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PubMed2003

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city, Brazil.

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.

Acute Disease↗
PubMed1980

Characterization of Clostridium difficile and its differentiation from Clostridium sporogenes by automatic head-space gas chromatography.

Although 47 strains of Clostridium difficile and Clostridium sporogenes were studied by gas chromatography. Acidic and neutral volatile products, formed after 96 h of incubation in glucose-containing peptone yeast-extract medium, were chromatographed. All strains produced appreciable amounts of fatty acids, which were tentatively identified by gas chromatographic retention data. Chromatograms obtained when analysing diethyl ether extracts of culture media of all 47 strains were virtually identical. However, analysis of the broth media by automatic head-space gas chromatography, employing a glass capillary column, gave chromatographic patterns which differentiated the two Clostridium species studied. C. sporogenes was characterized by chromatographic patterns containing a dominant peak of isovaleric acid. Strains of this species produced larger amounts of early eluting compounds and much smaller amounts of butyric and valeric acid than did strains of C. difficile. Automatic head-space gas chromatography provides an efficient means for differentiation of C. difficile and C. sporogenes. This gas chromatographic technique is easier and more rapidly performed than analysis of either extracts of culture media.

Chromatography, Gas↗
PubMed1992

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

Actin Cytoskeleton↗
PubMed1995

Cloning and characterization of the cytotoxin L-encoding gene of Clostridium sordellii: homology with Clostridium difficile cytotoxin B.

Hybridization of an oligodeoxyribonucleotide (oligo) probe, designed from a repeated sequence ('oligo rep') at the C terminus of the Clostridium difficile (Cd) cytotoxin (Cyt), revealed that homologies exist between the Cd cyt gene and the genomes of several other clostridia, including Clostridium sordellii (Cs), suggesting a common ancestral cyt amongst the Clostridium genus. This Cd 'oligo rep' probe was used to clone the Cs (strain 6018) cyt. The sequenced (7095 bp) region encodes 2364 amino acids (aa) and corresponds to a protein of 270,614 Da. Cs Cyt has 76% identity with the Cd Cyt and 47% identity with the Cd enterotoxin (Ent). The latter third of the protein consists of repeated units, similar to those found for Cd Cyt. A highly conserved hydrophobic domain can be delineated. Few structural differences are evident between Cd and Cs Cyt to explain their different cellular and sub-cellular effects. A small open reading frame (ORF) encoding a protein of 16,484 Da is located 210 bp downstream from cyt. No homology was evident with any known sequence. The first 30 aa of this ORF may correspond to a signal peptide.

Amino Acid Sequence↗
PubMed1998

Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases.

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.

3T3 Cells↗
PubMed2000

Clostridium akagii sp. nov. and Clostridium acidisoli sp. nov.: acid-tolerant, N2-fixing clostridia isolated from acidic forest soil and litter.

Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively. Both bacteria were spore-forming, motile rods with peritrichous flagella. The capacity to sporulate decreased with prolonged cultivation. Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath. Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T. These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T. Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products. Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively. CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1. CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs. The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively. CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum. The 16S rRNA gene sequence similarity values of CK58T and CK74T to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58T and CK74T be named Clostridium akagii CK58T (DSM 12554T) and Clostridium acidisoli CK74T (DSM 12555T), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N2 in the annoxic microzones of acidic forest soil and litter.

Anaerobiosis↗
PubMed2001

Transfer of thermobacteroides leptospartum and Clostridium thermolacticum as Clostridium stercorarium subsp. leptospartum subsp. thermolacticum subsp. nov., comb. nov. and C. stercorarium subsp. thermolacticum subsp. nov., comb. nov.

16S rRNA sequencing and sequence analysis of the sole member of the genus Thermobacteroides, Thermobacteroides leptospartum, revealed that it was related to members of cluster III (according to the scheme of Collins et al. 1994) represented exclusively by cellulolytic Clostridium species. Phenotypic studies indicated that Thermobacteroides leptospartum was also able to grow on cellulose, providing further evidence of its affiliation to members of cluster III. Its closest phylogenetic relatives, Clostridium thermolacticum and Clostridium stercorarium, were almost equidistantly placed with a similarity value of 99%. DNA hybridization studies also indicated that Thermobacteroides leptospartum, C. thermolacticum and C. stercorarium were closely related to each other (values of over 95% homology). Similarities based on the comparison of the 16S rRNA gene sequences and DNA homology are sufficiently high to regard all three strains as subspecies of a single species. It is therefore proposed that Thermobacteroides leptospartum and C. thermolacticum be transferred to cluster III as C. stercorarium subsp. leptospartum subsp. nov., comb. nov. and C. stercorarium subsp. thermolacticum subsp. nov., comb. nov., respectively, thus automatically creating C. stercorarium subsp. stercorarium subsp. nov., comb. nov. The transfer of the sole member of Thermobacteroides invalidates the taxonomic status of the genus.

Clostridium↗
PubMed2001

Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

Clostridium↗
PubMed2001

Clostridium felsineum and Clostridium acetobutylicum are two distinct species that are phylogenetically closely related.

The gene sequences encoding the 16S rRNA of Clostridium felsineum DSM 794T and NCIMB 10690T were determined. Both sequences exhibited a relatively very low degree of similarity to the previously determined 16S rRNA gene sequence from C. felsineum DSM 794T. C. felsineum is a member of the major Clostridium cluster, cluster I, and is phylogenetically closely related to Clostridium acetobutylicum. DNA-DNA hybridization results clearly indicated that C. felsineum and C. acetobutylicum belong to distinct species.

Clostridium↗
PubMedDate not supplied

[Extrachromosomal genetic elements of Clostridium botulinum. I. Plasmids of toxigenic and atoxigenic strains of Clostridium botulinum type A].

Plasmid profiles in fifteen toxigenic and six nontoxigenic strains of Clostridium botulinum type A were investigated. The electrophoretic patterns of nucleic acids from the bacterial lysates were shown to vary in relation to the mode of bacterial cells lysis and the stage of microbial population growth. Two toxigenic strains harbour a unique 3.9 MD plasmid, five strains have a plasmid of about 6 Md and seven other strains carry an 11.5 Md plasmid. The nontoxigenic derivatives of two toxigenic strains harbouring a 6 Md plasmid have lost this extrachromosomal element. The analysis of the derivatives has not indicated the 6 Md plasmid to be connected with bacteriocin production or antibiotic resistance in Clostridium botulinum. Nontoxigenic variants of three other strains possess an 11.5 Md plasmid similar to their toxigenic parents' one. One of the studied toxigenic strains harboured no plasmids, as well as its nontoxigenic derivative. The functions of all studied plasmids remain unknown. Physical maps of the 11.5 and 6 Md plasmids were constructed on the basis of restriction analysis. The obtained results can be used for identification and differentiation of Clostridium botulinum strains and in further molecular biological experiments with this organism.

Botulinum Toxins↗
PubMed2001

Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis.

The Clostridium septicum alpha toxin and the Clostridium perfringens beta toxin are examples of pore-forming toxins that exhibit several different features. The cell types that are targeted by these toxins reflect the effect these toxins have on the host during infection with either organism. Alpha toxin elicits a rapid shock-like syndrome, whereas beta toxin appears to induce a variety of neurological effects. The effects of the purified toxins appear to mimic some of the features of the animal and human diseases caused by C. septicum and C. perfringens. This review, examines the current state of knowledge for the cytolytic mechanism, role in pathogenesis and structure of these two toxins.

Bacterial Toxins↗
PubMed1975

Common mesophilic anaerobes, including Clostridium botulinum and Clostridium tetani, in 21 soil specimens.

A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 X 10-2 to 3.3 X 10-6 per g. The organisms most frequently isolated were Clostridium subterminate, C. sordelii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridium. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content.

Anaerobiosis↗
PubMed1979

Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains.

Clostridium difficile, C. perfringens, and C. tertium are very often present simultaneously in the feces of conventional diarrheic young hares, whereas these three bacterial species are rarely encountered and never present simultaneously in the feces of healthy young hares. When a strain of each of the three bacterial species was monoassociated with axenic young hares, the appearance of pathological disorders was only observed in animals monoassociated with C. difficile, when the number of C. difficile exceeded 10(8) per g of fresh feces. When a strain of C. perfringens or a strain of C. tertium, or both, was associated with C. difficile, diarrhea and death occurred more rapidly than in hares monoassociated with C. difficile. C. difficile and C. perfringens became established more rapidly when disassociated than when monoassociated with axenic hares. The association of C. perfringens and C. tertium with axenic hares did not bring about any pathological disorders. It may be concluded that C. difficile is the causal agent of neonatal diarrhea in conventional and gnotobiotic young hares and that other strains of Clostridium enhance its pathogenic effect. C difficile alone or associated with C. perfringens or C. tertium does not play any pathogenic role in young rats, mice, or rabbits.

Animals↗
PubMed1995

Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin.

The bacterial pathogen Clostridium difficle synthesizes two high-molecular-weight toxins (A and B), which exhibit toxic effects in vivo and in vitro. Here, we present evidence that the major intracellular targets of these two toxins are the Rho GTPases. Overexpression of RhoA, RhoB, or RhoC GTPases in transfected HeLa cells conferred an increased resistance to toxins A and B, indicating that these toxins cause their cytopathic effects primarily by affecting Rho proteins. In addition, toxin A and B treatment appeared to result in modification of Rho, since Rho isolated from toxin-treated cells had a decreased ability to be ADP-ribosylated by Clostridium botulinum C3 exoenzyme. In contrast, the lethal toxin (LT) of Clostridium sordellii, although structurally and immunologically related to C. difficile toxin B, appeared to induce cytopathic effects independently of the Rho GTPases. Overexpression of RhoA in transfected HeLa cells did not protect them from the effect of LT, and Rho isolated from lysates of LT-treated cells was not resistant to modification by C3. Immunofluorescence studies showed that LT treatment caused a cytopathic effect that was very different from those described for C. difficile toxins A and B, resulting in an increase in cortical F-actin, with a concomitant decrease in the number of stress fibers, and in the formation of numerous microvilli containing the actin-bundling protein fimbrin/plastin.

Bacterial Proteins↗
PubMed2001

Differentiation of Clostridium perfringens and Clostridium botulinum from non-toxigenic clostridia, isolated from prepared and frozen foods by PCR-DAN based methods.

During the elaboration process of prepared and frozen foods, Clostridium sp. have been reported. From those microorganisms, C. perfringens and C. botulinum may pose a high risk for the consumers. To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C. perfringens and C. boulinum should be established. In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C. perfringens from other Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characterization by API system. The restriction analysis of the 16S rDNA with Taq I and Rsa I showed at least the same sensitivity to differentiate C. perfringens from clostridial isolates as biochemical identification. However, the former method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6-8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.

Clostridium botulinum↗
PubMed1998

Conjugative transfer of the Escherichia coli-Clostridium perfringens shuttle vector pJIR1457 to Clostridium botulinum type A strains.

An RP4-oriT shuttle vector pJIR1457 originally developed for Clostridium perfringens was successfully transferred by conjugation from Escherichia coli to Clostridium botulinum type A strains and to a nontoxigenic C. botulinum type A-transposon Tn916 mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis of C. botulinum and will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.

Botulinum Toxins↗
PubMed1991

Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E.

Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.

Animals↗
PubMed2006

Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes.

Clostridium perfringens and Clostridium difficile are pathogenic clostridia potentially associated with gastrointestinal infections and allergy in infants. To enable the molecular detection and quantification of these species in the infant gut, two 16S rRNA oligonucleotide probes were developed: Cdif198 for C. difficile and Cperf191 for C. perfringens. We defined the probes in silico using the RDP sequence database. The probes were then validated using FISH combined with flow cytometry and a collection of target and non-target strains, and faecal samples inoculated with dilutions of C. difficile and C. perfringens strains. These new probes were used to assess the composition of the intestinal microbiota of 33 infants of 1.5 to 18.5 months of age, associated with a panel of 8 probes targeting the predominant faecal bacterial groups of humans. The probes designed allowed detection and quantification of the relative proportions of C. difficile (0.5+/-1.0%) and C. perfringens (2.1+/-2.3%) in the microbiota of infants.

Clostridioides difficile↗