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Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.

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Clostridium difficile PCR ribotype 106 (also identified as restriction endonuclease analysis [REA] group DH) recently emerged as the most common strain causing C. difficile infection (CDI) among US adults. We previously identified this strain predominating our pediatric cohort. Pediatric clinical CDI isolates previously characterized by REA underwent antibiotic resistance testing and whole genome sequencing. Of 134 isolates collected from children, 31 (23%) were REA group DH. We performed a comparative genomics analysis to identify DH-associated accessory genes. We identified five DH-associated genes that are associated with virulence in other bacterial species but not previously known to contribute to CDI. These genes are associated with intestinal mucosal adhesion (collagen-binding surface protein), sporulation (sporulation integral membrane protein YtvI), and protection from oxidative stress and foreign DNA (DNA phosphorothioation-dependent restriction proteins, sulfurtransferase, and DNA sulfur modification proteins). The association of these genes was validated in a cohort of 623 publicly available C. difficile sequences, 10 (1.6%) of which were monophyletic to REA group DH through in silico multilocus sequence typing and core genome phylogenetic analysis. Further investigation is required to determine the contribution of these genes to the emergence and virulence of this epidemic strain.

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Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene (clostridial myonecrosis), remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections.

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BACKGROUND: Although Clostridium difficile is a major cause of diarrhoea, its epidemiology in tropical settings is poorly understood. Strain characterisation requires work-up in specialised laboratories, often after prolonged storage without properly maintained cold chain. METHODS: We screened 298 human faecal samples from Côte d'Ivoire using a rapid test for C. difficile glutamate dehydrogenase (GDH). GDH-positive samples were aerobically stored at disrupted cold chain conditions (mean duration: 11 days) before transfer to a reference laboratory for anaerobic culture, susceptibility testing, PCR assays and ribotyping. RESULTS: Sixteen samples (5.4%) had a positive GDH screening test. C. difficile infection was confirmed in six specimens by culture and PCR, while no nucleic acids of C. difficile were detected in the culture-negative samples. Further analysis of stool samples harbouring toxigenic C. difficile strains confirmed that both GDH and toxins remained detectable for at least 28 days, regardless of storage conditions (aerobic storage at 4°C or 20°C). CONCLUSIONS: Storage conditions only minimally affect recovery of C. difficile and its toxins in stool culture. A rapid GDH screening test and subsequent transfer of GDH-positive stool samples to reference laboratories for in-depth characterisation may improve our understanding of the epidemiology of C. difficile in the tropics.

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We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

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A single intraperitoneal injection of a gram-positive pathogen Clostridium perfringens (Cp) causes a remarkable down-regulation the constitutive nitric oxide synthase (cNOS) with a simultaneous increase in the activity of inducible NOS (iNOS) and the level of reactive nitrogen species in the rat brain major regions (cortex, striatum, hippocampus and hypothalamus) at 48 h post-administration of Cp. Treatment by both a semiconductor laser (SCL) and/or a light-emitting diode (LED) with same wavelength, energy density and time exposure (continuous wave, λ=654 nm, fluence=1.27 J/cm(2), time exposure=600 s) could modulate brain nitrergic response following Cp-infection. Besides, unlike the LED, the SCL-irradiation prevents the cNOS inhibition in all the studied brain regions and might be useful in restoring its function in neurotransmission and cerebral blood flow, along with providing a protective effect against nitrosative stress-induced iNOS-mediated injury in the brain regions.

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Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.

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The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the alpha-toxin. Previously, mice have been protected against challenge with heterologous alpha-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa(247-370) or alpha-toxoid) of the alpha-toxin. In this study, we have determined the ability of the alpha-toxoid to protect against the lethal effects of a divergent C. perfringens alpha-toxin and against the PLCs of C. absonum or C. bifermentans, species which have been isolated from cases of clostridial myonecrosis. Protection against the C. perfringens alpha-toxin variant, the C. absonum alpha-toxin or the C. bifermentans PLC was elicited by immunisation with the alpha-toxoid in vivo.

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OBJECTIVE: To determine the epidemiology and relatedness of Clostridium difficile isolates in two geographically separated hospitals in a large metropolitan area, each with unique patients and personneL DESIGN: Observational descriptive molecular epidemiology of clinical C. difficile isolates. SETTING: Two tertiary-care hospitals in Chicago. METHODS: Consecutive C. difficile isolates from the clinical laboratory of a Veterans Affairs hospital during a 13-month period were typed by restriction endonuclease analysis (REA). During an overlapping 3-month period, stool specimens that tested positive for C. difficile toxin from patients at a nearby county hospital were cultured and the recovered isolates typed by the same method. RESULTS: Nineteen (68%) of 28 nosocomial isolates at the smaller, Veterans Affairs hospital belonged to REA group K. Within this group of closely related strains, 9 distinct REA types were recognized. Twenty-one (72%) of 29 nosocomial isolates at the larger, county hospital also belonged to group K. However, the predominant REA types within group K differed markedly at each institution. CONCLUSIONS: These findings demonstrate a high degree of similarity among nosocomial C. difficile strains from different hospitals in the same city and suggest the possibility of an extended outbreak of a prototype group K strain with subsequent genetic drift at the two different institutions.

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The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.

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Camel races have a long tradition in Arabia. Since the oil boom of the 1960s a tremendous revival of the old Bedouin tradition of camel racing has occurred in the United Arab Emirates. These camel races are comparable to horse races in Europe and the U.S.A. Since 1985 the most valuable racing camels of Dubai are routinely tested in the Central Veterinary Research Laboratory (CVRL) for their stamina and endurance. Blood and serum enzyme values, which have been statistically ascertained through testing of 10000 healthy racing camels, are now generally accepted as reference values. Besides these check-ups of healthy racing camels, hematological tests, enzyme and substrate estimations are performed on sick racing camels. These tests support the diagnosis, therapy and prognosis of sick camels. In this connection three diseases are discussed: B. cereus intoxication, Clostridium perfringens enterotoxemia and Trypanosomiasis.

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Two immunological methods, a sialidase-inhibition test and an ELISA, had been developed for a species-specific detection of sialidases (EC 3.2.1.18) secreted by growing cells of Clostridium perfringens, C. septicum, and C. sordellii. These assays were applied to samples from the infected tissues and cotton wool plugs soaked with wound exudate from patients suspected to be suffering from gas gangrene. The results from 72 patients were compared with bacteriological investigations of 67 homologous samples. From these, 35 patients were found to be infected with one or two of the clostridial species: thirty patients contained C. perfringens, six samples showed an immunologically positive reaction for C. septicum and eight for C. sordellii. In the case of C. perfringens, a high degree of coincidence between the immunological and the bacteriological analyses was obtained. However, in infections with C. septicum or C. sordellii the bacteriological and immunological tests did not agree well. The simple and rapid sialidase inhibition test or the slightly more time-consuming ELISA, which even detects low quantities of sialidase, can be applied for the specific diagnosis of infections caused by C. perfringens, thus allowing a species-specific detection of this bacterial species in two or six hours, respectively, even at an early stage of infection. This early diagnosis is a prerequisite for a specific and life-saving therapy of this severe disease.

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Following trauma, wound contamination with aerobic and/or anaerobic bacteria should always be suspected. Treatment with antibacterial antibiotics should begin immediately in the emergency room, particularly for those patients with fractures. Patients with serious trauma are best treated by a team of specialists including general surgeons, orthopedists, infectious disease specialists, and intensive care specialists. The author recommends transport of seriously injured patients to major hospitals specializing in the care of trauma. Particularly when gas gangrene secondary to clostridial infection is suspected, the patient should be moved to a major trauma center with the capability for hyperbaric oxygen therapy.

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An assay was devised to quantitate acute intestinal inflammation based on the assessment of myeloperoxidase activity. Myeloperoxidase is an enzyme found in neutrophils and, in much smaller quantities, in monocytes and macrophages. Myeloperoxidase was solubilized with hexadecyltrimethylammonium bromide and myeloperoxidase activity was measured with a dianisidine-H2O2 assay. In neutrophil suspensions, myeloperoxidase activity was directly related to cell number down to as few as 500 cells. Myeloperoxidase activity was assayed in two animal models of inflammation: acetic acid-induced colitis in rats and Clostridium difficile enterotoxin-induced enteritis in hamsters. In both models, the activity of myeloperoxidase solubilized from the inflamed tissue was directly proportional to the number of neutrophils seen in histologic sections. Histologic evaluation of neutrophil accumulation was performed by counting the number of neutrophils in a histologic section 0.18 mm long and 5 micron thick. In both animal models, myeloperoxidase activity was linearly related to neutrophil number from 400 and 4000 cells/mm. Myeloperoxidase activity from chronically inflamed colon, in which both neutrophils and histiocytes were present, was directly related to neutrophil content. Histiocytes did not contribute significantly to myeloperoxidase activity. The determination of myeloperoxidase activity in the intestine is a simple biochemical assay that can be used to quantitate inflammation.

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Daily oral administration of ampicillin to hamsters consistently resulted in fatal ileocecitis due to ampicillin-susceptible strains of Clostridium difficile. Ampicillin was not detected in the cecal contents of these hamsters once C. difficile appeared. Cecal contents obtained from hamsters with ampicillin-induced ileocecitis displayed beta-lactamase activity, whereas cecal contents obtained from untreated control hamsters did not. Colonization of the ceca with C. difficile corresponded to a decrease in the concentration of cecal ampicillin below the minimum inhibitory concentration effective against C. difficile in vitro. The concomitant administration of ampicillin and sulbactam, a nonabsorbable beta-lactamase inhibitor, protected hamsters from developing fatal ileocecitis. However, ileocecitis developed upon the discontinuation of treatment. beta-Lactamase produced by the cecal flora inactivates ampicillin present in the intestinal tract, thereby permitting ampicillin-sensitive C. difficile to multiply and cause disease.

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Red blood cells become polyagglutinable when the normally latent T-antigens of the red blood cell membrane are exposed. Unmasking of T-antigens results from removal of N-acetyl-neuraminic acid by neuraminidase, an enzyme commonly produced by a variety of bacteria. Red blood cells altered in this way are said to be T-activated. T-activated red blood cells can be agglutinated by anti-T, an antibody normally present in human serum, so that severe transfusion reactions may occur and have occurred, if T-antigen positive patients are transfused with normal whole blood or plasma. This can be avoided by transfusing only packed or washed red blood cells. From October 1978 to October 1980 we found T-activation in 16 pediatric surgical patients aged 3 days to 14 yr with severe anaerobic infections. This included patients with necrotizing enterocolitis, perforated appendicitis, megacolon, infected anal atresia and gas gangrene. The isolate neuraminidase-producing bacteria were Clostridium perfringens and Bacteroides fragilis. Clinical data of these 16 patients are briefly reviewed and the importance of T-antigen positivity for their management is discussed.

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Clostridium perfringens produces a variety of virulence factors. The mechanism of action of these factors usually falls into one of three groups. Some of these virulence factors, such as the alpha toxin, which is phospholipase C, and the kappa toxin, which is a collagenase, are enzymes that hydrolyze substances essential to the integrity of membranes or other body structures. Other virulence factors, such as the beta, episolon, and iota toxins, act primarily on the vascular endothelium, causing increased capillary permeability, especially in the brain. Still others, such as the delta and theta toxins, are essentially hemolysins. Theta toxin is similar in action and serologically related to streptolysin O.

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Clostridium perfringens septicemia with massive hemolysis is described in an elderly woman with refractory anemia. This case is highly unusual in that (1) the diagnosis was made during life by discovery of the organisms on a routine peripheral blood film, and that (2) phospholipase C activity was detected in the serum.

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