Flagellar glycosylation in Clostridium botulinum

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.     

Polar Flagellar Motility of the Vibrionaceae

Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 μm/s. The sodium motive force powers rotation of the filament, which acts as a propeller. The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane. The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus. The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species. This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae. In addition to its propulsive role, the single polar flagellum of V. parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression. Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces. Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery.     

Antibiotics and Clostridium difficile

Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy, and is particularly associated with clindamycin and third-generation cephalosporins. Depletion of the indigenous gut microflora by antibiotic therapy has long been established as a major factor in the disease. However, the direct influence of antimicrobials upon virulence mechanisms such as toxin production and adhesion in the bowel, and the exact mechanisms by which the organism causes disease remain to be elucidated.     

Gains of Bacterial Flagellar Motility in a Fungal World

The maintenance of energetically costly flagella by bacteria in non-water-saturated media, such as soil, still presents an evolutionary conundrum. Potential explanations have focused on rare flooding events allowing dispersal. Such scenarios, however, overlook bacterial dispersal along mycelia as a possible transport mechanism in soils. The hypothesis tested in this study is that dispersal along fungal hyphae may lead to an increase in the fitness of flagellated bacteria and thus offer an alternative explanation for the maintenance of flagella even in unsaturated soils. Dispersal along fungal hyphae was shown for a diverse array of motile bacteria. To measure the fitness effect of dispersal, additional experiments were conducted in a model system mimicking limited dispersal, using Pseudomonas putida KT2440 and its nonflagellated (ΔfliM) isogenic mutant in the absence or presence of Morchella crassipes mycelia. In the absence of the fungus, flagellar motility was beneficial solely under conditions of water saturation allowing dispersal, while under conditions limiting dispersal, the nonflagellated mutant exhibited a higher level of fitness than the wild-type strain. In contrast, in the presence of a mycelial network under conditions limiting dispersal, the flagellated strain was able to disperse using the mycelial network and had a higher level of fitness than the mutant. On the basis of these results, we propose that the benefit of mycelium-associated dispersal helps explain the persistence of flagellar motility in non-water-saturated environments.     

Clostridium difficile in seafood and fish

The objective of this study was to determine the prevalence of Clostridium difficile contamination in retail seafood and fish from Canadian grocery stores. C.?difficile was found in 4.8% (5/119) of the samples. This study, combined with studies of other food sources, suggests that widespread contamination of food is common.     

Sporulation studies in Clostridium difficile

Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called ‘hypervirulent’ C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.     

Clostridium difficile electrophoretic typing

Abstract Two typing schemes for Clostridium difficile based on slide agglutinations and polyacrylamide gel electrophoresis (PAGE) have been described. We compared the reference strains of each typing system with a simplified PAGE method using whole cells and Coomassie blue staining. The method was also applied to clinical isolates and immunoblots were performed with a monospecific serum directed against a major band of low molecular weight. The results indicated the great heterogeneity of Clostridium difficile strains complicated by antigenic subdivision for strains belonging to the same electrophoretic type.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.     

艰难梭菌的研究进展

艰难梭菌为革兰阳性厌氧芽胞杆菌,可引起艰难梭菌相关性腹泻,导致一系列肠道感染症状和相关临床表现。近年来由于高致病株的出现、菌株耐药性的增加,艰难梭菌感染在全球呈蔓延趋势。本文就艰难梭菌的耐药机制、检测技术、防治及国内感染现状等作一简要综述。     

Clostridium difficile in vegetables, Canada

Background: Clostridium difficile is an important gastrointestinal pathogen of humans and animals. It has been isolated from various foods, including meat and ready‐to‐eat salads, and concern has been expressed regarding food as a possible source of human C. difficile infection (CDI). Aims: We sought to isolate C. difficile from a variety of vegetables obtained from local grocery stores and to characterize these isolates. Materials and Methods: Vegetables were purchased from 11 different grocery stores in Guelph, Ontario, Canada between May and August 2009. Enrichment culture was performed and isolates were characterized by ribotyping, PFGE, toxinotyping and PCR detection of toxin genes. Results: Clostridium difficile was isolated from 4.5% (5/111) of retail vegetables. Two different ribotypes and two different toxinotypes were identified. Three isolates were ribotype 078/NAP 7/toxinotype V, possessing all three toxin genes. The other two isolates shared a ribotype with a toxigenic strain previously found in humans with CDI in this region. Discussion: Contamination of vegetables was found at relatively low levels, however, all isolates were toxigenic and belonging to ribotypes previously associated with CDI. Conclusions: Contamination of vegetables with CDI‐associated isolates can occur and although the implications for food safety practices remain elusive, the presence of toxigenic isolates suggests vegetables could be a source of C. difficile in humans.     

Thiosemicarbazones active against Clostridium difficile

A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activity against Clostridium difficile.     

Carbohydrate fermentation by Clostridium difficile

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.     

The Asymmetric Flagellar Distribution and Motility of Escherichia coli

Rod-shaped bacteria such as Escherichia coli divide by binary fission. They inherit an old pole from the parent cell. The new pole is recently derived from the septum. Because the chemoreceptor accumulates linearly with time on the cell pole, the old pole carries more receptors than does the new pole. Here, further evidence is provided that the old pole appears more frequently at the rear when bacteria swim. This phenomenon had been observed, yet not extensively explored in the literature. The biased swimming orientation is the consequence of the asymmetric distribution of flagella over the cell surface. On about 75% of cells, there are more flagella on the old-pole half of the cell than on the new-pole half, regardless of growth conditions. Most flagella are lateral, and few were found on the cell pole per se. The asymmetric flagellar distribution makes cells more efficient in chemotaxis. Both swimming orientation and receptor localization are components of chemotaxis, by which bacteria follow environmental stimuli. If unipolarly flagellated cells, such as the swarmer cells of Caulobacter crescentus, are regarded as 100% polar with respect to chemotaxis, E. coli is about 75%. The difference is quantitative. The peritrichous flagellation might enhance the motility and chemotaxis in the viscous environment of enteric bacteria.     

Clostridium difficile infection in adult hamsters.

Diarrhea was encountered in a group of adult female golden Syrian hamsters (Mesocricetus auratus) used for titrating the scrapie agent. Ninety percent of the cases occurred in animals over 210 days old even though animals of all age groups lived in the colony concurrently. The cause of diarrhea was investigated in both uninoculated animals and those receiving greater than a limiting dilution of scrapie infectivity, i.e., animals that were not expected to contract the experimental scrapie disease. Three forms of diarrhea were observed. The most commonly encountered was profuse and watery. A chronic form presented with semiformed, thin fecal material smearing the retroperitoneal region. Hemorrhagic diarrhea was observed rarely. Mortality was high among animals with acute watery or hemorrhagic diarrhea. Animals with semiformed soft stools were dehydrated, had a roughened hair-coat, and hunched back. Cardinal lesions were necrosis, inflammation, and mucosal hyperplasia of the cecum and colon and cholangiohepatitis with amyloid deposition. Diffuse renal amyloidosis was present in chronic cases. Toxigenic, cytotoxin B-positive Clostridium difficile was isolated from a majority of affected animals. Cytotoxin B was also present in cecal homogenates of diarrheic animals with C. difficile. The pathological and microbiologic findings indicated a typhlitis and colitis in adult hamsters that was associated with C. difficile infection.     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.