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PubMed2003

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

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.

Ampicillin↗
PubMed2001

Isolation of Clostridium innocuum from cases of recurrent diarrhea in patients with prior Clostridium difficile associated diarrhea.

Clostridium innocuum isolates resistant to vancomycin (MIC values of 16-24 microg/mL) were isolated from three patients with recurrent Clostridium difficile -associated diarrhea (CDAD). We discuss the clinical significance and problems associated with the identification and differentiation of these two clostridial species, which may result in misdiagnosis of patients.

Clostridioides difficile↗
PubMed1993

ADP-ribosylation of Drosophila indirect-flight-muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.

Purified Drosophila indirect-flight-muscle actin and arthrin, an actin-ubiquitin conjugate, were ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Phalloidin treatment inhibited the ADP-ribosylation of Drosophila actin and arthrin. Like actin, the ADP-ribose-arthrin linkage was sensitive towards hydroxylamine treatment, indicating arginine as the amino acid acceptor. Actin translated in vitro from the indirect-flight-muscle-specific gene Act88F was ADP-ribosylated by C. botulinum C2 toxin and C. perfringens iota toxin. Actin from the R177Q mutant of Act88F translated in vivo was not ADP-ribosylated confirming Arg-177 as the ADP-ribose acceptor. Mutant L176M actin was modified by both toxins, indicating that amino acid 176 of actin does not define the substrate specificity of C. botulinum C2 toxin. Whereas the gene products of various C-terminal mutants of Act88F translated in vitro (E334K, V339I, E364K, G368E, R372H) were substrates for ADP-ribosylation by C. botulinum C2 toxin and by C. perfringens iota toxin, neither toxin modified the N-terminal O-12 deletion mutant.

ADP Ribose Transferases↗
PubMed2001

Amplification of the 16S-23S rDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum.

Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.

Animals↗
PubMed2003

Detection of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin in cases of diarrhoea in the community.

Faecal specimens from 843 cases of diarrhoea in the community were tested for the presence of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin. C. difficile cytotoxin was detected in faecal specimens from 0.6 % of cases aged at least 2 years by using a Vero cell assay. Factors associated with detection of C. difficile cytotoxin were antibiotic therapy, age over 60 years and living in a home with other elderly people. Three methods were used for the detection of C. perfringens enterotoxin: a Vero cell assay, a commercial (TechLab) enzyme immunoassay (EIA) and an in-house EIA. The lower level of detection of pure C. perfringens enterotoxin in buffer was 0.01 micro g ml(-1) by the TechLab EIA and 1.0 micro g ml(-1) by the Vero cell assay. C. perfringens enterotoxin was detected by using the TechLab EIA in faecal specimens from 2.5 % of cases. This commercial EIA was less sensitive than the in-house EIA, detecting only 31 % of positive cases, but was specific and could be used for outbreak investigation by routine diagnostic laboratories. Age over 60 years was a factor associated with C. perfringens enterotoxin detection; this age group may be targeted for testing.

Adolescent↗
PubMed1982

The effect of oxidation-reduction potential on spore germination, outgrowth, and vegetative growth of Clostridium tetani, Clostridium butyricum, and Bacillus subtilis.

Spores of Clostridium tetani germinated in liver broth with +580 mV as the starting Eh value, and those of Clostridium butyricum germinated in liver broth with an initial Eh of +400 mV regardless of the presence or absence of a liquid paraffin covering. Spores of Bacillus subtilis germinated in liver broth with--100 mV as the starting Eh value. Also, it was found that there are two ranges of starting Eh values for germination and vegetative growth of Cl. tetani, Cl. butyricum, and B. subtilis. In the first range these spores germinated and grew, but in the second range they only germinated and then died without outgrowth.

Bacillus subtilis↗
PubMed1992

Cloning and whole nucleotide sequence of the gene for the light chain component of botulinum type E toxin from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike.

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.

Amino Acid Sequence↗
PubMed1992

[Distribution of Clostridium botulinum and Clostridium tetani in Okinawa Prefecture].

Soil samples from the sugar cane fields and muscovado samples from the manufacturing processes at sugar manufacturies in many parts of Okinawa Prefecture were collected, mainly in 1988 and 1989, and examined for Clostridium botulinum and Clostridium tetani. Of 290 soil samples 21 (7.2%) were positive for C. botulinum. Four (1.4%) and 17 (5.9%) contained types E and C respectively. C. botulinum type E was demonstrated in the North and South areas of Okinawa main island. Type A and B were not demonstrated from soil samples in Okinawa Prefecture. Of 53 samples of the manufacturing processes of muscovado 5 (9.4%) were positive for C. botulinum type C. Type A, B and E were not found from muscovado samples in Okinawa Prefecture. Of 290 soil samples 54 (18.6%) were positive for C. tetani. Among the 53 muscovado samples from the manufacturing processes of the sugar cane, 10 (18.9%) were positive for C. tetani.

Botulinum Toxins↗
PubMed1989

Hybridization analysis of three chloramphenicol resistance determinants from Clostridium perfringens and Clostridium difficile.

The chloramphenicol resistance determinant from a nonconjugative strain of Clostridium perfringens was cloned and shown to be expressed in Escherichia coli. Subcloning and deletion analysis localized the resistance gene, catQ, to within a 1.25-kilobase (kb) partial Sau3A fragment. The catQ gene contained internal HindII, HaeIII, and DraI restriction sites and was distinct from the catP gene, which was originally cloned (L. J. Abraham, A. J. Wales, and J. I. Rood Plasmid 14:37-46, 1985) from the conjugative C. perfringens R plasmid, pIP401. Hybridization studies were carried out with a 0.35-kb DraI-P fragment of pJIR260 as an internal catQ-specific probe and a 0.38-kb EcoRV-HinfI fragment of pJIR62 as an internal catP-specific gene probe. The results showed that the catP and catQ genes were not similar and that neither probe hybridized with cat genes from other bacterial genera. However, the catP gene was similar to the cloned catD gene from Clostridium difficile. Comparative studies with both catP and catD probes showed that these genes had significant restriction identity. We therefore suggest that these genes were derived from a common source.

Blotting, Southern↗
PubMed2005

Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum.

Clostridium acetobutylicum and Clostridium aminovalericum, both obligatory anaerobes, grow normally after growth conditions are changed from anoxic to microoxic, where the cells consume oxygen proficiently. In C. aminovalericum, a gene encoding a previously characterized H2O-forming NADH oxidase, designated noxA, was cloned and sequenced. The expression of noxA was strongly upregulated within 10 min after the growth conditions were altered to a microoxic state, indicating that C. aminovalericum NoxA is involved in oxygen metabolism. In C. acetobutylicum, genes suggested to be involved in oxygen metabolism and genes for reactive oxygen species (ROS) scavenging were chosen from the genome database. Although no clear orthologue of C. aminovalericum NoxA was found, Northern blot analysis identified many O2-responsive genes (e.g., a gene cluster [CAC2448 to CAC2452] encoding an NADH rubredoxin oxidoreductase-A-type flavoprotein-desulfoferrodoxin homologue-MerR family-like protein-flavodoxin, an operon [CAC1547 to CAC1549] encoding a thioredoxin-thioredoxin reductase-glutathione peroxidase-like protein, an operon [CAC1570 and CAC1571] encoding two glutathione peroxidase-like proteins, and genes encoding thiol peroxidase, bacterioferritin comigratory proteins, and superoxide dismutase) whose expression was quickly and synchronously upregulated within 10 min after flushing with 5% O2. The corresponding enzyme activities, such as NAD(P)H-dependent peroxide (H2O2 and alkyl hydroperoxides) reductase, were highly induced, indicating that microoxic growth of C. acetobutylicum is associated with the expression of a number of genes for oxygen metabolism and ROS scavenging.

Acclimatization↗
PubMed1974

Effect of potassium sorbate on salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in cooked, uncured sausage.

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.

Anti-Bacterial Agents↗
PubMed1989

Cloning and expression of the phospholipase C gene from Clostridium perfringens and Clostridium bifermentans.

The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined. It was found that the structural gene codes for a protein of 399 amino acid residues. The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion. Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space. Phospholipase C purified from E. coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously. The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated. The two phospholipase C genes were found to be 64% homologous in coding sequence. The C. bifermentans protein, however, was 50-fold less active enzymatically than the C. perfringens enzyme.

Amino Acid Sequence↗
PubMed1995

Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi.

The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported. A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes. Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C. perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al. (B. Canard, B. Saint-Joanis, and S. T. Cole, Mol. Microbiol. 6:1421-1429, 1992). Various C. perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned. All of the enzymes showed the same specific activity. On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C. perfringens strain to another. No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes. These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region. The evolution of C. perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes.

Amino Acid Sequence↗
PubMed1976

Electron capture gas chromatography study of the acid and alcohol products of Clostridium septicum and Clostridium chauvoei.

The metabolic products produced by several strains of Clostridium septicum obtained from patients and animals, along with strains of Clostridium chauvoei, were studied in chopped meat glucose medium by electron capture gas-liquid chromatography (EC-GLC). The strains of C. septicum and C. chauvoei were shown to comprise five different metabolic groups. Both the EC-GLC study and the O and H antigenic study performed previously showed that strains of C. septicum comprise a heterogeneous group. One type of metabolic profile was found only in strains of C. chauvoei. The O antigen types and EC-GLC metabolic types of C. septicum correlated fairly well in isolates from cancer patients but not in stock culture and animal isolates.

Alcohols↗
PubMed1994

Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms.

We studied the effectiveness of the PCR in detecting the type A, B, and E botulism neurotoxin genes in 209 strains of Clostridium botulinum and 29 strains of other Clostridium spp. All 79 strains that produced type A toxin, 77 strains that produced type B toxin, and 51 organisms that produced type E toxin (46 C. botulinum and 5 C. butyricum) were PCR positive in reactions with primers targeting sequences specific for their respective toxin genes. The PCR for type A toxin was positive for one type B toxin-producing strain that produced a small amount of type A toxin in addition to a large amount of type B toxin. Surprisingly, the type B toxin gene was detected in addition to the type A toxin gene in 43 type A toxin-producing strains, only 1 of which could be shown by bioassay to produce biologically active type B toxin in culture. The type B gene was also detected in two strains of C. subterminale, which were determined to be nontoxigenic by bioassay. While the PCR was sensitive and specific in detecting the neurotoxin genes, the discovery of unexpressed toxin genes indicates that PCR results may not be adequate for establishing type B neurotoxigenicity.

Base Sequence↗
PubMedDate not supplied

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs.

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.

Animals↗
PubMed1999

Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic.

OBJECTIVE: To determine prevalence of clostridial enterotoxins in feces of horses with diarrhea and colic, and to determine whether an association exists between detection of clostridial enterotoxins in feces and development of diarrhea as a complication of colic. DESIGN: Prospective case series and case-control study. ANIMALS: 174 horses with diarrhea, colic, or problems not related to the gastrointestinal tract. PROCEDURE: Horses were assigned to 1 of 4 groups: colic with diarrhea (group 1; n = 30); colic without diarrhea (group 2; 30); diarrhea without colic (group 3; 57); and control (group 4; 57). Feces were evaluated by use of ELISA to detect Clostridium perfringens enterotoxin (CPE) and C difficile toxin A (TOXA). Frequency of detection of CPE or TOXA in groups 1 and 3 was compared with that in groups 2 and 4, respectively. RESULTS: Prevalence of enteric clostridiosis in horses in group 3 was 25%. Clostridium perfringens enterotoxin was detected in 9 of 57 (16%), TOXA in 8 of 57 (14%), and both toxins in 3 of 57 (5%) fecal samples collected from these horses. Neither toxin was detected in feces of the age-matched horses in group 4. Clostridial enterotoxins were detected in feces of 7 of 60 (12%) horses with colic (groups 1 and 2), however, a significant association was not found between detection of enterotoxins in feces and development of diarrhea as a complication of colic. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridia are important etiologic agents of diarrhea in horses. Additionally, changes in intestinal flora of horses with colic may allow for proliferation of clostridia and elaboration of enterotoxins regardless of whether diarrhea develops.

Animals↗
PubMed1995

Sequencing and analysis of the gene encoding the alpha-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile.

A library of total Clostridium novyi DNA was established and screened for the alpha-toxin gene (tcn alpha) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcn alpha fragments were isolated and subsequently the total sequence of tcn alpha was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M(r) 250,166 and pI 5.9. The N-terminal alpha-toxin (Tcn alpha) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcn alpha and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcn alpha constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19-52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.

Amino Acid Sequence↗