Clostridium perfringens in the Environment

Clostridium perfringens was isolated from samples collected in Puget Sound in the state of Washington and areas considered as possible sources of these organisms to Puget Sound. The distribution of C. perfringens in the total Clostridium population was determined for fish gut contents and sediments collected in highly polluted and less polluted areas, sewage samples, freshwater sediments, and soils. The greatest numbers of C. perfringens were obtained from marine sediments collected near the sewage outfall at West Point. Fewer isolates were made from fish collected from less polluted stations, although the number of C. perfringens remained high in sediments from other Puget Sound stations. The proportion of C. perfringens in the total Clostridium populations varied between 56 and 71% for sewage samples and only 0.4 to 4.1% for freshwater sediments and soil samples. Only 25 C. perfringens isolates out of 137 from fish guts, or 18%, were identifiable serologically and these fell into 12 groups. C. perfringens were fed to fish and the fish were sacrificed after varying lengths of time. The number of C. perfringens increased slightly in the gut during the first 24 h and then the numbers decreased rapidly for the next 120 h.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.     

Nutrient cycling and productivity in a desert saline lake: observations from a dry,low-productivity year

Seasonal variability of nutrients and productivity were examined in Pyramid Lake, a hyposaline, N-deficient, terminal desert lake, during a dry period. River inflow and N-fixation during 1990 were minimal allowing internal nutrient cycling to be more closely studied. Nutrient cycling was strongly affected by seasonal thermal stratification that was typical for a warm monomictic lake. Concentrations of nitrate, phosphate, and silicate in surface waters were highest during winter mixing and decreased rapidly in the spring due to a diatom bloom. Maximum average chlorophyll concentration in surface waters was 2.7 ± 1.2 µg 1^–1 and occurred in April while surface nutrients were being depleted. In contrast to chlorophyll, maximum particulate carbon in surface waters occurred in July–August when areal productivity was highest (367–398 mg C m^–2 day^–1). Concurrent with spring nutrient depletion in surface waters was increasing N-deficiency in the plankton. After the spring bloom dissipated in May, particulate matter (POM) became increasingly N-deficient reaching maximum elemental C : N of > 18 during summer-fall. Profiles of the C : N ratio of POM were nearly constant with depth for individual sampling dates suggesting that the residence time of POM in the water column was short (< 1 month). While surface waters were nutrient depleted during summer stratification, nutrient concentrations of bottom waters progressively increased, presumably through the oxidation of POM sinking to the bottom (103 m). Converting the rate of oxygen depletion in bottom waters to carbon equivalents of POM suggests that 42 % of mean annual phytoplankton production in overlying waters during 1990 was mineralized in bottom waters.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal.

Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site. C. perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca. 48 km) and 50 nautical miles (ca. 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site. C. perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site. At stations where C. perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm. In some cases, C. perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation. We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media

In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method. Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work. The C. perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media. No statistically significant differences were found between C. perfringens spore counts on TSCF compared with those of other methods used. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C. perfringens spore recovery in groundwater samples. Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C. perfringens spores from groundwater samples.     

ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in feces.

As the second phase of an international comparative study for the enumeration of Clostridium perfringens, four methods were compared for "total" and spore counts of C. perfringens in fecal specimens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserine) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. In both the total and spore count procedures, the confirmed C. perfringens counts in method D were lower than in methods A, B, and C. Little differences among methods were found in the percentages of presumptive colonies confirmed as C. perfringens. The nonspecific counts in methods A and D were generally greater than in B and C, but nonspecific microorganisms did not interfere in the enumeration of C. perfringens spores by any of the four methods. In overall performance, methods B and C were superior to A and D. The mean C. perfringens spore count was only 0.17 log lower than the mean total count. Spore counts alone are, therefore, adequate in investigations of C. perfringens outbreaks.     

Investigation of the survival characteristics of Rhodococcus coprophilus and certain fecal indicator bacteria.

Rhodococcus coprophilus and Clostridium perfringens survived in fresh water samples held at 5, 20, and 30 degrees C for over 17 weeks, whereas Escherichia coli and fecal streptococci disappeared after 5 weeks at all three temperatures. R. coprophilus survived for more than 8 months in sterilized sewage and deionized water at all three temperatures, whereas in normal sewage held at 20 degrees C, the survival time was 12 to 26 weeks. In samples held at 30 degrees C, survival times were shorter, probably because of interbacterial competition or protozoal predation. The results indicate that R. coprophilus may be a useful indicator of the presence of remote fecal pollution of farm animal origin, but not of recent pollution, when enumerated alone in polluted waters or wastewaters.     

Activation and injury of Clostridium perfringens spores by alcohols

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Activation and injury of Clostridium perfringens spores by alcohols.

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Enumeration of Fecal Clostridium perfringens Spores in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin.     

Coat and enterotoxin-related proteins in Clostridium perfringens spores

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.     

EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5–15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis , and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens , Clostridium botulinum and Clostridium tetani , has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces ≤ 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli , and this protein catalysed DPA formation in vitro . The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.     

Detection and quantification of four species of the genus Clostridium in infant feces

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.     

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.     

Effects of periodic flooding on the water chemistry and primary production of the Mapire systems (Venezuela)

The Mapire river mouth forms a complex floodplain system, where the river behaves as a river during the dry season, but changes to a transient lake which partially covers the inundation forest during the rainy season. Thus, we expected changes in water chemistry and a gradual increase of primary production during high waters. The system was sampled monthly for one year; two floodplain lakes were also studied for comparative purposes. Variations in the concentration of macro- and micronutrients occurred in a pulse-like manner and seemed to relate to mechanisms at work in the transient lake. Dissolved oxygen showed a stratification with low values at the bottom, but never reached anoxia. Net and gross primary production and respiration did not show any clear spatial pattern, reflecting a mosaic of different biochemical states within the transient lake. Heterotrophy tended to prevail in the transient lake, while autotrophy dominated floodplain lakes.     

Spore coat protein and enterotoxin synthesis in Clostridium perfringens.

Polyacrylamide gel profiles of Clostridium perfringens spore coat protein revealed four and occasionally five components. Pulse-chase experiments indicated that synthesis of coat protein polypeptide and enterotoxin was an early sporulation event. However, maximum synthesis occurred coincident with the onset of heat resistance.     

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).     

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).