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PubMed2004

[An outbreak of diarrheal disease caused by enterotoxigenic Clostridium perfringens following exposure to a contaminated environment in a nursing home].

We herein report an outbreak of non-food-borne diarrhea which occurred in a nursing home due to enterotoxigenic Clostridium perfringens. The regional public health center in Gifu, Japan, recognized 7 patients with diarrhea in a nursing home, suspecting a food-borne illness. Bacteriological and epidemiological studies indicated that enterotoxigenic C. perfringens was the causative agent. However, suspected foods, the kitchen and the cooks carried no enteropathogenic bacteria, indicating that this outbreak was a non-food-borne diarrhea. The swab specimens obtained from the residential area of the nursing home were found to have enterotoxigenic C. perfringens. Isolates from the stool specimens of patients and environment were all serotype TW47, showing susceptibilities to ampicillin, levofloxacin, and clindamycin very similar to each other, and had banding patterns identical to each other by pulsed-field gel electrophoresis. These results strongly supported the existence of monoclonal spread of an enterotoxigenic C. perfringens among the environment of the nursing home and the residents. During 3 weeks 14 residents were involved in this outbreak. The extensive effort of keeping the residential area clean led to a prompt cease of this outbreak.

Aged↗
PubMed1993

Trypsin-dependent production of an antibacterial substance by a human Peptostreptococcus strain in gnotobiotic rats and in vitro.

An antibacterial substance appeared within 1 day in feces of gnotobiotic rats harboring a human intestinal Peptostreptococcus strain. It disappeared when the rat bile-pancreatic duct was ligatured or when the rats ingested a trypsin inhibitor. Anaerobic cultures of the Peptostreptococcus strain in a medium supplemented with trypsin also exhibited an antibacterial activity, which was also inhibited by the trypsin inhibitor. In vitro the antibacterial substance from both feces and culture medium was active against several gram-positive bacteria, including other Peptostreptococcus spp., potentially pathogenic Clostridium spp. such as C. perfringens, C. difficile, C. butyricum, C. septicum, and C. sordellii, Eubacterium spp., Bifidobacterium spp., and Bacillus spp. Whatever the order of inoculation of the strains, a sensitive strain of C. perfringens was eliminated within 1 day from the intestine of rats monoassociated with the Peptostreptococcus strain. These findings demonstrate for the first time that very potent antibacterial substances can be produced through a mechanism involving intestinal bacteria and exocrine pancreatic secretions.

Animals↗
PubMed1996

Sporulation-promoting ability of Clostridium perfringens culture fluids.

The culture supernatant fluids (CSFs) of 12 strains of Clostridium perfringens types A, B, C, and D stimulated sporulation of test strains NCTC 8238 and NCTC 8449 of this organism. The sporulation-promoting ability was present in vegetative and sporulating CSFs of both enterotoxin-positive (Ent+) and Ent- strains. The sporulation factor possessed a molecular weight between 1,000 and 5,000 and was heat and acid stable. This study suggests a potential role for Ent- strains in food-borne disease outbreaks caused by Ent+ strains of C. perfringens type A.

Clostridium Infections↗
PubMed1997

Ribotyping for strain characterization of Clostridium perfringens isolates from food poisoning cases and outbreaks.

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases.

Animals↗
PubMed2000

Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes.

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.

Blotting, Western↗
PubMed2001

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs.

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

Abattoirs↗
PubMed2001

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

Animals↗
PubMed2005

Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR.

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

Animals↗
PubMed1990

Toxigenic clostridia.

Toxigenic clostridia belonging to 13 recognized species are discussed in this review. Each species or group of organisms is, in general, introduced by presenting the historical aspects of its discovery by early investigators of human and animal diseases. The diseases caused by each species or group are described and usually discussed in relation to the toxins involved in the pathology. Morphological and physiological characteristics of the organisms are described. Finally, the toxins produced by each organism are listed, with a presentation of their biological activities and physical and biochemical characteristics. The complete amino acid sequences for some are known, and some of the genes have been cloned. The term toxin is used loosely to include the various antigenic protein products of these organisms with biological and serological activities which have served as distinguishing characteristics for differentiation and classification. Some of these factors are not truly toxic and have no known role in pathogenicity. Some of the interesting factors common to more than one species or group are the following: neurotoxins, lethal toxins, lecithinases, oxygen-labile hemolysins, binary toxins, and ADP-ribosyltransferases. Problems in bacterial nomenclature and designation of biologically active factors are noted.

Animals↗
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↗
PubMed1982

Quantitation of binding and subcellular distribution of Clostridium perfringens enterotoxin in rat liver cells.

Binding of enterotoxin from Clostridium perfringens type A was studied in suspensions of parenchymal and nonparenchymal cells from rat liver. In hepatocytes, 1.5 X 10(6) specific binding sites per cell with an association constant of 3.2 X 10(6) M-1 were found. About 1% of the added toxin was nonspecifically bound to the hepatocytes. At concentrations of toxin below 0.1 micrograms/ml, 80% of the toxin density of 7 X 10(6) cells per ml. Binding did not increase after the cells became permeable to the toxin. Subcellular fractionation in a sucrose gradient produced no evidence for binding to parts of the cell other than the plasma membrane. The degree of binding to nonparenchymal cells was less than 10% of the binding to hepatocytes.

Animals↗