Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

A Case of Adult Intestinal Toxemia Botulism During Prolonged Hospitalization in an Allogeneic Hematopoietic Cell Transplant Recipient.

We report a laboratory-confirmed case of adult intestinal toxemia botulism in an allogeneic hematopoietic stem cell transplantation (allo-HCT) recipient. Onset of symptoms occurred within the hospitalized setting, making this case particularly unique. Botulism may have arisen because of significant intestinal disruption and compromise, and not directly from immune compromise.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Adult Intestinal Toxemia Botulism.

Intoxication with botulinum neurotoxin can occur through various routes. Foodborne botulism results after consumption of food in which botulinum neurotoxin-producing clostridia (i.e., Clostridium botulinum or strains of Clostridiumbutyricum type E or Clostridiumbaratii type F) have replicated and produced botulinum neurotoxin. Infection of a wound with C. botulinum and in situ production of botulinum neurotoxin leads to wound botulism. Colonization of the intestine by neurotoxigenic clostridia, with consequent production of botulinum toxin in the intestine, leads to intestinal toxemia botulism. When this occurs in an infant, it is referred to as infant botulism, whereas in adults or children over 1 year of age, it is intestinal colonization botulism. Predisposing factors for intestinal colonization in children or adults include previous bowel or gastric surgery, anatomical bowel abnormalities, Crohn's disease, inflammatory bowel disease, antimicrobial therapy, or foodborne botulism. Intestinal colonization botulism is confirmed by detection of botulinum toxin in serum and/or stool, or isolation of neurotoxigenic clostridia from the stool, without finding a toxic food. Shedding of neurotoxigenic clostridia in the stool may occur for a period of several weeks. Adult intestinal botulism occurs as isolated cases, and may go undiagnosed, contributing to the low reported incidence of this rare disease.

Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury.

Corynebacterium parvum-treated mice produce large amounts of circulating nitrogen oxides and develop a severe liver injury in response to lipopolysaccharide (LPS). Concurrent administration of NG-monomethyl-L-arginine not only suppresses nitric oxide synthesis in these animals but also profoundly increases the hepatic damage following LPS. In this report, we present evidence that the increased hepatic damage from inhibition of nitric oxide synthesis is mediated in part by superoxide and hydroxyl radicals. The hepatic damage induced by suppressing nitric oxide production during endotoxemia could be reduced by treating mice with superoxide dismutase and deferoxamine, scavengers of superoxide and hydroxyl radicals, respectively. This damage could also be prevented by treating mice with the anticoagulant heparin sodium. The results suggest that nitric oxide synthesis during endotoxemia is important in preventing hepatic damage by reducing oxygen radical-mediated hepatic injury and preventing intravascular thrombosis.

Identification of the 1B vaccine strain of Chlamydia abortus in aborted placentas during the investigation of toxaemic and systemic disease in sheep.

CASE HISTORY: One hundred and forty Cheviot and 100 Suffolk cross Mule primiparous 1-2-year-old ewes, from a flock of about 700 ewes, were vaccinated with an attenuated live 1B strain Chlamydia abortus vaccine about 4 weeks before ram introduction (September 2011). Between 08 March and 01 April 2012, 50 2-year-old ewes aborted and 29 of these died, despite antimicrobial and anti-inflammatory treatment and supportive care. PATHOLOGICAL FINDINGS: Seven fetuses and three placentae from five 2-year-old ewes were submitted for pathological investigation. The aborted fetuses showed stages of autolysis ranging from being moderately fresh to putrefaction. Unusual, large multifocal regions of thickened membranes, with a dull red granular surface and moderate amounts of grey-white surface exudate were seen on each of the placentae. Intracellular, magenta-staining, acid fast inclusions were identified in Ziehl Neelsen-stained placental smears. Immunohistochemistry for Chlamydia-specific lipopolysaccharide showed extensive positive labelling of the placental epithelia. LABORATORY FINDINGS: Molecular analyses of the aborted placentae demonstrated the presence of the 1B vaccine-type strain of C. abortus and absence of any wild-type field strain. The vaccine strain bacterial load of the placental tissue samples was consistent with there being an association between vaccination and abortion. DIAGNOSIS: Initial laboratory investigations resulted in a diagnosis of chlamydial abortion. Further investigations led to the identification of the 1B vaccine strain of C. abortus in material from all three of the submitted aborted placentae. CLINICAL RELEVANCE: Timely knowledge and understanding of any potential problems caused by vaccination against C. abortus are prerequisites for sustainable control of chlamydial abortion. This report describes the investigation of an atypical abortion storm in sheep, and describes the identification of the 1B vaccine strain of C. abortus in products of abortion. The significance of this novel putative association between the vaccine strain of C. abortus and severe clinical disease is unknown. Aspects of the approach that is described are relevant to the investigation of all outbreaks of ovine abortion, irrespective of the diagnosis. Awareness of the changing role of C. abortus as a major global cause of abortion ought to reinforce the importance of monitoring of adequate biosecurity in those countries which are currently free from chlamydial abortion.

Historical review: Autointoxication and focal infection theories of dementia praecox.

The popularity of theories of autointoxication and focal infection in general medicine and dentistry in the late nineteenth and early twentieth centuries led Emil Kraepelin and others to speculate that dementia praecox was caused by a poisoning of the brain from toxins produced in other parts of the body, notably the sex glands, the intestines and the mouth. Emil Kraepelin's commitment to the autointoxication theory is ignored in the literature on the history of psychiatry due to the focus of historians and clinicians on the major contributions of Kraepelin's methods of clinical psychopathology. Besides heredity, autointoxication and focal infection were the other most dominant theories of the organic aetiology of dementia praecox in the first three decades of its existence as a nosological entity in psychiatry. Rational treatments for dementia praecox that followed logically from these aetio-logical theories were colonic irrigations and major abdominal surgeries such as appendicostomies, colectomies and the removal of presumably infected ovaries, testes and other organs associated with reproduction. Autointoxication and focal infection theories disappeared from psychiatry by the mid-1930s.

Death of an African elephant from probable toxemia attributed to chronic pulpitis.

A 31-year-old captive male African elephant (Loxodonta africana) of 5,000-kg body weight died suddenly in ventral recumbency. Lesions seen at necropsy were bilateral purulent pulpitis and periodontitis of both tusks, serous atrophy of coronary groove fat, Grammocephalus cholangitis, myocardial and skeletal lipofuscinosis, and scattered segmental necrosis in the pectoral muscles. Nonhemolytic streptococci, Corynebacterium sp, Peptostreptococcus anaerobius, Fusobacterium nucleatum, and Bacteroides sp, were recovered from the exudate around one or both tusks. We postulated that the elephant died of hypoxia from prolonged ventral recumbency because of weakness and inability to rise secondary to toxemia from bilateral pulpitis and periodontitis.

[The epidemiology of toxemic infections caused by Staphylococcus aureus in an intensive care setting]. / Epidémiologie des infections toxémiques à staphylocoque doré.

The mission of the National Reference Centre for Staphylococcal Toxemia (CNRTS) is to participate in the epidemiological surveillance of staphylococcal toxemia in France. As these syndromes do not have to be declared, the data collected remain incomplete. Comparison of the clinical data with the results of molecular analysis of the causal strains has nonetheless enabled advances in our understanding of the present-day epidemiology of these syndromes, a clearer knowledge of their pathophysiology and isolation of a hitherto unknown entity, staphylococcal necrotizing pneumonia.

[Pathology of the digestive organs and systemic endotoxinemia]. / Patologiia organov pishchevareniia i sistemnaia éndotoksinemiia.

The intestine being a natural depot of the gram-negative bacteria, may be the source of the systemic endotoxinemia in various pathologic processes followed by the increase of bacteria death, mucous membrane damage, pancreas endocrine function deficiency, liver barrier function depression, decrease of the portal circulation speed or its shunting. At the same time, the systemic endotoxinemia in itself may be one of the most important etiological factors of the various digestive organs injury. The authors observations and the literature suggest an important role of the endotoxin-positive granulocytes in the pathogenesis of the haemorrhagic infarct of the intestine, mesenteric vein thrombosis, destructive appendicitis and cholecystitis.

[Ofloxacin in comprehensive treatment of infections in burn patients]. / Ofloksatsin v kompleksnom lechenii infektsii u obozhzhennykh.

The experience with ofloxacin in the prophylaxis and treatment of infected burn wounds in 40 patients was investigated. High clinical and microbiological efficacy of the drug was stated (82.5 and 83 per cent respectively). The highest efficacy of ofloxacin was observed when the burned area did not exceed 25 per cent of the body surface. It was concluded that the prophylactic use of the drug during acute burn toxemia was not expedient.

Oxacillin or cefalotin treatment of hospitalized children with cellulitis.

Cellulitis is an important cause of hospitalization in pediatrics. Because Staphylococcus aureus is the main pathogen of cellulitis, medicinal therapeutics should take the changing resistance profile of this organism into consideration. The aim of this study was to evaluate the progression and outcomes of children hospitalized for cellulitis and treated with oxacillin or cefalotin. This retrospective cohort study enrolled 218 children, hospitalized between 2001 and 2008 in Salvador, Northeast Brazil. All were diagnosed with cellulitis and treated with oxacillin or cefalotin (≥100 mg/kg/day). The median age was 2 years and 56.9% were males. Frequencies of signs and symptoms used in the clinical diagnoses were as follows: swelling (91.3%), redness (81.7%), warmth (47.2%), and tenderness (31.7%). All patients were discharged due to clinical recovery and the mean length of hospitalization was 7 ± 4 days. None of the patients died, needed intensive care, or had sequelae. By comparing the daily frequency of clinical findings during hospitalization, significant decreases were found in the frequencies of fever (admission day [42.2%], first day [20.8%], second day [12.9%], third day [8.3%], fourth day [6.1%]), toxemia, irritability, somnolence, vomiting, tachycardia, and need for intravenous hydration. In conclusion, oxacillin or cefalotin remain the drugs of choice for treating uncomplicated cellulitis in regions where community-acquired methicillin-resistant S. aureus is infrequent (<10%).

[Genotyping on 47 Staphylococcus aureus strains associated with bloodstream infection.].

OBJECTIVE: To investigate the molecular epidemiological characteristics of Staphylococcus aureus associated with bloodstream infection in hospital. METHODS: 47 Staphylococcus aureus strains isolated from bloodstream in PLA General Hospital were collected from January 2006 to December 2008. Susceptibility of the strains to 11 antimicrobial agents was detected and DNA homology of them was analyzed with Rep-based DiversiLab(TM) Microbial Typing System. Panton-Valentine leukocidin (PVL) gene was determined by PCR. For methicillin-resistant Staphylococcus aureus (MRSA) strains, the genotypes of SCCmec were determined and ST239 clone was screened with multiplex PCR. Multilocus sequence typing (MLST) was used to determine the STs of the selected isolates. RESULTS: In the 47 Staphylococcus aureus isolated from blood, methicillin-resistant strains accounted for 51.1%, all belonged to SCCmec III type, with only 2 pvl gene positive strains identified. 12 different patterns (A-L) were found among 47 strains with Rep-PCR. All MRSA strains clustered in the A and B subtypes. CONCLUSION: Most MRSA strains isolated from blood in PLA General Hospital belonged to ST239-MRSA-SCCmec III clone. DiversiLab(TM) Microbial Typing System could provide a rapid and effective method to investigate the molecular epidemiological characteristics of Staphylococcus aureus in the hospital settings.

The HcaR regulatory protein of Photorhabdus luminescens affects the production of proteins involved in oxidative stress and toxemia.

Comparison of the proteomes of wild-type Photorhabdus luminescens and its hcaR derivative, grown in insect hemolymph, showed that hcaR disruption decreased the production of toxins (tcdA1, mcf, and pirAB) and proteins involved in oxidative stress response (SodA, AhpC, Gor). The disruption of hcaR did not affect growth rate in insects, but did delay the virulence of P. luminescens in Bombyx mori and Spodoptera littoralis larvae. This delayed virulence was associated with a lower toxemia rather than delay in bacteremia. The disruption of hcaR also increased bacterial sensitivity to hydrogen peroxide. A sodA mutant and an hcaR mutant had similar phenotypes in terms of sensitivity to hydrogen peroxide, virulence, toxin gene expression, and growth rate in insects. Thus, the two processes affected by hcaR disruption - toxemia and oxidative stress response - appear to be related. Besides, expression of toxin genes tcdA1, mcf, and pirAB was decreased by paraquat challenge. We provide here the first demonstration of the importance of toxemia for P. luminescens virulence. Our results also highlight the power of proteomic analysis for detecting unexpected links between different, concomitant processes in bacteria.

Sublethal exposure from microcystins to renal insufficiency patients in Rio de Janeiro, Brazil.

In November 2001, a cyanobacterial bloom dominated by Microcystis and Anabaena occurred in the Funil Reservoir and the Guandu River, both of which supply drinking water to Rio de Janeiro, Brazil. Using ELISA, microcystins were detected at a concentration of 0.4 microg/L in the drinking water, whereas a concentration of 0.32 microg/L was detected in activated carbon column-treated water for use at the renal dialysis center of Clementino Fraga Filho Hospital (HUCFF) at the Federal University of Rio de Janeiro. A total of 44 hemodialysis patients who received care at this center were believed to be exposed. Initial ELISA analyses confirmed the presence of serum microcystin concentrations > or = 0.16 ng/mL in 90% of serum samples collected from these patients. Twelve patients were selected for continued monitoring over the following 2-month period. Serum microcystin concentrations ranged from < 0.16 to 0.96 ng/mL during the 57 days after documented exposure. ELISA-positive samples were found throughout the monitoring period, with the highest values detected 1 month after initial exposure. ESI LC/MS analyses indicated microcystins in the serum; however, MS/MS fragmentation patterns typical of microcystins were not identified. LC/MS analyses of MMPB for control serum spiked with MCYST-LR. and patient sera revealed a peak at retention time of 8.4 min and a mass of 207 m/z. These peaks are equivalent to the peak observed in the MMPB standard analysis. Taken together ELISA, LC/MS, and MMPB results indicate that these renal dialysis patients were exposed to microcystins. This documents another incident of human microcystin exposure during hemodialysis treatment.