability when contact isolation compliance was ≥25 % (hospital perspective). Under some conditions screening led to cost savings per case averted (range, $53-272). Clostridium difficile screening, coupled with isolation precautions, may be a cost-effective intervention to hospitals and third party payers, based on prevalence. Limiting Clostridium difficile transmission can reduce the number of infections, thereby reducing its economic burden to the healthcare system. PMID:22752150

  18. Clostridium difficile infections among Jordanian adult hospitalized patients.

    PubMed

    Nasereddin, Lina M; Bakri, Fares G; Shehabi, Asem A

    2009-12-01

    This prospective study investigated the important epidemiologic aspects of Clostridium difficile infections (CDIs) among Jordanian adult hospitalized patients. A total of 300 stool specimens were investigated using culture and polymerase chain reaction methods for detection of C difficile, its toxins, and fluoroquinolone resistance. C difficile-positive cultures were found in 13.7% of the patients, and 73% of the isolates carried tcdA and/or tcdB toxin genes, and all C difficile isolates were negative for binary toxin. The isolates showed moderate level of resistance to both ciprofloxacin and levofloxacin, whereas metronidazole and vancomycin were highly susceptible. This study indicates the need for early detection of CDIs and prevention of its severe disease in hospitalized patients. PMID:19712999

  19. Organization and distribution of the cellulosome in Clostridium thermocellum.

    PubMed Central

    Bayer, E A; Setter, E; Lamed, R

    1985-01-01

    The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant. The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases. The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells. Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant. Images PMID:4019409

  20. Incidence of Clostridium botulinum in honey of various origins.

    PubMed

    Nakano, H; Okabe, T; Hashimoto, H; Sakaguchi, G

    1990-10-01

    By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one. Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C. Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F). The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%). It was estimated that most positive samples contained spores in one per gram or lower concentrations. One sample contained 4 type A spores per gram and another 36-60 type F spores per gram. No distinct biochemical properties were found with the honey isolates. PMID:2093130

  1. Activation and injury of Clostridium perfringens spores by alcohols.

    PubMed Central

    Craven, S E; Blankenship, L C

    1985-01-01

    The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation. PMID:2864897

  2. Cytology of Spore Formation in Clostridium perfringens1

    PubMed Central

    Hoeniger, Judith F. M.; Stuart, Philip F.; Holt, Stanley C.

    1968-01-01

    The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits. Images PMID:4302300

  3. Rabbit Ileal Loop Response to Strains of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Sugiyama, H.; Strong, Dorothy H.

    1968-01-01

    The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops. Images PMID:4297020

  4. Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.

    PubMed Central

    Brumm, P J; Fried, J; Friedmann, H C

    1983-01-01

    A blue bile pigment, possessing four acetic and four propionic acid side chains has been isolated from extracts of the anaerobic microorganism Clostridium tetanomorphum and in smaller amounts from Propionibacterium shermanii. The compound could be prepared in larger amounts by incubation of C. tetanomorphum enzyme extracts with added delta-aminolevulinic acid. The ultraviolet-visible, infrared, and proton magnetic resonance spectra of the pigment indicate a chromophore of the biliverdin type. Field-desorption mass spectrometry of the purified methyl ester showed a strong molecular ion at m/e = 962. This corresponds to the molecular weight expected for the octamethyl ester of a bilatriene type of bile pigment structurally derived from uroporphyrin III or I. Of the five possible structures, two could be eliminated by proton magnetic resonance spectroscopy. The name bactobilin is proposed for this previously unreported bile pigment. PMID:6575387

  5. Clostridium difficile: emergence of hypervirulence and fluoroquinolone resistance.

    PubMed

    Razavi, B; Apisarnthanarak, A; Mundy, L M

    2007-10-01

    Clostridium difficile is a well-known cause of sporadic and healthcare-associated diarrhea. Multihospital outbreaks due to a single strain and outbreaks associated with antibiotic selective pressure, especially clindamycin, have been well documented. Severe cases and fatalities from C. difficile are uncommon. The recent global emergence of a hypervirulent strain containing binary toxin (Toxinotype III ribotype 027), with or without deletion in a regulatory gene (tcdC gene), together with high-level resistance to third generation fluoroquinolones, has been associated with increased morbidity and mortality. Although the defective regulatory gene locus is associated with increased toxin production in vitro, the in vivo significance of this mutation and of the binary toxin remains undefined. To date, treatment strategies have not evolved in response to the emergence of this hypervirulaent strain. We provide a critical, quantitative summary of the evolving clinical and molecular epidemiology of C. difficile along with implications relevant to future treatment strategies. PMID:17885732

  6. Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

    PubMed Central

    Edwards, Adrianne N.; Suárez, Jose M.; McBride, Shonna M.

    2013-01-01

    Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation. PMID:24084491

  7. Interactions Between the Gastrointestinal Microbiome and Clostridium difficile

    PubMed Central

    Theriot, Casey M.; Young, Vincent B.

    2016-01-01

    Antibiotics have significant and long-lasting effects on the intestinal microbiota and consequently reduce colonization resistance against pathogens, including Clostridium difficile. By altering the community structure of the gut microbiome, antibiotics alter the intestinal metabolome, which includes both host- and microbe-derived metabolites. The mechanisms by which antibiotics reduce colonization resistance against C. difficile are unknown yet important for development of preventative and therapeutic approaches against this pathogen. This review focuses on how antibiotics alter the structure of the gut microbiota and how this alters microbial metabolism in the intestine. Interactions between gut microbial products and C. difficile spore germination, growth, and toxin production are discussed. New bacterial therapies to restore changes in bacteria-driven intestinal metabolism following antibiotics will have important applications for treatment and prevention of C. difficile infection. PMID:26488281

  8. Recurrent Clostridium difficile infection: From colonization to cure.

    PubMed

    Shields, Kelsey; Araujo-Castillo, Roger V; Theethira, Thimmaiah G; Alonso, Carolyn D; Kelly, Ciaran P

    2015-08-01

    Clostridium difficile infection (CDI) is increasingly prevalent, dangerous and challenging to prevent and manage. Despite intense national and international attention the incidence of primary and of recurrent CDI (PCDI and RCDI, respectively) have risen rapidly throughout the past decade. Of major concern is the increase in cases of RCDI resulting in substantial morbidity, morality and economic burden. RCDI management remains challenging as there is no uniformly effective therapy, no firm consensus on optimal treatment, and reliable data regarding RCDI-specific treatment options is scant. Novel therapeutic strategies are critically needed to rapidly, accurately, and effectively identify and treat patients with, or at-risk for, RCDI. In this review we consider the factors implicated in the epidemiology, pathogenesis and clinical presentation of RCDI, evaluate current management options for RCDI and explore novel and emerging therapies. PMID:25930686

  9. Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii

    PubMed Central

    Molitor, Bastian; Kirchner, Kristina; Henrich, Alexander W.; Schmitz, Simone; Rosenbaum, Miriam A.

    2016-01-01

    Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction. PMID:27527841

  10. Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii.

    PubMed

    Molitor, Bastian; Kirchner, Kristina; Henrich, Alexander W; Schmitz, Simone; Rosenbaum, Miriam A

    2016-01-01

    Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction. PMID:27527841

  11. In Vivo Kinetics of Nitrogenase Formation in Clostridium pasteurianum

    PubMed Central

    Seto, Belinda; Mortenson, L. E.

    1974-01-01

    Clostridium pasteurianum exhibits diauxic growth when grown in the presence of both NH3 and N2; no nitrogenase activity or formation was detected either serologically or by activity during growth on NH3. During the 60-min lag that ensued after NH3 was consumed and before growth resumed, molybdoferredoxin and azoferredoxin were first detected by activity measurements and serologically at 25 and 40 min, respectively. With the use of rifampin and dactinomycin, it was found that azoferredoxin messenger ribonucleic acid was initiated between 25 and 30 min after the inception of the lag and was completed by 38 min. An explanation of these results and their relation to possible models for the regulation of nitrogenase is given. Images PMID:4218235

  12. Clostridium difficile Infection: Epidemiology, Pathogenesis, Risk Factors, and Therapeutic Options

    PubMed Central

    Seyedjavadi, Sima Sadat; Goudarzi, Hossein; Mehdizadeh Aghdam, Elnaz; Nazeri, Saeed

    2014-01-01

    The incidence and mortality rate of Clostridium difficile infection have increased remarkably in both hospital and community settings during the last two decades. The growth of infection may be caused by multiple factors including inappropriate antibiotic usage, poor standards of environmental cleanliness, changes in infection control practices, large outbreaks of C. difficile infection in hospitals, alteration of circulating strains of C. difficile, and spread of hypervirulent strains. Detection of high-risk populations could be helpful for prompt diagnosis and consequent treatment of patients suffering from C. difficile infection. Metronidazole and oral vancomycin are recommended antibiotics for the treatment of initial infection. Current treatments for C. difficile infection consist of supportive care, discontinuing the unnecessary antibiotic, and specific antimicrobial therapy. Moreover, novel approaches include fidaxomicin therapy, monoclonal antibodies, and fecal microbiota transplantation mediated therapy. Fecal microbiota transplantation has shown relevant efficacy to overcome C. difficile infection and reduce its recurrence. PMID:24991448

  13. Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

    PubMed

    Kovacs-Simon, Andrea; Leuzzi, Rosanna; Kasendra, Magdalena; Minton, Nigel; Titball, Richard W; Michell, Stephen L

    2014-07-15

    Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease. PMID:24482399

  14. Prevalence of diverticulosis in recurrent Clostridium difficile infection

    PubMed Central

    Lipp, Michael J; Pagovich, Odelya E; Rabin, David; Min, Albert D; Bernstein, Brett B

    2010-01-01

    AIM: To re-evaluate the theory that colonic diverticulosis is associated with relapse of Clostridium difficile associated disease (CDAD) in light of data suggesting increasing rates of CDAD infection and relapse. METHODS: Charts were reviewed for patients with recurrent CDAD who had also had a prior colonoscopy or flexible sigmoidoscopy. An age and gender matched control group was used to compare the prevalence of diverticulosis. RESULTS: Twenty-two patients met the study criteria, and the prevalence of diverticulosis in patients with CDAD relapse was 23% compared to 32% in age and sex matched controls (P = 0.44). A significant proportion of patients with CDAD relapse had co-morbidities associated with immune suppression. CONCLUSION: Diverticulosis does not appear to be associated with CDAD relapse. PMID:20082480

  15. Dentists, antibiotics and Clostridium difficile-associated disease.

    PubMed

    Beacher, N; Sweeney, M P; Bagg, J

    2015-09-25

    Dentists prescribe significant volumes of antimicrobial drugs within primary care settings. There is good evidence that many of the prescriptions are not justified by current clinical guidance and that that there is considerable misuse of these drugs in dentistry. One of the risks associated with antibiotic administration is Clostridium difficile-associated disease (CDAD), an entity of which many healthcare workers, including dentists, have little knowledge or understanding. This review seeks to identify the extent and nature of the problem and provides an up to date summary of current views on CDAD, with particular reference to community acquired disease. As for all healthcare workers, scrupulous attention to standard infection control procedures and reducing inappropriate antibiotic prescribing are essential to reduce the risks of CDAD, prevent emergence of further resistant strains of microorganisms and maintain the value of the arsenal of antibiotics currently available to us. PMID:26404991

  16. Structural Determinants of Clostridium difficile Toxin A Glucosyltransferase Activity

    SciTech Connect

    Pruitt, Rory N.; Chumbler, Nicole M.; Rutherford, Stacey A.; Farrow, Melissa A.; Friedman, David B.; Spiller, Ben; Lacy, D. Borden

    2012-03-28

    The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.

  17. The complete genome sequence of Clostridium indolis DSM 755T

    PubMed Central

    Leschine, Susan; Huntemann, Marcel; Han, James; Chen, Amy; Kyrpides, Nikos; Markowitz, Victor; Palaniappan, Krishna; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Schaumberg, Andrew; Pati, Amrita; Stamatis, Dimitrios; Reddy, Tatiparthi; Lobos, Elizabeth; Goodwin, Lynne; Nordberg, Henrik P.; Cantor, Michael N.; Hua, Susan X.; Woyke, Tanja; Blanchard, Jeffrey L.

    2014-01-01

    Clostridium indolis DSM 755T is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755T reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species. PMID:25197485

  18. Structure of methionine γ-lyase from Clostridium sporogenes.

    PubMed

    Revtovich, Svetlana; Anufrieva, Natalya; Morozova, Elena; Kulikova, Vitalia; Nikulin, Alexey; Demidkina, Tatyana

    2016-01-01

    Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the γ-elimination reaction of L-methionine. The enzyme is a promising target for therapeutic intervention in some anaerobic pathogens and has attracted interest as a potential cancer treatment. The crystal structure of MGL from Clostridium sporogenes has been determined at 2.37 Å resolution. The fold of the protein is similar to those of homologous enzymes from Citrobacter freundii, Entamoeba histolytica, Pseudomonas putida and Trichomonas vaginalis. A comparison of these structures revealed differences in the conformation of two flexible regions of the N- and C-terminal domains involved in the active-site architecture. PMID:26750487

  19. [Tetanus and Clostridium tetani--a brief review].

    PubMed

    Stock, Ingo

    2015-02-01

    Tetanus is an acute, often fatal, disease caused by an exotoxin (tetanospasmin) produced by the anaerobic, gram-positive spore-forming bacterium Clostridium tetani. It is characterized by generalized rigidity and convulsive spasms of skeletal muscles. In most industrialized countries, tetanus is a rare disease. However, in many tropical and subtropical countries with low vaccination coverage and poor medical care, it is still widely distributed. This applies in particular for neonatal tetanus. About 50 000 newborns and infants die each year from consequences from this severe illness. Management of tetanus involves neutralization of free circulating toxin, adequate antibacterial and symptomatic therapy as well as intensive care of the patient. For prophylaxis of the disease, active tetanus toxoid vaccination is the method of choice. PMID:26376540

  20. [Clostridium tetani isolated from patients with systemic tetanus].

    PubMed

    Onuki, Tomoyo; Nihonyanagi, Shin; Nakamura, Masaki; Ide, Toshimitsu; Hattori, Jun; Kanoh, Yuhsaku; Soma, Kazui

    2013-01-01

    Clostridium tetani is widely distributed in ground or mud, especially in field and pond-shore surface layers. C. tetani is rarely isolated from specimens of patients with tetanus, and is generally diagnosed based on clinical symptoms such as trismus or general tonic spasms. This means that positive C. tetani infection is rarely diagnosed bacterially. Using gram straing, we identified C. tetani in specimens from patients suspected of C. tetani infection brought to the Kitasato University Hospital emergency center. Rapid gram staining information in the bacteriology laboratory is expected to improve recovery from C. tetani infection. It is therefore necessary to ensure clinical specimen quality control, and to keep standard strains of rare bacteria for isolation and identification. PMID:23484376

  1. Clostridium botulinum type C in the Mersey estuary.

    PubMed Central

    Smith, G. R.; Oliphant, J. C.; White, W. R.

    1982-01-01

    Nineteen of 98 samples of mud or sand taken from the Mersey estuary in 1981 contained Clostridium botulinum type C, the organism almost always responsible for botulism in water birds. In the Dungeon and Score Bank areas, where many dead and dying birds were found during the period September-December 1979, almost half the samples contained type C. Most of the positive samples were essentially muddy rather than sandy. The findings do not prove that botulism contributed to the 1979 mortality but are nonetheless thought-provoking, particularly because type C--unlike type B--is by no means ubiquitous in Britain. Type B was present in 12.2% of samples from the Mersey estuary. PMID:6759578

  2. Collagenase Clostridium histolyticum for the pharmacological management of Peyronie's disease.

    PubMed

    Anaissie, J; Powers, M K; Hellstrom, W J; Yafi, F A

    2015-08-01

    Peyronie's disease (PD) is defined as the abnormal accumulation of connective tissue in the tunica albuginea of the penis, and is an ongoing physical and psychological challenge for thousands of Americans. In vitro studies in the 1950s uncovered the potential of collagenase Clostridium histolyticum (CCH) to disrupt the collagen-containing plaques in PD, and opened the door to more in-depth clinical trials. Results indicated that with multiple dosage cycles followed by plaque modeling, penile curvature can be corrected, on average, in up to 35% of cases, with the majority of patients achieving ≥ 25% improvement in penile curvature. Most studies also indicated an improvement in patient-reported symptoms from the Peyronie's Disease Questionnaire. Adverse events from treatment with CCH included penile bruising, pain and edema, but most were mild to moderate in severity and usually resolved without intervention, suggesting that CCH is an effective and safe treatment for PD. PMID:26380384

  3. Hazard analysis of Clostridium perfringens in the Skylab Food System

    NASA Technical Reports Server (NTRS)

    Bourland, C. T.; Huber, C. S.; Kiser, P. R.; Heidelbaugh, N. D.; Rowley, D. B.

    1974-01-01

    The Skylab Food System presented unique microbiological problems because food was warmed in null-gravity and because the heat source was limited to 69.4 C (to prevent boiling in null-gravity). For these reasons, the foods were manufactured using critical control point techniques of quality control coupled with appropriate hazard analyses. One of these hazard analyses evaluated the threat from Clostridium perfringens. Samples of food were inoculated with C. perfringens and incubated for 2 h at temperatures ranging from 25 to 55 C. Generation times were determined for the foods at various temperatures. Results of these tests were evaluated taking into consideration: food-borne disease epidemiology, the Skylab food manufacturing procedures, and the performance requirements of the Skylab Food System. Based on this hazard analysis, a limit for C. perfringens of 100/g was established for Skylab foods.

  4. Treatment of Clostridium difficile infection in pediatric patients.

    PubMed

    Esposito, Susanna; Umbrello, Giulia; Castellazzi, Luca; Principi, Nicola

    2015-06-01

    Clostridium difficile causes infections that can either remain asymptomatic or manifest as clinical disease. In this report, problems, possible solutions, and future perspectives on the treatment of C. difficile infections (CDIs) in pediatric patients are discussed. CDI, despite increasing as a consequence of the overuse and misuse of antibiotics, remains relatively uncommon in pediatrics mainly because younger children are poorly susceptible to the action of C. difficile toxins. In most such cases, C. difficile disease is mild to moderate and discontinuation of the administered antibiotics in patients receiving these drugs when CDI develops, or administration of metronidazole, is sufficient to solve this problem. In severe or frequently relapsing cases, vancomycin is the drug of choice. Probiotics do not seem to add significant advantages. Other treatment options must be reserved for severe cases and be considered as a salvage treatment, although potential advantages in pediatric patients remain unclear. PMID:25912469

  5. Clear, defined medium for the sporulation of Clostridium perfringens.

    PubMed Central

    Sacks, L E; Thompson, P A

    1978-01-01

    A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated. Images PMID:25045

  6. [New treatment options for infections with Clostridium difficile].

    PubMed

    van Nood, Els; Keller, Josbert J; Kuijper, Ed J; Speelman, Peter

    2013-01-01

    Currently available broad spectrum antibiotics are not sufficiently effective against recurrent Clostridium difficile infections (CDI). Donor faecal microbiota transplantation is a very effective treatment for second and recurrent infection but is time-consuming and requires careful screening of donors. The new narrow spectrum antibiotic fidaxomicin is a good alternative in a first CDI or a first recurrence, but treatment is expensive and there are no data on its effectiveness in a second or later recurrence. Fidaxomicin is less effective against infections caused by the Ribotype 027 strain, a virulent strain that is regularly encountered in the Netherlands. The effectiveness of various other promising narrow spectrum antibiotics is currently being investigated. Medications that support the gut flora or the immune system seem to offer new perspectives. Expectations for the currently available probiotic preparations and toxin binders are not high. PMID:24279951

  7. Recurrent Clostridium difficile Infection: From Colonization to Cure

    PubMed Central

    Shields, Kelsey; Araujo-Castillo, Roger V.; Theethira, Thimmaiah G.; Alonso, Carolyn D.; Kelly, Ciaran

    2015-01-01

    Clostridium difficile infection (CDI) is increasingly prevalent, dangerous and challenging to prevent and manage. Despite intense national and international attention the incidence of primary and of recurrent CDI (PCDI and RCDI, respectively) have risen rapidly throughout the past decade. Of major concern is the increase in cases of RCDI resulting in substantial morbidity, morality and economic burden. RCDI management remains challenging as there is no uniformly effective therapy, no firm consensus on optimal treatment, and reliable data regarding RCDI-specific treatment options is scant. Novel therapeutic strategies are critically needed to rapidly, accurately, and effectively identify and treat patients with, or at-risk for, RCDI. In this review we consider the factors implicated in the epidemiology, pathogenesis and clinical presentation of RCDI, evaluate current management options for RCDI and explore novel and emerging therapies. PMID:25930686

  8. Recurrent Clostridium difficile infections: The importance of the intestinal microbiota

    PubMed Central

    Zanella Terrier, Marie Céline; Simonet, Martine Louis; Bichard, Philippe; Frossard, Jean Louis

    2014-01-01

    Clostridium difficile infections (CDI) are a leading cause of antibiotic-associated and nosocomial diarrhea. Despite effective antibiotic treatments, recurrent infections are common. With the recent emergence of hypervirulent isolates of C. difficile, CDI is a growing epidemic with higher rates of recurrence, increasing severity and mortality. Fecal microbiota transplantation (FMT) is an alternative treatment for recurrent CDI. A better understanding of intestinal microbiota and its role in CDI has opened the door to this promising therapeutic approach. FMT is thought to resolve dysbiosis by restoring gut microbiota diversity thereby breaking the cycle of recurrent CDI. Since the first reported use of FMT for recurrent CDI in 1958, systematic reviews of case series and case report have shown its effectiveness with high resolution rates compared to standard antibiotic treatment. This article focuses on current guidelines for CDI treatment, the role of intestinal microbiota in CDI recurrence and current evidence about FMT efficacy, adverse effects and acceptability. PMID:24966611

  9. Clostridium difficile infection: New insights into therapeutic options.

    PubMed

    Kachrimanidou, Melina; Sarmourli, Theopisti; Skoura, Lemonia; Metallidis, Symeon; Malisiovas, Nikolaos

    2016-09-01

    Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings and represents a major social and economic burden. The major virulence determinants are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), encoded within the pathogenicity locus. Traditional therapies, such as metronidazole and vancomycin, frequently lead to a vicious circle of recurrences due to their action against normal human microbiome. New disease management strategies together with the development of novel therapeutic and containment approaches are needed in order to better control outbreaks and treat patients. This article provides an overview of currently available CDI treatment options and discusses the most promising therapies under development. PMID:25955884

  10. Recent insights into Clostridium perfringens beta-toxin.

    PubMed

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-02-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin. PMID:25654787

  11. Cattle enterotoxaemia and Clostridium perfringens: description, diagnosis and prophylaxis.

    PubMed

    Lebrun, M; Mainil, J G; Linden, A

    2010-07-01

    Cattle enterotoxaemia is one of numerous pathologies caused by Clostridium perfringens. These anaerobic Gram-positive bacteria are naturally present in the intestinal flora of mammals, but their uncontrolled multiplication under certain conditions results in the overproduction of toxins in the intestinal tract. Major clinical signs are induced by the systemic spread of these toxins in the blood and tissues. Enterotoxaemia may be acute or peracute, and sudden death is often reported in rapidly growing, apparently healthy cattle. Enterotoxaemia can be prevented only with better understanding of its risk factors and pathogenesis. This paper provides an up-to-date overview of knowledge concerning the aetiology of the syndrome, its epidemiological context, pathogenesis, clinical signs and lesions, the diagnostic procedures and prophylactic tools, with specific attention to field aspects that are directly relevant to practitioners and clinical researchers. PMID:20605954

  12. Recent Insights into Clostridium perfringens Beta-Toxin

    PubMed Central

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-01-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin. PMID:25654787

  13. Clostridium difficile infection: management strategies for a difficult disease

    PubMed Central

    Pardi, Darrell S.

    2014-01-01

    Clostridium difficile was first described as a cause of diarrhea in 1978 and in the last three decades has reached an epidemic state with increasing incidence and severity in both healthcare and community settings. There also has been a rise in severe outcomes from C. difficile infection (CDI). There have been tremendous advancements in the field of CDI with the identification of newer risk factors, recognition of CDI in populations previously thought not at risk and development of better diagnostic modalities. Several treatment options are available for CDI apart from metronidazole and vancomycin, and include new drugs such as fidaxomicin and other options such as fecal microbiota transplantation. This review discusses the epidemiology, risk factors and outcomes from CDI, and focuses primarily on existing and evolving treatment modalities. PMID:24587820

  14. Protein phosphorylation in response to stress in Clostridium acetobutylicum

    SciTech Connect

    Balodimos, I.A.; Rapaport, E.; Kashket, E.R. )

    1990-07-01

    The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with {sup 32}P{sub i} or cell extracts with ({gamma}-{sup 32}P)ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5{prime},5{double prime}{prime}-P{sup 1},P{sup 4}-tetraphosphate had no influence on protein phosphorylation.

  15. Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation

    PubMed Central

    Arai, Takamitsu; Matsuoka, Satoshi; Cho, Hee-Yeon; Yukawa, Hideaki; Inui, Masayuki; Wong, Sui-Lam; Doi, Roy H.

    2007-01-01

    The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon “intercellular complementation.” Interesting implications concerning bacterial cooperation are suggested from these results. PMID:17244702

  16. Clostridium difficile infection in the twenty-first century.

    PubMed

    Ghose, Chandrabali

    2013-09-01

    Clostridium difficile is a spore-forming gram-positive bacillus, and the leading cause of antibiotic-associated nosocomial diarrhea and colitis in the industrialized world. With the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027), the epidemiology of C. difficile infection has rapidly changed in the last decade. C. difficile infection, once thought to be an easy to treat bacterial infection, has evolved into an epidemic that is associated with a high rate of mortality, causing disease in patients thought to be low-risk. In this review, we discuss the changing face of C .difficile infection and the novel treatment and prevention strategies needed to halt this ever growing epidemic. PMID:26038491

  17. Integration of metabolism and virulence in Clostridium difficile

    PubMed Central

    Bouillaut, Laurent; Dubois, Thomas; Sonenshein, Abraham L.; Dupuy, Bruno

    2015-01-01

    Synthesis of the major toxin proteins of the diarrheal pathogen, Clostridium difficile, is dependent on the activity of TcdR, an initiation (sigma) factor of RNA polymerase. The synthesis of TcdR and the activation of toxin gene expression are responsive to multiple components in the bacterium’s nutritional environment, such as the presence of certain sugars, amino acids, and fatty acids. This review summarizes current knowledge about the mechanisms responsible for repression of toxin synthesis when glucose or branched-chain amino acids or proline are in excess and the pathways that lead to synthesis of butyrate, an activator of toxin synthesis. The regulatory proteins implicated in these mechanisms also play key roles in modulating bacterial metabolic pathways, suggesting that C. difficile pathogenesis is intimately connected to the bacterium’s metabolic state. PMID:25445566

  18. Clostridium difficile-associated reactive arthritis in two children.

    PubMed

    Löffler, Helga A; Pron, Benedicte; Mouy, Richard; Wulffraat, Nico M; Prieur, Anne-Marie

    2004-01-01

    In adults, reactive arthritis (ReA) following Clostridium difficile-enterocolitis has been documented. In children, only one case of C. difficile-associated ReA has been reported. We now describe two other cases of ReA associated with C. difficile in children. The characteristics of ReA due to C. difficile appear to be similar in adults and children. Both children show polyarthritis after an episode of diarrhoea with positive stool cultures for C. difficile. Arthritis is asymmetrical with a self-limiting course. Nonsteroidal antiinflammatory drug (NSAID) therapy is sufficient. One case is remarkable because of its prolonged course of ReA despite NSAID therapy, and its association with the presence of HLA-B27 antigen. PMID:14769523

  19. Clostridium perfringens Sepsis and Fetal Demise after Genetic Amniocentesis

    PubMed Central

    Hendrix, Nancy W.; Mackeen, A. Dhanya; Weiner, Stuart

    2011-01-01

    Clostridium perfringens is a rare cause of intrauterine infection. There have been five case reports concerning infection associated with invasive procedures. We report a woman who underwent a genetic amniocentesis due to her history of chronic granulomatous disease. She presented to the hospital ∼38 hours after the amniocentesis complaining of fever and chills. Due to acute decompensation, she underwent an emergent dilatation and evacuation. During her stay, blood cultures came back positive for C. perfringens. Gradual improvement with intensive monitoring led to hospital discharge 4 days after the procedure. Uterine infection due to C. perfringens leading to maternal sepsis is associated with a high morbidity and mortality rate. Our patient was able to survive without a hysterectomy due to the rapid administration of antibiotics and surgical intervention while being evaluated. PMID:23705080

  20. Metronidazole-triazole conjugates: Activity against Clostridium difficile and parasites

    PubMed Central

    Jarrad, Angie M.; Karoli, Tomislav; Debnath, Anjan; Tay, Chin Yen; Huang, Johnny X.; Kaeslin, Geraldine; Elliott, Alysha G.; Miyamoto, Yukiko; Ramu, Soumya; Kavanagh, Angela M.; Zuegg, Johannes; Eckmann, Lars; Blaskovich, Mark A.T.; Cooper, Matthew A.

    2015-01-01

    Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window. PMID:26117821

  1. Clostridium difficile Genome Editing Using pyrE Alleles.

    PubMed

    Ehsaan, Muhammad; Kuehne, Sarah A; Minton, Nigel P

    2016-01-01

    Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. PMID:27507332

  2. Characterization of thermostable cyclodextrinase from Clostridium thermohydrosulfuricum 39E.

    PubMed Central

    Saha, B C; Zeikus, J G

    1990-01-01

    Clostridium thermohydrosulfuricum 39E produced a cell-bound cyclodextrin (CD)-degrading enzyme (cyclodextrinase). It was partially purified 205-fold (specific activity, 14.5 U/mg of protein) by solubilizing with Triton X-100, ammonium sulfate treatment, and DEAE-Sepharose CL-6B column chromatography. The enzyme activity was found to be stable at pH 5.5 and 60 degrees C and optimally active at pH 6.0 and 65 degrees C. The enzyme preparation hydrolyzed CDs, with alpha-CD greater than beta-CD greater than gamma-CD, and displayed a putative multiple attack pattern. The enzyme activity was inhibited by p-chloromercuribenzoate but not by N-bromosuccinimide. PMID:2275540

  3. Clostridium difficile infection: molecular pathogenesis and novel therapeutics

    PubMed Central

    Rineh, Ardeshir; Kelso, Michael J; Vatansever, Fatma; Tegos, George P; Hamblin, Michael R

    2015-01-01

    The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed. PMID:24410618

  4. Clostridium piliforme encephalitis in a weaver bird (Ploceus castaneiceps).

    PubMed

    Mete, Asli; Eigenheer, Andrea; Goodnight, Andrea; Woods, Leslie

    2011-11-01

    A juvenile Taveta golden weaver (Ploceus castaneiceps) bird housed in a zoo aviary developed a sudden onset of neurological signs. The bird demonstrated head tilt, rolling onto its back, and inability to perch. Euthanasia was elected due to lack of response to intensive care, and a necropsy was performed. There were no significant abnormal findings on gross examination. On histopathology, the remarkable findings were localized to the brain and consisted of multifocal cerebral microabscesses and rarefaction. Filamentous rod-shaped bacteria were present within and at the periphery of the necrotic foci, and dispersed throughout the neuroparenchyma and intracellularly in neurons. The bacteria were Gram negative, and Warthin-Starry stain demonstrated characteristic "hay stacking." Real-time polymerase chain reaction analysis of the brain identified the agent as Clostridium piliforme. An additional microscopic finding was severe crypt enteritis; however, the bacteria were not observed in the intestinal sections. PMID:22362811

  5. Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

    PubMed Central

    Kim, A Y; Attwood, G T; Holt, S M; White, B A; Blaschek, H P

    1994-01-01

    Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E). Images PMID:8117087

  6. Prevalence and Risk Factors for Asymptomatic Clostridium difficile Carriage

    PubMed Central

    Alasmari, Faisal; Seiler, Sondra M.; Hink, Tiffany; Burnham, Carey-Ann D.; Dubberke, Erik R.

    2014-01-01

    Background. Clostridium difficile infection (CDI) incidence has increased dramatically over the last decade. Recent studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We sought to identify the prevalence and risk factors for asymptomatic C. difficile carriage on admission to the hospital. Methods. Patients admitted to Barnes-Jewish Hospital without diarrhea were enrolled from June 2010 through October 2011. Demographic information and healthcare and medication exposures 90 days prior to admission were collected. Stool specimens or rectal swabs were collected within 48 hours of admission and stored at −30°C until cultured. Clostridium difficile isolates were typed and compared with isolates from patients with CDI. Results. A stool/swab specimen was obtained for 259 enrolled subjects on admission. Two hundred four (79%) were not colonized, 40 (15%) had toxigenic C. difficile (TCD), and 15 (6%) had nontoxigenic C. difficile. There were no differences between TCD-colonized and -uncolonized subjects for age (mean, 56 vs 58 years; P = .46), comorbidities, admission from another healthcare facility (33% vs 24%; P = .23), or recent hospitalization (50% vs 50%; P = .43). There were no differences in antimicrobial exposures in the 90 days prior to admission (55% vs 56%; P = .91). Asymptomatic carriers were colonized with strains similar to strains from patients with CDI, but the relative proportions were different. Conclusions. There was a high prevalence of TCD colonization on admission. In contrast to past studies, TCD colonization was not associated with recent antimicrobial or healthcare exposures. Additional investigation is needed to determine the role of asymptomatic TCD carriers on hospital-onset CDI incidence. PMID:24755858

  7. Clostridium difficile Spore-Macrophage Interactions: Spore Survival

    PubMed Central

    Paredes-Sabja, Daniel; Cofre-Araneda, Glenda; Brito-Silva, Christian; Pizarro-Guajardo, Marjorie; Sarker, Mahfuzur R.

    2012-01-01

    Background Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment. Methodology/Principal Findings In this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1. Conclusions/Significance These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells. PMID:22952726

  8. Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy

    PubMed Central

    Weigand, Michael R.; Pena-Gonzalez, Angela; Shirey, Timothy B.; Broeker, Robin G.; Ishaq, Maliha K.; Konstantinidis, Konstantinos T.

    2015-01-01

    Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified as Clostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains of C. botulinum group I and C. sporogenes clearly separated these two species into discrete clades which showed ∼93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143 C. sporogenes clade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bont/B2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-bearing plasmid (pCLD), while two different strains belonging to the C. botulinum clade carried bont/B2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification. PMID:26048939

  9. Clostridium perfringens and Clostridium difficile in cooked beef sold in Côte d'Ivoire and their antimicrobial susceptibility.

    PubMed

    Kouassi, Kra Athanase; Dadie, Adjéhi Thomas; N'Guessan, Kouadio Florent; Dje, Koffi Marcellin; Loukou, Yao Guillaume

    2014-08-01

    The aim of this study was to evaluate the prevalence of Clostridium difficile and Clostridium perfringens in cooked beef sold in the streets in Côte d'Ivoire and their antimicrobial susceptibility. A total of 395 kidney and flesh samples of cooked beef were collected from vendors at Abidjan and subjected to C. difficile and C. perfringens isolation and identification by using biochemical tests, API 20A system and PCR detection. Subsequently, the antimicrobial susceptibility test was performed for confirmed isolates. Our results showed the prevalence of 12.4% for C. difficile (11.04% in kidney and 13.45% in flesh) and 5.06% for C. perfringens (2.32% in kidney and 7.17% in flesh). Metronidazole and vancomycin remained the most potent antimicrobial agents against C. difficile while metronidazole and penicillin G were the most potent agents against C. perfringens. The resistance rates to tetracycline, doxycycline, chloramphenicol and erythromycin against C. difficile and C. perfringens isolates ranged from 2.05% to 8.16% and from 20% to 50%, respectively. Among all antimicrobial agents tested against C. difficile, percentages of resistance to quinolones ciprofloxacin, norfloxacin and nalidixic acid as well as to gentamicin and cefotaxime were the highest. Eight resistant phenotypes were defined for C. difficile isolates and eleven resistant phenotypes for C. perfringens isolates. Clindamycin/gentamicin/cefotaxime/ciprofloxacin/norfloxacin/nalidixic acid resistance was the most common phenotype for C. difficile (55.10% of isolates) while norfloxacin/nalidixic acid resistance was the most common phenotype for C. perfringens (20% of isolates). PMID:24944124

  10. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    PubMed Central

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  11. Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

    PubMed

    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

    The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. PMID:23816139

  12. Catabolite pathway for the production of skatole and indole acetic acid by the acetogen Clostridium drakei, Clostridium scatologenes, and swine manure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skatole (3-methyl-indole) is a malodorous chemical in stored swine manure and is implicated as a component of foul tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei...

  13. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus.

    PubMed

    Wang, Han; Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J

    2015-01-01

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. PMID:25908129

  14. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  15. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus

    PubMed Central

    Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J.

    2015-01-01

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. PMID:25908129

  16. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium

    PubMed Central

    Pyne, Michael E.; Bruder, Mark R.; Moo-Young, Murray; Chung, Duane A.; Chou, C. Perry

    2016-01-01

    Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. PMID:27157668

  17. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium.

    PubMed

    Pyne, Michael E; Bruder, Mark R; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2016-01-01

    Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium. PMID:27157668

  18. Clostridium botulinum type E occurs and grows in the alga Cladophora glomerata

    USGS Publications Warehouse

    Byappanahalli, M.N.; Whitman, R.L.

    2009-01-01

    In recent years, massive avian die-offs from Clostridium botulinum type E infection have occurred in the Sleeping Bear Dunes National Lakeshore (SLBE) area of Lake Michigan. These outbreaks have been coincidental with massive blooms of the green algae Cladophora, mostly Cladophora glomerata. We tested the hypothesis that Clostridium botulinum type E can grow under suitable conditions in these algal mats. In a lab mesocosm study, Cladophora from four outbreak-impacted beaches from SLBE were compared with four unimpacted beaches in the Milwaukee–Racine area for bontE gene of Clostridium botulinum. Frequency of the bontE gene was higher after incubation (25 °C for up to 6 weeks) of Cladophora from impacted vs. the unimpacted area. Since no type E gene was detected initially in Cladophora from any of the eight locations, we infer that the increased occurrence of type E gene arose from spore germination or vegetative Clostridium growth within the existing algal mats of SLBE. Moreover, we found that the congener Clostridium perfringens readily grows in mesocosms containing Cladophora.

  19. Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

    PubMed

    Tseng, Shawn; Abramzon, Nina; Jackson, James O; Lin, Wei-Jen

    2012-03-01

    Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance. PMID:22075631

  20. Role of Pectinolytic Enzymes Identified in Clostridium thermocellum Cellulosome

    PubMed Central

    Chakraborty, Soumyadeep; Fernandes, Vania O.; Dias, Fernando M. V.; Prates, Jose A. M.; Ferreira, Luis M. A.; Fontes, Carlos M. G. A.; Goyal, Arun; Centeno, Maria S. J.

    2015-01-01

    The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes. PMID:25658912

  1. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    PubMed

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  2. Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

    PubMed Central

    Ueki, Toshiyuki; Nevin, Kelly P.; Lovley, Derek R.

    2014-01-01

    The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis. PMID:24509933

  3. Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

    SciTech Connect

    Banerjee, A; Leang, C; Ueki, T; Nevin, KP; Lovley, DR

    2014-03-25

    The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

  4. Conditions associated with Clostridium sporogenes growth as a surrogate for Clostridium botulinum in nonthermally processed canned butter.

    PubMed

    Taylor, R H; Dunn, M L; Ogden, L V; Jefferies, L K; Eggett, D L; Steele, F M

    2013-05-01

    The objective of this study was to better understand the effect of butter composition and emulsion structure on growth and survival of Clostridium sporogenes, used as a surrogate for C. botulinum in canned butter. The lack of a thermal process step in commercially available canned butter raises questions of potential safety, because it is hermetically sealed and generally exhibits anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or hurdles, within butter include: reduced water activity, acidity in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal, intact butter emulsion would have sufficient hurdles to prevent C. botulinum growth, whereas a broken butter emulsion would result in a coalesced aqueous phase that would allow for C. botulinum growth. Batch-churned butter was inoculated with C. sporogenes; butter samples with varying salt contents (0, 0.8, 1.6, and 2.4% wt/wt NaCl) were prepared and stored in coated steel cans for varying times (1 or 2 wk) and temperatures (22 or 41°C) to determine temperature and emulsion structure effects on C. sporogenes growth. Samples stored at 41°C showed a significant increase in C. sporogenes growth compared with those stored at 22°C. Furthermore, NaCl addition was found to have a significant effect on C. sporogenes growth, with 0.8% NaCl promoting more growth than 0%, but with decreases in growth observed at 1.6 and 2.4%. Uninoculated control plates were also found to have bacterial growth; this growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the hurdle created by the micro-droplet size of the emulsion aqueous phase could result in C. botulinum growth even at elevated salt

  5. Comparison of methods for the enumeration of Clostridium perfringens spores in water.

    PubMed

    Junqueira, Valéria Christina Amstalden; Neto, Romeu Cantúsio; da Silva, Neusely; Terra, Juliana Hirata

    2012-01-01

    Four methods for enumerating Clostridium perfringens spores in water were evaluated: (1) the IMM (Iron Milk Medium) method (MPN); (2) the LS (Lactose Sulfite Broth) method (MPN); (3) the m-CP (membrane filtration Clostridium perfringens Agar) method (membrane filtration); and (4) the TSC (Tryptose Sulfite Cycloserine Agar) method (membrane filtration). The performance of these methods was compared with that of the DRCM (Differential Reinforced Clostridium Medium) method (MPN) as adopted by CETESB (Brazil's Environmental Sanitation Technology Company) for the analysis of C. perfringens spores in water. Statistical analysis was performed according to ISO 17994:2004 (Water Quality - Criteria for Establishing Equivalence between Microbiological Methods). The LS, m-CP, and TSC methods were considered not equivalent to the DRCM method, as they gave significantly lower results. The IMM showed inconclusive results and, according to ISO 17994:2004, analysis of a greater number of samples is needed to draw definitive conclusions comparing IMM and DRCM. PMID:22233899

  6. Non-contiguous finished genome sequence and description of Clostridium dakarense sp. nov.

    PubMed Central

    Lo, Cheikh Ibrahima; Mishra, Ajay Kumar; Padhmanabhan, Roshan; Samb, Bissoume; Sow, Amy Gassama; Robert, Catherine; Couderc, Carine; Faye, Ngor; Raoult, Didier; Fournier, Pierre-Edouard; Fenollar, Florence

    2013-01-01

    Clostridium dakarense strain FF1T, is the type strain of Clostridium dakarense sp. nov., a new species within the genus Clostridium. This strain, whose genome is described here, was isolated from the fecal flora of a 4-month-old Senegalese child suffering from gastroenteritis. C. dakarense sp. nov. strain FF1T is an obligate anaerobic Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,735,762 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 27.98% and contains 3,843 protein-coding and 73 RNA genes, including 8 rRNA genes. PMID:24501642

  7. Non-contiguous finished genome sequence and description of Clostridium senegalense sp. nov.

    PubMed Central

    Mishra, Ajay Kumar; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier; Fournier, Pierre-Edouard

    2012-01-01

    Clostridium senegalense strain JC122T, is the type strain of Clostridium senegalense sp. nov., a new species within the genus Clostridium. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. C. senegalense strain JC122T is an obligate anaerobic Gram-positive rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,893,008 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 26.8% and contains 3,704 protein-coding and 57 RNA genes, including 6 rRNA genes. PMID:23408737

  8. Spore Coat Architecture of Clostridium novyi-NT spores

    SciTech Connect

    Plomp, M; McCafferey, J; Cheong, I; Huang, X; Bettegowda, C; Kinzler, K; Zhou, S; Vogelstein, B; Malkin, A

    2007-05-07

    Spores of the anaerobic bacterium Clostridium novyi-NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Towards this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of dormant as well as germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers as well as the underlying spore coat and undercoat layers sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi-NT, these studies document the presence of proteinaceous growth spirals in a biological organism.

  9. Sporicidal activity of two disinfectants against Clostridium difficile spores.

    PubMed

    Wheeldon, L J; Worthington, T; Hilton, A C; Lambert, P A; Elliott, T S J

    The sporicidal activity of an odour-free peracetic acid-based disinfectant (Wofasteril) and a widely-used dichloroisocyanurate preparation (Chlor-clean) was assessed against spores of the hyper-virulent strain of Clostridium difficile (ribotype 027), in the presence and absence of organic matter. In environmentally clean conditions, dichloroisocyanurate achieved a >3 log10 reduction in 3 minutes, but a minimum contact time of 9 minutes was required to reduce the viable spore load to below detection levels. Peracetic acid achieved a >3 log10 reduction in 30 minutes and was overall significantly less effective (P<0.05). However, in the presence of organic matter - which reflects the true clinical environment - there was no significant difference between the sporicidal activity of dichloroisocyanurate and peracetic acid over a 60-minute period (P=0.188). Given the greater occupational health hazards generally associated with chlorine-releasing agents, odour-free peracetic acid-based disinfectants may offer a suitable alternative for environmental disinfection. PMID:18414294

  10. Contributions to the taxonomy and biology of Clostridium difficile.

    PubMed

    Bittner, J; Macovei, A; Lemeni, D; Arboreanu, D; Potorac, E

    1992-01-01

    Clostridium difficile was incriminated by Hughes and Jarvis (1987) as a cause of intestinal infections in USA in the 1980-1984 period in 45 p. 100 of cases, whereas Salmonellae only in 12 p. 100. Four strains of this organism are studied in this paper in comparison with ten strains of C. bifermentans and six of C. sordellii, because the three species share a common antigen and have other common characteristics, as well. However, spores of C. difficile swell the bacteria and those of other bacteria (C. bifermentans and C. sordellii) do not; C. difficile does not produce indole, whereas the other species produce it. We confirmed the selective capacity of the medium of George et al. (1979) using the "alcohol shock" and as selective agents cycloserine D and cefoxitin. C. difficile proved to be most susceptible to metronidazole and rifampin. Whereas the former antibiotic was considered as a cause of post-antibiotic intestinal infections by different authors, the second was not, to our knowledge. The strain 10463 has a considerable toxicity (1000 DLM/ml for the white mouse, and pathogenicity--2000--5000 DCL for the white mouse, as compared to 25 DCL of the other three strains). Using this toxin an antitoxic serum was obtained in horse, with a capacity of neutralizing the action of the toxin up to a dilution of 1 p. 1000. PMID:1304829

  11. Bench-to-bedside review: Clostridium difficile colitis

    PubMed Central

    Gould, Carolyn V; McDonald, L Clifford

    2008-01-01

    In recent years, the incidence and severity of Clostridium difficile-associated disease (CDAD) have increased dramatically. Beginning in 2000, widespread regional outbreaks associated with a previously uncommon hypervirulent strain of C. difficile have occurred in North America and Europe. Most likely because of increased toxin production as well as other virulence factors, this epidemic strain has caused more severe and refractory disease leading to complications, including intensive care unit admission, colectomies, and death. Worldwide increasing use of fluoroquinolones and cephalosporins has likely contributed to the proliferation of this epidemic strain, which is highly resistant to both. The elderly have been disproportionately affected by CDAD, but C. difficile has also recently emerged in populations previously considered to be at low risk, including healthy outpatients and peripartum women, although it is unknown if these cases are related to the epidemic strain. Nevertheless, transmission within hospitals is the major source of C. difficile acquisition, and previous or concurrent antimicrobial use is almost universal among cases. Applying current evidence-based strategies for management and prevention is critically important, and clinicians should maintain an awareness of the changing epidemiology of CDAD and take measures to reduce the risk of disease in patients. PMID:18279531

  12. Quantifying Transmission of Clostridium difficile within and outside Healthcare Settings

    PubMed Central

    Olsen, Margaret A.; Dubberke, Erik R.; Galvani, Alison P.; Townsend, Jeffrey P.

    2016-01-01

    To quantify the effect of hospital and community-based transmission and control measures on Clostridium difficile infection (CDI), we constructed a transmission model within and between hospital, community, and long-term care-facility settings. By parameterizing the model from national databases and calibrating it to C. difficile prevalence and CDI incidence, we found that hospitalized patients with CDI transmit C. difficile at a rate 15 (95% CI 7.2–32) times that of asymptomatic patients. Long-term care facility residents transmit at a rate of 27% (95% CI 13%–51%) that of hospitalized patients, and persons in the community at a rate of 0.1% (95% CI 0.062%–0.2%) that of hospitalized patients. Despite lower transmission rates for asymptomatic carriers and community sources, these transmission routes have a substantial effect on hospital-onset CDI because of the larger reservoir of hospitalized carriers and persons in the community. Asymptomatic carriers and community sources should be accounted for when designing and evaluating control interventions. PMID:26982504

  13. Comparison of media for enumeration of Clostridium perfringens from foods.

    PubMed

    de Jong, A E I; Eijhusen, G P; Brouwer-Post, E J F; Grand, M; Johansson, T; Kärkkäinen, T; Marugg, J; in't Veld, P H; Warmerdam, F H M; Wörner, G; Zicavo, A; Rombouts, F M; Beumer, R R

    2003-09-01

    Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar] were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial. Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial. Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (+/-30%) of IS, which was set as a reference medium. Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological-analytical relevance. Food matrices did not affect the recovery of C. perfringens in general. DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common. Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C. perfringens from foods. PMID:12842482

  14. Growth of Clostridium perfringens in cooked chili during cooling.

    PubMed Central

    Blankenship, L C; Craven, S E; Leffler, R G; Custer, C

    1988-01-01

    U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili. PMID:2898919

  15. Metabolic Adaption of Ethanol-Tolerant Clostridium thermocellum

    PubMed Central

    Zhu, Xinshu; Cui, Jiatao; Feng, Yingang; Fa, Yun; Zhang, Jingtao; Cui, Qiu

    2013-01-01

    Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production. PMID:23936233

  16. Genetic and biochemical analysis of solvent formation in Clostridium acetobutylicum

    SciTech Connect

    Bennett, G.N.; Rudolph, F.B.

    1998-05-01

    The anaerobic organism Clostridium acetobutylicum has been used for commercial production of important organic solvents due to its ability to convert a wide variety of crude substrates to acids and alcohols. Current knowledge concerning the molecular genetics, cell regulation and metabolic engineering of this organism is still rather limited. The objectives are to improve the knowledge of the molecular genetics and enzymology of Clostridia in order to make genetic alterations which will more effectively channel cell metabolism toward production of desired products. Two factors that limit butanol production in continuous cultures are: (1) The degeneration of the culture, with an increase in the proportion of cells which are incapable of solvent production. Currently isolated degenerate strains are being evaluated to analyze the molecular mechanism of degeneration to determine if it is due to a genetic loss of solvent related genes, loss of a regulatory element, or an increase in general mutagenesis. Recent studies show two general types of degenerates, one which seems to have lost essential solvent pathway genes and another which has not completely lost all solvent production capability and retains the DNA bearing solvent pathway genes. (2) The production of hydrogen which uses up reducing equivalents in the cell. If the reducing power were more fully directed to the reduction reactions involved in butanol production, the process would be more efficient. The authors have studied oxidation reduction systems related to this process. These studies focus on ferredoxin and rubredoxin and their oxidoreductases.

  17. Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum.

    PubMed Central

    Cardenas, J; Mortenson, L E

    1975-01-01

    The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W. Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM. Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity. In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected. Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur. Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C. pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein. It is concluded that in C. pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase. PMID:1158853

  18. Clostridium difficile ribotypes in humans and animals in Brazil

    PubMed Central

    Silva, Rodrigo Otávio Silveira; Rupnik, Maja; Diniz, Amanda Nádia; Vilela, Eduardo Garcia; Lobato, Francisco Carlos Faria

    2015-01-01

    Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen. PMID:26676318

  19. Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile

    PubMed Central

    Pye, Hayley; Kohoutova, Darina; Mosse, Charles A.; Yahioglu, Gokhan; Stamati, Ioanna; Deonarain, Mahendra; Battah, Sinan; Ready, Derren; Allan, Elaine; Mullany, Peter; Lovat, Laurence B.

    2015-01-01

    Background Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. Methods PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. Results Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. Conclusion This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon. PMID:26313448

  20. Comparison of Media for the Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 104 organisms per g, but with 106 organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested. PMID:4324885

  1. Clostridium welchii and Bacillus cereus infection and intoxication

    PubMed Central

    Hobbs, Betty C.

    1974-01-01

    Clostridium welchii type A is a common agent of food poisoning when allowed to proliferate to large numbers in cooked foods, usually meat and poultry. The main factors of importance are survival of the spores, frequently found on raw products, through the cooking process, and possible contamination of cooked meats transferred to unclean containers; subsequent germination of spores and rapid multiplication of the vegetative cells during long slow cooling and non-refrigerated storage lead to heavy contamination. The toxin responsible is different from the soluble antigens, and its formation in the intestine is associated with sporulation. Large numbers of Cl. welchii of the same serological types in food and faeces is the main diagnostic factor. Important preventive measures are rapid cooling and cold storage to prevent growth. Bacillus cereus is an aerobic sporulating organism commonly found in cereals. Outbreaks described from Europe have a different aetiology with regard to food vehicles, incubation period and symptoms from those that have been reported recently in the U.K. from fried and boiled rice. The spores survive through cooking procedures and grow out to cells which sporulate readily in the cooked food and which are assumed to produce toxin in the food. Large numbers of B. cereus are found in foods causing illness and, as with Cl. welchii, the main preventive measure is inhibition of growth by quick cooling and cold storage of foods cooked ahead of requirements. A comparative table of the characteristics and clinical symptoms of Cl. welchii and B. cereus is given. PMID:4377580

  2. Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

    PubMed Central

    Nishida, S.; Seo, N.; Nakagawa, M.

    1969-01-01

    A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting. Images PMID:4304763

  3. Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments

    PubMed Central

    Leuzzi, Rosanna; Spencer, Janice; Buckley, Anthony; Brettoni, Cecilia; Martinelli, Manuele; Tulli, Lorenza; Marchi, Sara; Luzzi, Enrico; Irvine, June; Candlish, Denise; Veggi, Daniele; Pansegrau, Werner; Fiaschi, Luigi; Savino, Silvana; Swennen, Erwin; Cakici, Osman; Oviedo-Orta, Ernesto; Giraldi, Monica; Baudner, Barbara; D'Urzo, Nunzia; Maione, Domenico; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

    2013-01-01

    Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection. PMID:23716610

  4. Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

    PubMed Central

    Yan, Weiwei; Shin, Kang-Soon; Wang, Shih-Jon; Xiang, Hua; Divers, Thomas; McDonough, Sean; Bowman, James; Rowlands, Anne; Akey, Bruce; Mohamed, Hussni

    2014-01-01

    Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 107 C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses. PMID:24136208

  5. Case of Clostridium perfringens bacteremia after routine colonoscopy and polypectomy.

    PubMed

    Kunz, Anjali N; Riera, Diana; Hickey, Patrick

    2009-10-01

    Bacteremia is an uncommon complication after polypectomy and colonoscopy. We report one of the first cases of Clostridium perfringens bacteremia after polypectomy. Our patient was a four years old boy with congenital polyposis, who underwent colonoscopy and polypectomy without complication. Approximately 12h later he developed a fever and tachycardia with no other clinical symptoms. His blood cultures grew out penicillin susceptible C. perfringens and Enterococcus faecalis. He responded to antibiotic therapy and remained clinically asymptomatic for the duration of his course. There are a few reports of bacteremia after routine polypectomy, but no reported cases of C. perfringens bacteremia in the pediatric population. Clostridial sp. bacteremia can be fatal with devastating consequences if appropriate antibiotics and/or surgical debridement are delayed. Polymicrobial infection, as illustrated in our patient, is also common and can be a poor prognostic risk factor. Therefore, for patients with a history of polypectomy and new onset fever, anaerobic infections should be considered and empiric antibiotic therapy should include coverage for these organisms. PMID:19324098

  6. Genotypic investigation of Clostridium difficile in Prince Edward Island.

    PubMed

    Martin, H; Abbott, L P; Low, D E; Willey, B; Mulvey, M; Weese, J Scott

    2008-11-01

    Clostridium difficile is an important cause of disease in Canada; however, little information is available about the disease in the Maritime provinces. The objective of the present study was to characterize C difficile isolates obtained from people hospitalized with C difficile infection in Prince Edward Island. One hundred twenty-six C difficile ELISA toxin-positive stool samples were obtained and cultured using an enrichment protocol. C difficile was isolated from 105 of 126 (83%) samples. Twenty-two different ribotypes were identified. The most common ribotype, ribotype W, was a North American pulsotype 2 (NAP2), toxinotype 0 strain, which represented 18% of isolates. The next most common ribotype was a NAP1, toxinotype III strain, which accounted for 11% of isolates. Ribotype 027/NAP1 only accounted for five (4.7%) isolates. Forty-five per cent of isolates possessed genes encoding production of binary toxin. Three different ribotypes, all NAP1, toxinotype III strains, had a frameshift mutation in the tcdC gene (Delta117), while one isolate (ribotype 078, NAP4, toxinotype V) had a truncating mutation (C184T) in the tcdC gene. PMID:19436570

  7. Detection of virulence genes of Clostridium difficile by multiplex PCR.

    PubMed

    Antikainen, Jenni; Pasanen, Tanja; Mero, Sointu; Tarkka, Eveliina; Kirveskari, Juha; Kotila, Saara; Mentula, Silja; Könönen, Eija; Virolainen-Julkunen, Anni-Riitta; Vaara, Martti; Tissari, Päivi

    2009-08-01

    Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027. PMID:19664132

  8. Bacteriophage-mediated toxin gene regulation in Clostridium difficile.

    PubMed

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D; Dupuy, Bruno; Fralick, Joe A

    2009-12-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by PhiCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in PhiCD119 lysogens. During this study we found that repR, a putative repressor gene of PhiCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in PhiCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen. PMID:19776116

  9. Characteristics of patients with Clostridium difficile infection in Taiwan.

    PubMed

    Lin, Y-C; Huang, Y-T; Lee, T-F; Lee, N-Y; Liao, C-H; Lin, S-Y; Ko, W-C; Hsueh, P-R

    2013-10-01

    The medical records of 84 patients with stool cultures positive for Clostridium difficile during the period August 2007 to June 2009 were retrospectively reviewed. A case of confirmed (toxigenic)C. difficile infection (CDI) was defined by the presence of symptoms (fever, diarrhoea, abdominal discomfort or distension, ileus) and the presence of toxigenic C. difficile. Patients with compatible clinical symptoms and stool cultures positive for non-toxigenic C. difficile isolates were defined as probable (non-toxigenic) CDI cases. Of these 84 patients, 50 (59.5%) were diagnosed as confirmed CDI and 34 (40.5%) as probable CDI. Thirteen (15.5%) of the 84 patients died during their hospital stay. Usage of proton pump inhibitors was a significant independent risk factor for CDI (OR 3.21, P=0.014). Of the 50 isolates associated with confirmed CDI, seven (8.3%) carried binary toxin genes (cdtAB), and six (7.1%) had a deletion in the tcdC gene. The mortality rate in confirmed CDI patients with isolates exhibiting deletion in the tcdC gene (2/6, 33.3%), those with isolates harbouring binary toxin genes (2/7, 28.6%), and those with isolates containing mutations in gyrA (2/7, 28.6%) and gyrB (1/2, 50%) was higher than the overall mortality rate (10/50, 20%) in patients with confirmed CDI. PMID:23218131

  10. An Alkaline Phosphatase Reporter for use in Clostridium difficile

    PubMed Central

    Edwards, Adrianne N.; Pascual, Ricardo A.; Childress, Kevin O.; Nawrocki, Kathryn L.; Woods, Emily C.; McBride, Shonna M.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia. PMID:25576237

  11. Toxin Synthesis by Clostridium difficile Is Regulated through Quorum Signaling

    PubMed Central

    DuPont, Herbert L.; Norris, Steven J.; Kaplan, Heidi B.

    2015-01-01

    ABSTRACT Clostridium difficile infection (CDI) is dramatically increasing as a cause of antibiotic- and hospital-associated diarrhea worldwide. C. difficile, a multidrug-resistant pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Consequently, it produces toxins A and B that directly cause disease. Despite the enormous public health problem posed by this pathogen, the molecular mechanisms that regulate production of the toxins, which are directly responsible for disease, remained largely unknown until now. Here, we show that C. difficile toxin synthesis is regulated by an accessory gene regulator quorum-signaling system, which is mediated through a small (<1,000-Da) thiolactone that can be detected directly in stools of CDI patients. These findings provide direct evidence of the mechanism of regulation of C. difficile toxin synthesis and offer exciting new avenues both for rapid detection of C. difficile infection and development of quorum-signaling-based non-antibiotic therapies to combat this life-threatening emerging pathogen. PMID:25714717

  12. Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype

    PubMed Central

    Trovato, Alberto; Bianchini, Valentina; Biancardi, Anna; Cichero, Paola; Mazzotti, Maria; Nizzero, Paola; Moro, Matteo; Ossi, Cristina; Scarpellini, Paolo

    2015-01-01

    Clostridium difficile infection (CDI) became a public health problem for the global spreading of the so-called hypervirulent PCR ribotypes (RTs) 027 and 078, associated with increases in the transmission and severity of the disease. However, especially in Europe, several RTs are prevalent, and the concept of hypervirulence is currently debated. We investigated the toxin and resistance profiles and the genetic relatedness of 312 C. difficile strains isolated in a large Italian teaching hospital during a 5-year period. We evaluated the role of CDI-related antibiotic consumption and infection control practices on the RT predominance in association with their molecular features and transmission capacity. Excluding secondary cases due to nosocomial transmission, RT018 was the predominant genotype (42.4%) followed by RT078 (13.6%), while RT027 accounted for 0.8% of the strains. RT078 was most frequently isolated from patients in intensive care units. Its prevalence significantly increased over time, but its transmission capacity was very low. In contrast, RT018 was highly transmissible and accounted for 95.7% of the secondary cases. Patients with the RT018 genotype were significantly older than those with RT078 and other RTs, indicating an association between epidemic RT and age. We provide here the first epidemiological evidence to consider RT018 as a successful epidemic genotype that deserves more attention in clinical practice. PMID:26041894

  13. Clostridium butyricum: from beneficial to a new emerging pathogen.

    PubMed

    Cassir, N; Benamar, S; La Scola, B

    2016-01-01

    Clostridium butyricum, a strictly anaerobic spore-forming bacillus, is a common human and animal gut commensal bacterium, and is also frequently found in the environment. Whereas non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. In terms of the latter, within the same species, different strains have antagonist effects on the intestinal mucosa. In particular, short-chain fatty acids, which are products of carbohydrate fermentation, have a dose-dependent paradoxical effect. Moreover, toxin genes have been identified by genome sequencing in pathological strains. Asymptomatic carriage of these strains has also been reported. Herein, we provide an overview of the implications of C. butyricum for human health, from the beneficial to the pathogenic. We focus on pathogenic strains associated with the occurrence of necrotizing enterocolitis. We also discuss the need to use complementary microbiological methods, including culture, in order to better assess gut bacterial diversity and identify new emergent enteropathogens at the strain level. PMID:26493849

  14. Genomic characterization of Italian Clostridium botulinum group I strains.

    PubMed

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Azarnia Tehran, Domenico; Ciammaruconi, Andrea; Spagnolo, Ferdinando; Pittiglio, Valentina; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Lista, Florigio

    2015-12-01

    Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8. PMID:26341861

  15. Heat treatment adaptations in Clostridium perfringens vegetative cells.

    PubMed

    Novak, J S; Tunick, M H; Juneja, V K

    2001-10-01

    Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238. and H6 were grown at 37 degrees C followed by a 60-min exposure to 28 degrees C or 46 degrees C. D10-values, as a measure of thermal resistance at 60 degrees C, were significantly lower for 28 degrees C exposures as compared with cultures given 37 and 46 degrees C exposures. Following refrigeration at 4 degrees C for 24 h, D10-values for the 37 and 46 degrees C samples could not be differentiated from 28 degrees C samples. Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC. Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 +/- 0.5 degrees C. Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions. Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C. perfringens. N-terminal amino acid analyses identified two of the proteins as glyceraldehyde 3-phosphate dehydrogenase and rubrerythrin. Both appear to have roles in this anaerobe under stressful conditions. PMID:11601701

  16. Immune-based treatment and prevention of Clostridium difficile infection

    PubMed Central

    Zhao, Song; Ghose-Paul, Chandrabali; Zhang, Keshan; Tzipori, Saul; Sun, Xingmin

    2015-01-01

    Clostridium difficile (C. difficile) causes over 500,000 infections per year in the US, with an estimated 15,000 deaths and an estimated cost of $1–3 billion. Moreover, a continual rise in the incidence of severe C. difficile infection (CDI) has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15–35%. Prevention of recurrence is one of the most challenging aspects in the field. A better knowledge of the molecular mechanisms of the disease, the host immune response and identification of key virulence factors of C. difficilenow permits the development of immune-based therapies. Antibodies specific for C. difficile toxins have been shown to effectively treat CDI and prevent disease relapse in animal models and in humans. Vaccination has been recognized as the most cost-effective treatment/prevention for CDI. This review will summarize CDI transmission, epidemiology, major virulent factors and highlights the rational and the development of immune-based approaches against this remerging threat. PMID:25668664

  17. Motility and Flagellar Glycosylation in Clostridium difficile▿ †

    PubMed Central

    Twine, Susan M.; Reid, Christopher W.; Aubry, Annie; McMullin, David R.; Fulton, Kelly M.; Austin, John; Logan, Susan M.

    2009-01-01

    In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein. PMID:19749038

  18. Flooding and Clostridium difficile Infection: A Case-Crossover Analysis.

    PubMed

    Lin, Cynthia J; Wade, Timothy J; Hilborn, Elizabeth D

    2015-06-01

    Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospitalized and/or receiving antibiotics; however, community-associated infections affecting otherwise healthy individuals have become more commonly reported. A case-crossover study was used to assess emergency room (ER) and outpatient visits for C. difficile infection following flood events in Massachusetts from 2003 through 2007. Exposure status was based on whether or not a flood occurred prior to the case/control date during the following risk periods: 0-6 days, 7-13 days, 14-20 days, and 21-27 days. Fixed-effects logistic regression was used to estimate the risk of diagnosis with C. difficile infection following a flood. There were 129 flood events and 1575 diagnoses of C. difficile infection. Among working age adults (19-64 years), ER and outpatient visits for C. difficile infection were elevated during the 7-13 days following a flood (Odds Ratio, OR = 1.69; 95% Confidence Interval, CI: 0.84, 3.37). This association was more substantial among males (OR = 3.21; 95% CI: 1.01-10.19). Associations during other risk periods were not observed (p < 0.05). Although we were unable to differentiate community-associated versus nosocomial infections, a potential increase in C. difficile infections should be considered as more flooding is projected due to climate change. PMID:26090609

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

    PubMed Central

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

    2016-01-01

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

  20. Clostridium difficile infection: a review of current and emerging therapies

    PubMed Central

    Ofosu, Andrew

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the most common cause of ­healthcare-associated infections in US hospitals. The epidemic strain NAP1/BI/ribotype 027 accounts for outbreaks worldwide, with increasing mortality and severity. CDI is acquired from an endogenous source or from spores in the environment, most easily acquired during the hospital stay. The use of antimicrobials disrupts the intestinal microflora enabling C. difficile to proliferate in the colon and produce toxins. Clinical diagnosis in symptomatic patients requires toxin detection from stool specimens and rarely in combination with stool culture to increase sensitivity. However, stool culture is essential for epidemiological studies. Oral metronidazole is the recommended therapy for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases. Treatment of first recurrence involves the use of the same therapy used in the initial CDI. In the event of a second recurrence oral vancomycin often given in a tapered dose or intermittently, or fidaxomicin may be used. Fecal transplantation is playing an immense role in therapy of recurrent CDI with remarkable results. Fulminant colitis and toxic megacolon warrant surgical intervention. Novel approaches including new antibiotics and immunotherapy against CDI or its toxins appear to be of potential value. PMID:27065726

  1. Beneficial effect of catalase treatment on growth of Clostridium perfringens.

    PubMed Central

    Harmon, S M; Kautter, D A

    1976-01-01

    Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media. PMID:185958

  2. Colonization Resistance of the Gut Microbiota against Clostridium difficile

    PubMed Central

    Pérez-Cobas, Ana Elena; Moya, Andrés; Gosalbes, María José; Latorre, Amparo

    2015-01-01

    Antibiotics strongly disrupt the human gut microbiota, which in consequence loses its colonization resistance capacity, allowing infection by opportunistic pathogens such as Clostridium difficile. This bacterium is the main cause of antibiotic-associated diarrhea and a current problem in developed countries, since its incidence and severity have increased during the last years. Furthermore, the emergence of antibiotic resistance strains has reduced the efficiency of the standard treatment with antibiotics, leading to a higher rate of relapses. Here, we review recent efforts focused on the impact of antibiotics in the gut microbiome and their relationship with C. difficile colonization, as well as, in the identification of bacteria and mechanisms involved in the protection against C. difficile infection. Since a healthy gut microbiota is able to avoid pathogen colonization, restoration of the gut microbiota seems to be the most promising approach to face C. difficile infection, especially for recurrent cases. Therefore, it would be possible to design probiotics for patients undergoing antimicrobial therapies in order to prevent or fight the expansion of the pathogen in the gut ecosystem. PMID:27025628

  3. Clostridium difficile ribotypes in humans and animals in Brazil.

    PubMed

    Silva, Rodrigo Otávio Silveira; Rupnik, Maja; Diniz, Amanda Nádia; Vilela, Eduardo Garcia; Lobato, Francisco Carlos Faria

    2015-12-01

    Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies about C. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficile ribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen. PMID:26676318

  4. Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

    PubMed Central

    Barker, Gary C.; Malakar, Pradeep K.; Plowman, June

    2016-01-01

    We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg−1. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments. PMID:26729721

  5. Toxinotype V Clostridium difficile in humans and food animals.

    PubMed

    Jhung, Michael A; Thompson, Angela D; Killgore, George E; Zukowski, Walter E; Songer, Glenn; Warny, Michael; Johnson, Stuart; Gerding, Dale N; McDonald, L Clifford; Limbago, Brandi M

    2008-07-01

    Clostridium difficile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. difficile-associated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identified as toxinotype V; before 2001, 7 (<0.02%) of approximately 6,000 isolates were identified as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. difficile, including the potential of foodborne transmission to humans. PMID:18598622

  6. Analysis of the pathogenicity locus in Clostridium difficile strains.

    PubMed

    Cohen, S H; Tang, Y J; Silva, J

    2000-02-01

    The genes for Clostridium difficile toxins A and B (tcdA and tcdB) are part of a 19.6-kb pathogenicity locus (PaLoc) that includes the genes tcdD, tcdE, and tcdC. To determine whether the C. difficile PaLoc is a stable and conserved genetic unit in toxigenic strains, a multiplex polymerase chain reaction was used to analyze 50 toxigenic, 39 nontoxigenic, and 2 toxin-defective isolates. The respective amplicons were identified for tcdA-E in the toxigenic isolates; these were absent in the nontoxigenic isolates. C. difficile P-829 lacked at least a fragment of tcdD, tcdB, tcdE, and tcdC, but tcdA was present. C. difficile 8864 had deletions in the tcdA and tcdC genes. These data suggest that the PaLoc is highly stable in toxigenic C. difficile, nontoxigenic isolates lack the unit, and isolates with a defective PaLoc can still cause clinical disease. Further studies are needed to define the role of individual genes in the pathogenesis of C. difficile-associated diarrhea. PMID:10669352

  7. Flooding and Clostridium difficile Infection: A Case-Crossover Analysis

    PubMed Central

    Lin, Cynthia J.; Wade, Timothy J.; Hilborn, Elizabeth D.

    2015-01-01

    Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospitalized and/or receiving antibiotics; however, community-associated infections affecting otherwise healthy individuals have become more commonly reported. A case-crossover study was used to assess emergency room (ER) and outpatient visits for C. difficile infection following flood events in Massachusetts from 2003 through 2007. Exposure status was based on whether or not a flood occurred prior to the case/control date during the following risk periods: 0–6 days, 7–13 days, 14–20 days, and 21–27 days. Fixed-effects logistic regression was used to estimate the risk of diagnosis with C. difficile infection following a flood. There were 129 flood events and 1575 diagnoses of C. difficile infection. Among working age adults (19–64 years), ER and outpatient visits for C. difficile infection were elevated during the 7–13 days following a flood (Odds Ratio, OR = 1.69; 95% Confidence Interval, CI: 0.84, 3.37). This association was more substantial among males (OR = 3.21; 95% CI: 1.01–10.19). Associations during other risk periods were not observed (p < 0.05). Although we were unable to differentiate community-associated versus nosocomial infections, a potential increase in C. difficile infections should be considered as more flooding is projected due to climate change. PMID:26090609

  8. Clostridium perfringens enterotoxicosis in two Amur leopards (Panthera pardus orientalis).

    PubMed

    Neiffer, D L

    2001-03-01

    Two 6-yr-old male sibling Amur leopards (Panthera pardus orientalis) housed together at the Pittsburgh Zoo presented for acute onset of diarrhea with no changes in appetite or behavior. Heat-fixed modified Wright-stained and Gram-stained fecal smears revealed a mixed bacterial population with a large number of gram-positive Clostridium perfringens-like spores (>20 per high-power oil immersion field). In addition, C. perfringens enterotoxin was isolated from one leopard at 1:256, confirming the presence of C. perfringens enterotoxicosis. Treatment with oral metronidazole, tylosin tartrate, and psyllium fiber was prescribed, with return of more normal stool by the third day of treatment. Fecal consistency steadily improved and was considered normal by the time all prescribed treatments were complete. Diarrhea has not recurred. Partially thawed meat in the leopards' diet may have precipitated the production of an endogenous clostridial enterotoxicosis by disrupting digestive tract flora with resultant clostridial overgrowth and sporulation. PMID:12790411

  9. Action of egg white lysozyme on Clostridium tyrobutyricum.

    PubMed Central

    Wasserfall, F; Teuber, M

    1979-01-01

    A 500-U ml-1 portion of egg white lysozyme was able to kill 99% of 5 X 10(5) resting vegetative cells of Clostridium tyrobutyricum within 24 h of incubation at 25 degrees C. Spores were completely resistant to lysozyme. Proliferating vegetative cells were severely inhibited, although lysozyme-resistant cells developed in growing cultures in the presence of lysozyme. Whereas early stages of spore germination (loss of optical refractility and heat resistance) were not inhibited by lysozyme, the overall outgrowth of spore cells into vegetative cells was delayed by 1 day in the presence of 500 U of lysosyme ml-1. This delay was independent of the lysozyme sensitivity or resistance of the mother culture of the used spores. It is suggested that this inhibition by lysozyme of the outgrowth of spore cells into vegetative cells of the lactate-fermenting C. tyrobutyricum is the basis for the observation that lysozyme can substitute for nitrate in preventing the "late gas" defect of Edam- and Gouda-type cheeses. PMID:518083

  10. The Regulatory Networks That Control Clostridium difficile Toxin Synthesis.

    PubMed

    Martin-Verstraete, Isabelle; Peltier, Johann; Dupuy, Bruno

    2016-01-01

    The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. PMID:27187475

  11. Novel actin-like filament structure from Clostridium tetani.

    PubMed

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Tanaka, Toshitsugu; Robinson, Robert C

    2012-06-15

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines. PMID:22514279

  12. The Regulatory Networks That Control Clostridium difficile Toxin Synthesis

    PubMed Central

    Martin-Verstraete, Isabelle; Peltier, Johann; Dupuy, Bruno

    2016-01-01

    The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. PMID:27187475

  13. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    SciTech Connect

    Guss, Adam M; Olson, Daniel G.; Caiazza, Nicky; Lynd, Lee R

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  14. The Systemic Inflammatory Response to Clostridium difficile Infection

    PubMed Central

    Rao, Krishna; Erb-Downward, John R.; Walk, Seth T.; Micic, Dejan; Falkowski, Nicole; Santhosh, Kavitha; Mogle, Jill A.; Ring, Cathrin; Young, Vincent B.; Huffnagle, Gary B.; Aronoff, David M.

    2014-01-01

    Background The systemic inflammatory response to Clostridium difficile infection (CDI) is incompletely defined, particularly for patients with severe disease. Methods Analysis of 315 blood samples from 78 inpatients with CDI (cases), 100 inpatients with diarrhea without CDI (inpatient controls), and 137 asymptomatic outpatient controls without CDI was performed. Serum or plasma was obtained from subjects at the time of CDI testing or shortly thereafter. Severe cases had intensive care unit admission, colectomy, or death due to CDI within 30 days after diagnosis. Thirty different circulating inflammatory mediators were quantified using an antibody-linked bead array. Principal component analysis (PCA), multivariate analysis of variance (MANOVA), and logistic regression were used for analysis. Results Based on MANOVA, cases had a significantly different inflammatory profile from outpatient controls but not from inpatient controls. In logistic regression, only chemokine (C-C motif) ligand 5 (CCL5) levels were associated with cases vs. inpatient controls. Several mediators were associated with cases vs. outpatient controls, especially hepatocyte growth factor, CCL5, and epithelial growth factor (inversely associated). Eight cases were severe and associated with elevations in IL-8, IL-6, and eotaxin. Conclusions A broad systemic inflammatory response occurs during CDI and severe cases appear to differ from non-severe infections. PMID:24643077

  15. Emerging therapies for Clostridium difficile infection – focus on fidaxomicin

    PubMed Central

    Chaparro-Rojas, Fredy; Mullane, Kathleen M

    2013-01-01

    The epidemiology of Clostridium difficile infections (CDI) has evolved during the last decades, with an increase in the reported incidence, severity of cases, and rate of mortality and relapses. These increases have primarily affected some special populations including the elderly, patients requiring concomitant antibiotic therapy, patients with renal failure, and patients with cancer. Until recently, the treatment of CDI was limited to either metronidazole or vancomycin. New therapeutic options have emerged to address the shortcomings of current antibiotic therapy. Fidaxomicin stands out as the first-in-class oral macrocyclic antibiotic with targeted activity against C. difficile and minimal collateral damage on the normal colonic flora. Fidaxomicin has demonstrated performance not inferior to what is considered the “gold standard” available therapy for CDI, vancomycin, in two separate Phase III clinical trials, but with significant advantages, including fewer recurrences and higher rates of sustained clinical cures. Fidaxomicin constitutes an important development in targeted antibiotic therapy for CDI and must be considered as a first-line agent for patients with risk factors known to portend relapse and severe infection. PMID:23843696

  16. Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.

    PubMed

    Martin, Jessica S H; Monaghan, Tanya M; Wilcox, Mark H

    2016-04-01

    Clostridium difficile infection (CDI) continues to affect patients in hospitals and communities worldwide. The spectrum of clinical disease ranges from mild diarrhoea to toxic megacolon, colonic perforation and death. However, this bacterium might also be carried asymptomatically in the gut, potentially leading to 'silent' onward transmission. Modern technologies, such as whole-genome sequencing and multi-locus variable-number tandem-repeat analysis, are helping to track C. difficile transmission across health-care facilities, countries and continents, offering the potential to illuminate previously under-recognized sources of infection. These typing strategies have also demonstrated heterogeneity in terms of CDI incidence and strain types reflecting different stages of epidemic spread. However, comparison of CDI epidemiology, particularly between countries, is challenging due to wide-ranging approaches to sampling and testing. Diagnostic strategies for C. difficile are complicated both by the wide range of bacterial targets and tests available and the need to differentiate between toxin-producing and non-toxigenic strains. Multistep diagnostic algorithms have been recommended to improve sensitivity and specificity. In this Review, we describe the latest advances in the understanding of C. difficile epidemiology, transmission and diagnosis, and discuss the effect of these developments on the clinical management of CDI. PMID:26956066

  17. Expression and delivery of an endolysin to combat Clostridium perfringens.

    PubMed

    Gervasi, Teresa; Horn, Nikki; Wegmann, Udo; Dugo, Giacomo; Narbad, Arjan; Mayer, Melinda J

    2014-03-01

    Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract. PMID:23942878

  18. Carbohydrate Transport by the Anaerobic Thermophile Clostridium thermocellum LQRI

    PubMed Central

    Strobel, H. J.; Caldwell, F. C.; Dawson, K. A.

    1995-01-01

    Clostridium thermocellum is an anaerobic thermophilic bacterium which degrades cellulose and ferments the resulting glucose, cellobiose, and cellodextrins predominantly to ethanol. However, relatively little information was available on carbohydrate uptake by this bacterium. Washed cells internalized intact oligomers as large as cellopentaose. Since cellobiose and cellodextrin phosphorylase activities were detected in the cytosol and were not associated with cell membranes, phosphorylation of carbohydrates occurred intracellularly. Kinetic studies indicated that cellobiose and larger cellodextrins were taken up by a common uptake system while glucose entered via a separate mechanism. When cells were treated with metabolic inhibitors including iodoacetate and arsenate, the uptake of radiolabeled glucose or cellobiose was reduced by as much as 90%, and this reduction was associated with a 95% decline in intracellular ATP content. A combination of the ionophores nigericin and valinomycin abolished the proton-motive force but only slightly decreased transport and ATP. These results suggested that the two modes of carbohydrate transport in C. thermocellum were ATP dependent. This work is the first demonstration of cellodextrin transport by a cellulolytic bacterium. PMID:16535164

  19. Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production.

    PubMed

    Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R; McClane, Bruce A

    2016-06-01

    The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold, and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involve their production of a variant small acid-soluble protein-4 that binds more tightly to spore DNA than to the small acid-soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the United States. During this foodborne disease, C. perfringens is ingested with food and then, by using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

  20. Survey of Clostridium difficile infection surveillance systems in Europe, 2011.

    PubMed

    Kola, Axel; Wiuff, Camilla; Akerlund, Thomas; van Benthem, Birgit H; Coignard, Bruno; Lyytikäinen, Outi; Weitzel-Kage, Doris; Suetens, Carl; Wilcox, Mark H; Kuijper, Ed J; Gastmeier, Petra

    2016-07-21

    To develop a European surveillance protocol for Clostridium difficile infection (CDI), existing national CDI surveillance systems were assessed in 2011. A web-based electronic form was provided for all national coordinators of the European CDI Surveillance Network (ECDIS-Net). Of 35 national coordinators approached, 33 from 31 European countries replied. Surveillance of CDI was in place in 14 of the 31 countries, comprising 18 different nationwide systems. Three of 14 countries with CDI surveillance used public health notification of cases as the route of reporting, and in another three, reporting was limited to public health notification of cases of severe CDI. The CDI definitions published by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Centre for Disease Prevention and Control (ECDC) were widely used, but there were differing definitions to distinguish between community- and healthcare-associated cases. All CDI surveillance systems except one reported annual national CDI rates (calculated as number of cases per patient-days). Only four surveillance systems regularly integrated microbiological data (typing and susceptibility testing results). Surveillance methods varied considerably between countries, which emphasises the need for a harmonised European protocol to allow consistent monitoring of the CDI epidemiology at European level. The results of this survey were used to develop a harmonised EU-wide hospital-based CDI surveillance protocol. PMID:27469420

  1. Clostridium difficile infection in a French university hospital

    PubMed Central

    Khanafer, Nagham; Oltra, Luc; Hulin, Monique; Dauwalder, Olivier; Vandenesch, Francois; Vanhems, Philippe

    2016-01-01

    Abstract The epidemiology of Clostridium difficile infection (CDI) has changed with an increase in incidence and severity. Prospective surveillance was therefore implemented in a French university hospital to monitor the characteristics of patients at risk and to recognize local trends. Between 2007 and 2014, all hospitalized patients (≥18 years) with CDI were included. During the survey, the mean incidence rate of CDI was 2.9 per 10,000 hospital-days. In all, 590 patients were included. Most of the episodes were healthcare-associated (76.1%). The remaining cases were community-acquired (18.1%) and unknown (5.9%). The comparison with healthcare-associated cases showed that the community-acquired group had a lower rate of antimicrobial exposure (P < 0.001), proton pump inhibitor (P < 0.001), and immunosuppressive drugs (P = 0.02). Over the study period, death occurred in 61 patients (10.3%), with 18 (29.5%) being related to CDI according to the physician in charge of the patient. Active surveillance of CDI is required to obtain an accurate picture of the real dimensions of CDI. PMID:27281101

  2. Fidaxomicin - the new drug for Clostridium difficile infection

    PubMed Central

    Vaishnavi, Chetana

    2015-01-01

    Clostridium difficile is one of the many aetiological agents of antibiotic associated diarrhoea and is implicated in 15-25 per cent of the cases. The organism is also involved in the exacearbation of inflammatory bowel disease and extracolonic manifestations. Due to increase in the incidence of C. difficile infection (CDI), emergence of hypervirulent strains, and increased frequency of recurrence, the clinical management of the disease has become important. The management of CDI is based on disease severity, and current antibiotic treatment options are limited to vancomycin or metronidazole in the developing countries. this review article briefly describes important aspects of CDI, and the new drug, fidaxomicin, for its treatment. Fidaxomicin is particularly active against C. difficile and acts by inhibition of RNA synthesis. Clinical trials done to compare the efficacy and safety of fidaxomicin with that of vancomycin in treating CDI concluded that fidaxomicin was non-inferior to vancomycin for treatment of CDI and that there was a significant reduction in recurrences. The bactericidal properties of fidaxomicin make it an ideal alternative for CDI treatment. However, fidaxomicin use should be considered taking into account the potential benefits of the drug, along with the medical requirements of the patient, the risks of treatment and the high cost of fidaxomicin compared to other treatment regimens. PMID:26112840

  3. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    PubMed Central

    Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko; Takehara, Masaya; Nagahama, Masahiro

    2016-01-01

    Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2). All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis. PMID:26807591

  4. Metabolic adaption of ethanol-tolerant Clostridium thermocellum.

    PubMed

    Zhu, Xinshu; Cui, Jiatao; Feng, Yingang; Fa, Yun; Zhang, Jingtao; Cui, Qiu

    2013-01-01

    Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production. PMID:23936233

  5. The potential for emerging therapeutic options for Clostridium difficile infection

    PubMed Central

    Mathur, Harsh; Rea, Mary C; Cotter, Paul D; Ross, R Paul; Hill, Colin

    2014-01-01

    Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibiotic-associated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. Recently, advancements in next generation sequencing technology (NGS) have highlighted the extent of damage to the gut microbiota caused by broad-spectrum antibiotics, often resulting in C. difficile infection (CDI). Currently the treatment of choice for CDI involves the use of metronidazole and vancomycin. However, recurrence and relapse of CDI, even after rounds of metronidazole/vancomycin administration is a problem that must be addressed. The efficacy of alternative antibiotics such as fidaxomicin, rifaximin, nitazoxanide, ramoplanin and tigecycline, as well as faecal microbiota transplantation has been assessed and some have yielded positive outcomes against C. difficile. Some bacteriocins have also shown promising effects against C. difficile in recent years. In light of this, the potential for emerging treatment options and efficacy of anti-C. difficile vaccines are discussed in this review. PMID:25564777

  6. Effect of cooling on Clostridium perfringens in pea soup.

    PubMed

    de Jong, A E I; Rombouts, F M; Beumer, R R

    2004-02-01

    Foods associated with Clostridium perfringens outbreaks are usually abused after cooking. Because of their short generation times, C. perfringens spores and cells can grow out to high levels during improper cooling. Therefore, the potential of C. perfringens to multiply in Dutch pea soup during different cooling times was investigated. Tubes of preheated pea soup (50 degrees C) were inoculated with cocktails of cells or heat-activated spores of this pathogen. The tubes were linearly cooled to 15 degrees C in time spans of 3, 5, 7.5, and 10 h and were subsequently stored in a refrigerator at 3 or 7 degrees C for up to 84 h. Cell numbers increased by 1-log cycle during the 3-h cooling period and reached their maximum after 10 h of cooling. Subsequent refrigeration hardly reduced cell numbers. Cooling of 3.75 liters of pea soup in an open pan showed that this amount of pea soup cooled from 50 to 15 degrees C in 5 h, which will allow a more than 10-fold increase in cell numbers. These findings emphasize the need of good hygienic practices and quick cooling of heated foods after preparation. PMID:14968969

  7. Fecal microbiota transplant for Clostridium difficile infection in older adults

    PubMed Central

    Tauxe, William M.; Haydek, John P.; Rebolledo, Paulina A.; Neish, Emma; Newman, Kira L.; Ward, Angela; Dhere, Tanvi; Kraft, Colleen S.

    2015-01-01

    Background: The objective of this study was to describe the safety of fecal microbiota transplant (FMT) for Clostridium difficile infection (CDI) among older adults. Methods: We performed a case review of all FMT recipients aged 65 or older treated at Emory University Hospital, a tertiary care and referral center for Georgia and surrounding states. Results: CDI resolved in 27 (87%) of 31 respondents, including three individuals who received multiple FMTs. Among four whose CDI was not resolved at follow up, three respondents did well initially before CDI recurred, and one individual never eradicated his CDI despite repeating FMT. During the study, five deaths and eight serious adverse events requiring hospitalization were reported within the study group during the follow-up period. Fecal transplant was not a causative factor in these events. The most common adverse event reported in 4 (13%) of 31 respondents was subjective worsening of arthritis. Conclusion: FMT is a generally safe and effective treatment option for older adults with CDI. PMID:27134658

  8. Fidaxomicin in Clostridium difficile infection: latest evidence and clinical guidance

    PubMed Central

    2014-01-01

    The incidence of Clostridium difficile infection (CDI) has risen 400% in the last decade. It currently ranks as the third most common nosocomial infection. CDI has now crossed over as a community-acquired infection. The major failing of current therapeutic options for the management of CDI is recurrence of disease after the completion of treatment. Fidaxomicin has been proven to be superior to vancomycin in successful sustained clinical response to therapy. Improved outcomes may be due to reduced collateral damage to the gut microflora by fidaxomicin, bactericidal activity, inhibition of Clostridial toxin formation and inhibition of new sporulation. This superiority is maintained in groups previously reported as being at high risk for CDI recurrence including those: with relapsed infection after a single treatment course; on concomitant antibiotic therapy; aged >65 years; with cancer; and with chronic renal insufficiency. Because the acquisition cost of fidaxomicin far exceeds that of metronidazole or vancomycin, in order to rationally utilize this agent, it should be targeted to those populations who are at high risk for relapse and in whom the drug has demonstrated superiority. In this manuscript is reviewed the changing epidemiology of CDI, current treatment options for this infection, proposed benefits of fidaxomicin over currently available antimicrobial options, available analysis of cost effectiveness of the drug, and is given recommendations for judicious use of the drug based upon the available published literature. PMID:24587892

  9. Prevalence of Clostridium difficile colonization among healthcare workers

    PubMed Central

    2013-01-01

    Background Clostridium difficile infection (CDI) has increased to epidemic proportions in recent years. The carriage of C. difficile among healthy adults and hospital inpatients has been established. We sought to determine whether C. difficile colonization exists among healthcare workers (HCWs) in our setting. Methods A point prevalence study of stool colonization with C. difficile among doctors, nurses and allied health staff at a large regional teaching hospital in Geelong, Victoria. All participants completed a short questionnaire and all stool specimens were tested by Techlab® C.diff Quik Check enzyme immunoassay followed by enrichment culture. Results Among 128 healthcare workers, 77% were female, of mean age 43 years, and the majority were nursing staff (73%). Nineteen HCWs (15%) reported diarrhoea, and 12 (9%) had taken antibiotics in the previous six weeks. Over 40% of participants reported having contact with a patient with known or suspected CDI in the 6 weeks before the stool was collected. C. difficile was not isolated from the stool of any participants. Conclusion Although HCWs are at risk of asymptomatic carriage and could act as a reservoir for transmission in the hospital environment, with the use of a screening test and culture we were unable to identify C. difficile in the stool of our participants in a non-outbreak setting. This may reflect potential colonization resistance of the gut microbiota, or the success of infection prevention strategies at our institution. PMID:24090343

  10. Fecal Transplantation for the Treatment of Recurrent Clostridium Difficile Infection

    PubMed Central

    Karadsheh, Zeid; Sule, Sachin

    2013-01-01

    Clostridium difficile infection (CDI) is currently a leading cause of antibiotic and health care-related diarrhea. The incidence and the severity of CDI-related diarrhea have increased dramatically in the USA and Europe in the past few decades. The emergence of multidrug-resistant hypervirulent strains of C. difficile has led to an increase in mortality. Fecal microbiota transplantation (FMT) (also known as fecal bacteriotherapy) has been utilized sporadically since the 1950s; and currently, the interest in using FMT has grown again in the past few years for the treatment of CDI and other chronic gastrointestinal diseases. FMT has shown to be effective, cheap, and has very few side effects. It is believed to manipulate and restore the gut microbiota, and therefore enhances the growth of “healthy” bacteria that break the cycle of recurrent CDI. This article focus on the recent case reports on FMT, and general approach to patients undergoing this therapy. Data were obtained through a literature search via PubMed and Google. PMID:23923106

  11. How to eradicate Clostridium difficile from the environment.

    PubMed

    Barbut, F

    2015-04-01

    During the last decade, Clostridium difficile has emerged as a major cause of healthcare-associated diarrhoea and death. Transmission of this spore-forming bacterium is thought to occur via the hands of healthcare providers or via the contaminated environment. Therefore, enhanced environmental cleaning/disinfection of the rooms housing C. difficile-infected patients is warranted. Guidelines from various scientific bodies have been published. They recommend performing environmental decontamination of rooms of patients with C. difficile infection (CDI) using hypochlorite (diluted 1/10) or a sporicidal product. Compliance with cleaning and disinfection is a critical point and is often suboptimal. Novel 'no-touch' methods for room disinfection have recently been introduced. Ultraviolet (UV) light or hydrogen peroxide systems are most widely used. In-vitro studies suggest that hydrogen peroxide vapour (from 30% hydrogen peroxide) methods achieve a >6 log10 reduction in C. difficile spores placed on carriers, and that aerosolized hydrogen peroxide systems (from 5% to 6% hydrogen peroxide) achieve ∼4 log10 reduction, whereas UV-based methods achieve ∼2 log10 reduction. Very few studies have assessed the impact of these devices on the transmission of C. difficile. Major limitations of these devices include the fact that they can only be used after the patient's discharge, because patients and staff must be removed from the room. The new no-touch methods for room disinfection supplement, but do not replace, daily cleaning. PMID:25638358

  12. Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile▿

    PubMed Central

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D.; Dupuy, Bruno; Fralick, Joe A.

    2009-01-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ΦCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ΦCD119 lysogens. During this study we found that repR, a putative repressor gene of ΦCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ΦCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen. PMID:19776116

  13. Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats.

    PubMed

    Veschi, Josir Laine A; Bruzzone, Octavio A; Losada-Eaton, Daniela M; Dutra, Iveraldo S; Fernandez-Miyakawa, Mariano E

    2008-09-15

    Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies. PMID:18538416

  14. Pathology of Clostridium perfringens type C enterotoxemia in horses.

    PubMed

    Diab, S S; Kinde, H; Moore, J; Shahriar, M F; Odani, J; Anthenill, L; Songer, G; Uzal, F A

    2012-03-01

    Clostridium perfringens type C is an important cause of enteritis and enterocolitis in foals and occasionally in adult horses. The disease is a classic enterotoxemia, and the enteric lesions and systemic effects are caused primarily by beta toxin, 1 of 2 major toxins produced by C. perfringens type C. Until now, only sporadic cases of C. perfringens type C equine enterotoxemia have been reported. We present a comprehensive description of the lesions in 8 confirmed cases of type C enterotoxemia in foals and adult horses. Grossly, multifocal to segmental hemorrhage and thickening of the intestinal wall were most common in the small intestine, although the colon and cecum were also frequently affected. All horses had variable amounts of fluid, often hemorrhagic intestinal contents. The most characteristic microscopic lesion was necrotizing or necrohemorrhagic enteritis, with mucosal and/or submucosal thrombosis. Numerous gram-positive rods were occasionally seen in affected mucosa. A definitive diagnosis of C. perfringens type C enterotoxemia in all 8 cases was based on the clinical history, gross and histologic lesions, and detection of the beta toxin in intestinal contents. PMID:21502373

  15. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

  16. A survey of Clostridium spiroforme antimicrobial susceptibility in rabbit breeding.

    PubMed

    Agnoletti, Fabrizio; Ferro, Tiziana; Guolo, Angela; Marcon, Barbara; Cocchi, Monia; Drigo, Ilenia; Mazzolini, Elena; Bano, Luca

    2009-04-14

    Rabbit meat breeding may be heavily affected by enterotoxaemia due to Clostridium spiroforme. Data on its antimicrobial susceptibility are insufficient, presumably because of difficulties in cultivating and identifying the pathogen. Our aim is therefore to provide this information to veterinary practitioners by focusing on a panel of therapeutics used in intensive rabbit units. Lincomycin was also checked in order to investigate the origin of resistance to macrolides. Minimal inhibitory concentrations (MICs) were determined with the agar dilution method according to the CLSI M11-A7 protocol (2007). MIC(50) and MIC(90) were, respectively, 64 and 64microg/ml for tiamulin, 32 and 32microg/ml for norfloxacin, 0.063 and 0.125microg/ml for amoxicillin, and 8 and 16microg/ml for doxycycline. MIC(50) and MIC(90) were 256microg/ml for sulphadimethoxine, spiramycin and lincomycin. Our results have shown that intrinsic or acquired antimicrobial resistances are diffuse in the C. spiroforme population and suggest focusing on prevention rather than on treatment of clostridial overgrowth, by reducing risk factors and using antimicrobials prudently. PMID:19058929

  17. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats.

    PubMed

    Uzal, Francisco A; Songer, J Glenn

    2008-05-01

    Clostridium perfringens produces enteric diseases, generically called enterotoxemias, in sheep, goats, and other animals. This microorganism can be a normal inhabitant of the intestine of most animal species, including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates and produces potent toxins that act locally or are absorbed into the general circulation with usually devastating effects on the host. History, clinical signs, and gross postmortem findings are useful tools for establishing a presumptive diagnosis of clostridial enterotoxemia in sheep and goats. Definitive diagnosis requires laboratory confirmation. Isolation of some types of C. perfringens (e.g., B and C) can be of diagnostic value, but other types (e.g., A) are so commonly found in the intestine of normal animals that isolation is meaningless from a diagnostic point of view. The most accepted criterion in establishing a definitive diagnosis of enterotoxemia is detection of C. perfringens toxins in intestinal contents. Also, histopathological examination of brain is very useful for diagnosis of type D disease, as lesions produced by epsilon toxin in the brains of sheep and goats are pathognomonic for type D enterotoxemia. Ancillary tests, such as measuring urine glucose or observing Gram-stained smears of intestinal mucosa, can be used. However, although such tests have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxemia when negative. PMID:18460610

  18. Regulation of toxin gene expression in Clostridium perfringens.

    PubMed

    Ohtani, Kaori; Shimizu, Tohru

    2015-05-01

    The Gram-positive, anaerobic, spore-forming, rod-shaped Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tract of humans and animals. C. perfringens causes clostridial myonecrosis (or gas gangrene), enteritis and enterotoxemia in humans and livestock by producing numerous extracellular toxins and enzymes. The toxin gene expression is regulated by a two-component regulatory system and regulatory RNA VirR/VirS-VR-RNA cascade. The VirR/VirS system was originally found in a type A strain, but a recent report showed that it is also important for the toxin gene regulation in other types of strains. Two types of cell-cell signaling, i.e., agr-system and AI-2 signaling, are also important for the regulation of toxin genes. Several regulatory systems independent from the VirR/VirS system, including virX, the orphan histidine kinase ReeS and orphan response regulator RevR, are also involved in the regulation of toxin genes. In addition, the expression of toxin genes is upregulated after contact with Caco-2 cells. C. perfringens has a complex regulatory network for toxin gene expression and thus the coordination of toxin gene expression is important for the process of infection. PMID:25303832

  19. Clostridium sordellii in a brown bear (Ursus arctos) from Spain.

    PubMed

    Balseiro, Ana; Oleaga, Álvaro; Polledo, Laura; Aduriz, Gorka; Atxaerandio, Raquel; Kortabarria, Nekane; Marín, Juan F García

    2013-10-01

    Clostridium sordellii is found in the environment and occasionally in animal (including human) intestines and may cause myonecrosis and large outbreaks of enterotoxemia. A few cases of fatal clostridial infection in bears (Ursus spp.) have been described worldwide but none attributed to C. sordellii. We describe a fatal case of septicemia caused by C. sordellii in an illegally trapped brown bear (Ursus arctos). At necropsy, acute gangrenous myositis was the primary lesion. Serohemorrhagic edema was observed in the abdominal cavity, thorax, pericardium, and skeletal muscle, mostly affecting femoral, humeral, and scapular muscles. Hemorrhage was observed in the heart, skeletal muscles, stomach, and intestine. Liver, spleen, and kidney appeared with loss of consistency, hemorrhages, and edema. Microscopically, primary lesions were in skeletal muscle, stomach, and small intestine, with gram-positive, clostridial-like bacilli. Biochemical and molecular tests identified C. sordellii in cultures from liver, muscle, and intestine. Sequences showed a homology of >99% with the 16S rRNA gene sequence of C. sordellii. The severity of effects of the C. sordellii infection reveal the importance of this pathogen as a wildlife health risk with conservation concerns, as well as the need to consider possible infection with this pathogen in management actions involving immobilization, stress, or severe muscular activity of wild brown bears. PMID:24502739

  20. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

    PubMed

    Losada-Eaton, D M; Fernandez-Miyakawa, M E

    2010-12-01

    Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia. PMID:20434186

  1. Clostridium perfringens Iota-Toxin b Induces Rapid Cell Necrosis▿

    PubMed Central

    Nagahama, Masahiro; Umezaki, Mariko; Oda, Masataka; Kobayashi, Keiko; Tone, Shigenobu; Suda, Taiji; Ishidoh, Kazumi; Sakurai, Jun

    2011-01-01

    Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis. PMID:21911469

  2. Toxinotype V Clostridium difficile in Humans and Food Animals

    PubMed Central

    Thompson, Angela D.; Killgore, George E.; Zukowski, Walter E.; Songer, Glenn; Warny, Michael; Johnson, Stuart; Gerding, Dale N.; McDonald, L. Clifford; Limbago, Brandi M.

    2008-01-01

    Clostridium difficile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. difficile–associated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identified as toxinotype V; before 2001, 7 (<0.02%) of ≈6,000 isolates were identified as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. difficile, including the potential of foodborne transmission to humans. PMID:18598622

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

    PubMed

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

    2016-01-01

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

  4. Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis.

    PubMed

    Makroczyová, Jana; Jamroškovič, Ján; Krascsenitsová, Eva; Labajová, Nad'a; Barák, Imrich

    2016-06-01

    In rod-shaped bacteria, the proper placement of the division septum at the midcell relies, at least partially, on the proteins of the Min system as an inhibitor of cell division. The main principle of Min system function involves the formation of an inhibitor gradient along the cell axis; however, the establishment of this gradient differs between two well-studied gram-negative and gram-positive bacteria. While in gram-negative Escherichia coli, the Min system undergoes pole-to-pole oscillation, in gram-positive Bacillus subtilis, proper spatial inhibition is achieved by the preferential attraction of the Min proteins to the cell poles. Nevertheless, when E.coli Min proteins are inserted into B.subtilis cells, they still oscillate, which negatively affects asymmetric septation during sporulation in this organism. Interestingly, homologs of both Min systems were found to be present in various combinations in the genomes of anaerobic and endospore-forming Clostridia, including the pathogenic Clostridium difficile. Here, we have investigated the localization and behavior of C.difficile Min protein homologs and showed that MinDE proteins of C.difficile can oscillate when expressed together in B.subtilis cells. We have also investigated the effects of this oscillation on B.subtilis sporulation, and observed decreased sporulation efficiency in strains harboring the MinDE genes. Additionally, we have evaluated the effects of C.difficile Min protein expression on vegetative division in this heterologous host. PMID:26817670

  5. Growth potential of Clostridium perfringens during cooling of cooked meats.

    PubMed

    Taormina, Peter J; Dorsa, Warren J

    2004-07-01

    Many meat-based food products are cooked to temperatures sufficient to inactivate vegetative cells of Clostridium perfringens, but spores of this bacterium can survive, germinate, and grow in these products if sufficient time, temperature, and other variables exist. Because ingestion of large numbers of vegetative cells can lead to concomitant sporulation, enterotoxin release in the gastrointestinal tract, and diarrhea-like illness, a necessary food safety objective is to ensure that not more than acceptable levels of C. perfringens are in finished products. As cooked meat items cool they will pass through the growth temperature range of C. perfringens (50 to 15 degrees C). Therefore, an important step in determining the likely level of C. perfringens in the final product is the estimation of growth of the pathogen during cooling of the cooked product. Numerous studies exist dealing with just such estimations, yet consensual methodologies, results, and conclusions are lacking. There is a need to consider the bulk of C. perfringens work relating to cooling of cooked meat-based products and attempt to move toward a better understanding of the true growth potential of the organism. This review attempts to summarize observations made by researchers and highlight variations in experimental approach as possible explanations for different outcomes. An attempt is also made here to identify and justify optimal procedures for conducting C. perfringens growth estimation in meat-based cooked food products during cooling. PMID:15270517

  6. Diversity and Evolution in the Genome of Clostridium difficile

    PubMed Central

    Knight, Daniel R.; Elliott, Briony; Chang, Barbara J.; Perkins, Timothy T.

    2015-01-01

    SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen. PMID:26085550

  7. Fecal Microbiota Transplantation for the Treatment of Clostridium difficile Infection

    PubMed Central

    Rao, Krishna; Safdar, Nasia

    2016-01-01

    Clostridium difficile, a major cause of healthcare-associated diarrhea due to perturbation of the normal gastrointestinal microbiome, is responsible for significant morbidity, mortality, and healthcare expenditures. The incidence and severity of C. difficile infection (CDI) is increasing and recurrent disease is common. Recurrent infection can be difficult to manage with conventional antibiotic therapy. Fecal microbiota transplantation (FMT), which involves instillation of stool from a healthy donor into the gastrointestinal tract of the patient, restores the gut microbiome to a healthy state. FMT has emerged as a promising new treatment for CDI. There are limited data on FMT for treatment of primary CDI, but FMT appears safe and effective for recurrent CDI. The safety and efficacy of FMT in patients with severe primary or recurrent CDI has not been established. Patients with inflammatory bowel disease (IBD) who undergo FMT for CDI may be at increased risk of IBD flare and caution should be exercised with use of FMT in that population. The long-term safety of FMT is unknown; thus, rigorously conducted prospective studies are needed. PMID:26344412

  8. Clostridium difficile-associated diarrhea in an ocelot (Leopardus pardalis).

    PubMed

    Silva, Rodrigo Otávio Silveira; D'elia, Mirella Lauria; de Magalhães Soares, Danielle Ferreira; Cavalcanti, Álvaro Roberto; Leal, Rodrigo Costa; Cavalcanti, Guilherme; Pereira, Pedro Lúcio Lithg; Lobato, Francisco Carlos Faria

    2013-04-01

    The aim of this study is to report a case of Clostridium difficile-associated diarrhea in an ocelot (Leopardus pardalis) in the state of Mato Grosso do Sul, Brazil. The animal, a 24-month-old male, was referred to the Centro de Reabilitação de Animais Silvestres (CRAS) with a history of having been run over and tibia and fibula fractures. After a surgery to repair the fractures, the ocelot underwent antibiotic therapy with two doses of sodium cefovecin, during which he presented with diarrhea. A stool sample was positive for A/B toxins by a cytotoxicity assay, and a toxigenic strain of C. difficile was isolated. No other enteropathogens were detected. The association between the history, clinical signs and laboratory exams confirmed the diagnosis of C. difficile-associated diarrhea. The present report confirms C. difficile as a potential pathogen for wild felids and suggests that the C. difficile-associated diarrhea should be considered in diarrhea cases, especially when the clinical signs began after antimicrobial use. PMID:23467074

  9. Clostridium ljungdahlii represents a microbial production platform based on syngas

    PubMed Central

    Köpke, Michael; Held, Claudia; Hujer, Sandra; Liesegang, Heiko; Wiezer, Arnim; Wollherr, Antje; Ehrenreich, Armin; Liebl, Wolfgang; Gottschalk, Gerhard; Dürre, Peter

    2010-01-01

    Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO2/H2 and synthesis gas (CO/H2). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO2, thus combining industrial needs with sustained reduction of CO and CO2 in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures. PMID:20616070

  10. Purification of Clostridium botulinum type G progenitor toxin.

    PubMed

    Nukina, M; Mochida, Y; Sakaguchi, S; Sakaguchi, G

    1988-04-01

    Clostridium botulinum type G cultured for 6 days at 30 degrees C in proteose peptone-yeast extract-glucose medium produced toxin of 1.3 x 10(4) LD50/ml. The toxin was precipitated at pH 4.0, extracted with 0.2 M phosphate buffer, pH 6.0, and activated with trypsin. Sonic treatment and trypsinization of the residual precipitate released additional toxin, the toxicity of which corresponded to that detected in whole culture. Activated toxin obtained from the first extract and that from the residual precipitate were combined and purified by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second gel filtration on Sephadex G-200. The yield of purified toxin from 10 liters of culture was 22.9 mg an 1.1 X 10(8) mouse ip LD50 with a specific toxicity of 3.0 X 10(7) mouse ip LD50/mg nitrogen. The molecular weight of the toxin was about 500,000, corresponding to that of L toxin of the other types. No M nor LL toxin was detected. PMID:3293334

  11. Thermostable amylolytic enzymes from a new Clostridium isolate

    SciTech Connect

    Madi, E.; Antranikian, G.; Ohmiya, K.; Gottschalk, G.

    1987-07-01

    A new Clostridium strain was isolated on starch at 60 degrees C. Starch, pullulan, maltotriose, and maltose induced the synthesis of alpha-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of alpha-amylase and pullulanase into the culture broth; but also by a decrease of total activity. Alpha-amylase, pullulanase, and alpha-glucosidase were active in a broad temperature range (40 to 85 degrees C) and displayed temperature optima for activity at 60 to 70 degrees C. During incubation with starch under aerobic conditions at 75 degrees C for 2 hours, the activity of both enzymes decreased to only 90 or 80%. The apparent Km values of alpha-amylase, pullulanase, and alpha-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively. (Refs. 31).

  12. Comparative transcriptomic analysis of Clostridium acetobutylicum biofilm and planktonic cells.

    PubMed

    Liu, Dong; Xu, Jiahui; Wang, Yanyan; Chen, Yong; Shen, Xiaoning; Niu, Huanqing; Guo, Ting; Ying, Hanjie

    2016-01-20

    Biofilm-based immobilization of solventogenic Clostridia has been extensively exploited to overcome traditional bottlenecks in biobutanol production like solvent toxicity and low productivities. However, the molecular basis of solventogenic Clostridia biofilm is rarely explored. Here, for the first time, we report DNA array-based study of Clostridium acetobutylicum biofilm cells to elucidate the transcriptional modulation. Results showed that 16.2% of the C. acetobutylicum genome genes within the biofilm cells were differentially expressed, with most genes being up-regulated. The most dramatic changes occurred with amino acid biosynthesis, with sulfur uptake and cysteine biosynthesis being the most up-regulated and histidine biosynthesis being the most down-regulated in the biofilm cells. It was demonstrated that C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes. Furthermore, genes involved in sporulation, granulose formation, extracellular polymer degradation, pentose catabolisms, and various other processes were also notably regulated, indicating that the biofilm mode of growth rendered the cells a distinct phenotype. This study provides valuable insights into the transcriptional regulation in C. acetobutylicum biofilm cells and should be highly useful for understanding and developing the biofilm-based processes. PMID:26621081

  13. Antimicrobial susceptibility of equine and environmental isolates of Clostridium difficile.

    PubMed

    Båverud, V; Gunnarsson, A; Karlsson, M; Franklin, A

    2004-01-01

    The antimicrobial susceptibility of 50 Clostridium difficile isolates, 36 of them from horse feces and 14 from environmental sites, was determined by broth microdilution. The antimicrobial agents tested were avilamycin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, neomycin, oxacillin, oxytetracycline, penicillin, spiramycin, streptomycin, trimethoprim/sulfamethoxazole, vancomycin, and virginiamycin. All isolates were susceptible to vancomycin (MIC 16 microg/ml), oxytetracycline (MIC >/=32 microg/ml), spiramycin (MIC > 16 microg/ml), and virginiamycin (MIC 8-16 microg/ml) were higher for 18 isolates. Those were mainly isolated from horses at animal hospitals and further from environmental sites at a stud farm. In contrast, all isolates, except one, from healthy foals had low MICs of erythromycin, spiramycin, virginiamycin, and oxytetracycline. The isolates from soil in public parks had also low MICs of these antimicrobial agents. Broth microdilution appeared both reliable and reproducible for susceptibility testing of C. difficile. The method was also readily performed and the MIC endpoints were easily read. PMID:15140395

  14. Occurrence of Clostridium perfringens from different cultivated soils.

    PubMed

    Voidarou, C; Bezirtzoglou, E; Alexopoulos, A; Plessas, S; Stefanis, C; Papadopoulos, I; Vavias, S; Stavropoulou, E; Fotou, K; Tzora, A; Skoufos, I

    2011-12-01

    The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole. When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality. PMID:21621626

  15. Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

    PubMed

    Perz, Veronika; Baumschlager, Armin; Bleymaier, Klaus; Zitzenbacher, Sabine; Hromic, Altijana; Steinkellner, Georg; Pairitsch, Andris; Łyskowski, Andrzej; Gruber, Karl; Sinkel, Carsten; Küper, Ulf; Ribitsch, Doris; Guebitz, Georg M

    2016-05-01

    Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site. Biotechnol. Bioeng. 2016;113: 1024-1034. © 2015 Wiley Periodicals, Inc. PMID:26524601

  16. Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

    PubMed Central

    Matano, Y; Park, J S; Goldstein, M A; Doi, R H

    1994-01-01

    Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly. Images PMID:7961457

  17. Cell wall proteome of Clostridium thermocellum and detection of glycoproteins.

    PubMed

    Yu, Tingting; Xu, Xinping; Peng, Yanfeng; Luo, Yuanming; Yang, Keqian

    2012-06-20

    Clostridium thermocellum, a thermophilic anaerobe, has the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. In this work, the cell wall proteome of C. thermocellum was investigated. The proteins in the cell wall fraction of C. thermocellum prepared by the boiling SDS method were released by mutanolysin digestion and resolved on two-dimensional (2D) gel. One hundred and thirty-two proteins were identified by mass spectrometry, among which the extracellular solute-binding protein (CbpB/cthe_1020), enolase, glyceraldehyde-3-phosphate dehydrogenase and translation elongation factor EF-Tu were detected as highly abundant proteins. Besides the known surface localized proteins, including FtsZ, MinD, GroEL, DnaK, many enzymes involved in bioenergetics, such as alcohol dehydrogenases and hydrogenases were also detected. By glycan stain and MS analysis of glycopeptides, we identified CbpB as a glycoprotein, which is the second glycoprotein from C. thermocellum characterized. The fact that CbpB was highly abundant in the cell wall region and glycosylated, reflects its importance in substrate assimilation. Our results indicate cell wall proteins constitute a significant portion of cellular proteins and may play important physiological roles (i.e. bioenergetics) in this bacterium. The insights described are relevant for the development of C. thermocellum as a biofuel producer. PMID:22494898

  18. Reannotation of the genome sequence of Clostridium difficile strain 630.

    PubMed

    Monot, Marc; Boursaux-Eude, Caroline; Thibonnier, Marie; Vallenet, David; Moszer, Ivan; Medigue, Claudine; Martin-Verstraete, Isabelle; Dupuy, Bruno

    2011-08-01

    A regular update of genome annotations is a prerequisite step to help maintain the accuracy and relevance of the information they contain. Five years after the first publication of the complete genome sequence of Clostridium difficile strain 630, we manually reannotated each of the coding sequences (CDSs), using a high-level annotation platform. The functions of more than 500 genes annotated previously with putative functions were reannotated based on updated sequence similarities to proteins whose functions have been recently identified by experimental data from the literature. We also modified 222 CDS starts, detected 127 new CDSs and added the enzyme commission numbers, which were not supplied in the original annotation. In addition, an intensive project was undertaken to standardize the names of genes and gene products and thus harmonize as much as possible with the HAMAP project. The reannotation is stored in a relational database that will be available on the MicroScope web-based platform (https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?S_id=752&wwwpkgdb=a78e3466ad5db29aa8fe49e8812de8a7). The original submission stored in the (International Nucleotide Sequence Database Collaboration) INSDC nucleotide sequence databases was also updated. PMID:21349987

  19. Clostridium perfringens Type E Virulence Traits Involved in Gut Colonization

    PubMed Central

    Redondo, Leandro M.; Carrasco, Juan M. Díaz; Redondo, Enzo A.; Delgado, Fernando; Miyakawa, Mariano E. Fernández

    2015-01-01

    Clostridium perfringens type E disease in ruminants has been characterized by hemorrhagic enteritis or sudden death. Although type E isolates are defined by the production of alpha and iota toxin, little is known about the pathogenesis of C. perfringens type E infections. Thus far, the role of iota toxin as a virulence factor is unknown. In this report, iota toxin showed positive effects on adherence and colonization of C. perfringens type E while having negative effect on the adherence of type A cells. In-vitro and in-vivo models suggest that toxinotype E would be particularly adapted to exploit the changes induced by iota toxin in the surface of epithelial cells. In addition, type E strains produce metabolites that affected the growth of potential intra-specific competitors. These results suggest that the alteration of the enterocyte morphology induced by iota toxin concomitantly with the specific increase of type E cell adhesion and the strong intra-specific growth inhibition of other strains could be competitive traits inherent to type E isolates that improve its fitness within the bovine gut environment. PMID:25799452

  20. Genomic analyses of Clostridium perfringens isolates from five toxinotypes.

    PubMed

    Hassan, Karl A; Elbourne, Liam D H; Tetu, Sasha G; Melville, Stephen B; Rood, Julian I; Paulsen, Ian T

    2015-05-01

    Clostridium perfringens can be isolated from a range of environments, including soil, marine and fresh water sediments, and the gastrointestinal tracts of animals and humans. Some C. perfringens strains have attractive industrial applications, e.g., in the degradation of waste products or the production of useful chemicals. However, C. perfringens has been most studied as the causative agent of a range of enteric and soft tissue infections of varying severities in humans and animals. Host preference and disease type in C. perfringens are intimately linked to the production of key extracellular toxins and on this basis toxigenic C. perfringens strains have been classified into five toxinotypes (A-E). To date, twelve genome sequences have been generated for a diverse collection of C. perfringens isolates, including strains associated with human and animal infections, a human commensal strain, and a strain with potential industrial utility. Most of the sequenced strains are classified as toxinotype A. However, genome sequences of representative strains from each of the other four toxinotypes have also been determined. Analysis of this collection of sequences has highlighted a lack of features differentiating toxinotype A strains from the other isolates, indicating that the primary defining characteristic of toxinotype A strains is their lack of key plasmid-encoded extracellular toxin genes associated with toxinotype B to E strains. The representative B-E strains sequenced to date each harbour many unique genes. Additional genome sequences are needed to determine if these genes are characteristic of their respective toxinotypes. PMID:25445567