Strains Of Clostridium Perfringens Research Articles

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).

Cloning of tetracycline-resistance genes from various strains of Clostridium perfringens and expression in Escherichia coli.

One hundred strains of Clostridium perfringens and 52 strains of other clostridia of human and animal origins were screened for tetracycline resistance. Fifty-six strains were resistant to tetracycline in the C. perfringens group. Ten strains were selected for their high level of resistance. In all of them, the tetracycline-resistance genes were found to be residing in large plasmids of about 50 kb, all showing homologies. Several tetracycline-resistance genes from plasmids of various strains of C. perfringens were cloned in plasmid pUC19 and the resistance was expressed in Escherichia coli. Hybridization analysis showed these genes to be homologous among themselves and also to tetP gene from the PCW3-type plasmid.

An outbreak of enterotoxaemia in cats.

Eighteen local breed cats suffered from enterotoxaemia after scavenging on chicken-remnants. They suffered from vomitus and diarrhoea and collapsed dead after a few hours. An untypable strain of Clostridium perfringens was isolated in pure form and was suspected to be the cause of the outbreak.

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.

Growth, sporulation and enterotoxin production by Clostridium perfringens type A in the presence of human bile salts

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.

Growth, sporulation and enterotoxin production byClostridium perfringenstype A in the presence of human bile salts

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.

The presence of enterotoxigenic Clostridium perfringens strains in faeces of various animals

The presence of Clostridium perfringens in faeces of horses, cattle, poultry and pigs was determined. C. perfringers was detected in 24%, 36%, 80% and 2% of the faecal samples, respectively. Faecal samples containing enteroxigenic strains as assessed by colony hybridization amounted to 14%, 22%, 10% and 0% respectively.

Description d’un protocole pour l’isolement et l’identification de <em>Clostridium perfringens</em> et des bactéries apparentées

The properties of 220 strains of Clostridium perfringens and Clostridium perfringens-like organisms were studied. A scheme was designed for the identification of these strains. The scheme was based on the presence/or absence of lecithinase enzyme, synergestic haemolysis with Streptococcus group B toxin, their inhibition with appropriate antisera and reaction in the lactose gelatin nitrate motility test (LGNM) with the fermentation of a few sugars.

Alpha Toxin Production by Enterotoxin-Producing Strains of Clostridium perfringens Derived from Patients of Food Poisoning Outbreaks, Food Handlers and Foods

Alpha toxin is produced by C. perfringens and forms a white muddy zone on egg yolk-CW agar. It is known that the amount of α-toxin produced varies from strain to strain. Four hundred and thirty-six strains derived from patients of food poiosning outbreaks, food handlers and foods were therefore tested for enterotoxin and α-toxin production.1. Duncan and Strong medium and fructose-chopped meat medium were equally effective for enterotoxin production and sporulation of strains of C. perfringens.2. The majority of enterotoxin-producing strains derived from patients of food poisoning outbreaks and food handlers produced small amounts of α-toxin (less than 0.05 unit). However, the amounts of α-toxin produced by enterotoxin-producing strains derived from foods varied from strain to strain, and ranged from less than 0.05 to 1.5 units.3. The amounts of α-toxin produced by enterotoxin-nonproducing strains derived from patients of food poisoning outbreaks, food handlers and food varied, ranging from less than 0.05 to 1.5 units.4. The amounts of α-toxin produced in fructose-chopped meat medium were not closely related to the size of the white muddy zone around the colonies grown on egg yolk-CW agar. Enterotoxin-producing strains derived from seven outbreaks of food poisoning formed white muddy zones of 3 to 5mm around colonies grown on egg yolk-CW agar.

Gnotobiotic, athymic mice; a possible system for the study of the role of bacteria in human amoebiasis

Two groups of 12 + 14 gnotobiotic, athymic mice were intracaecally injected with Entamoeba histolytica strain HK9 and NIH:200, respectively. Two groups of 16 and 15 mice were given amoebae together with a pure strain of Escherichia coli and a further two groups of 16 and 27 were given amoebae with a pure strain of Clostridium perfringens. Batches of 3-7 mice from each group were killed at intervals of 1-4 weeks. All the mice given NIH:200 alone were found to be infected with trophozoites. Of those given HK9 alone, 20% of the first and 57% of the second group to be examined were infected. Groups of mice given either strain of amoeba monocontaminated with E. coli were all found to be infected at post-mortem examination with no apparent clinical signs and little histological change. The group given HK9 and C. perfringens, although all were infected, failed to produce clinical signs or histological lesions, though some died expectedly. In the group given NIH:200 with C. perfringens the amoebae showed a change of activity and there was evidence of both caecal and liver lesions after 120 days. The usefulness of the system in studying the effect of individual species of bacteria on invasive amoebae is discussed.

A54145 a new lipopeptide antibiotic complex. Microbiological evaluation.

A54145 complex is made up of eight factors; A, A1, B, B1, C, D, E, and F which were active in vitro (MIC 0.25 approximately greater than 32 micrograms/ml) against Gram-positive aerobic organisms. The complex, factor B and B1 were found to be active against two strains of Clostridium perfringens. A calcium dependence study on some of the factors showed that their in vitro antibacterial activity was greatly enhanced by the presence of calcium (50 mg/liter) in the media. Resistance build-up was seen when Staphylococcus sp. and Streptococcus sp. were passed seven times in the presence of sublethal concentrations of A54145 antibiotics. This resistance disappeared immediately when the resistant organisms were passed in the absence of the antibiotics. Factor A was very effective against Staphylococcus aureus and Streptococcus pyogenes infections in mice (sc ED50s of 3.3 approximately 2.4 mg/kg x 2, respectively). Factor B was more active against S. pyogenes in vivo (sc ED50, 0.9 mg/kg x 2). Acute mouse toxicities were determined with these antibiotics. Semisynthetic derivatives were evaluated.

Comparative in vitro Bactericidal Activity of 24 Antimicrobial Drugs against <i>Clostridium perfringens</i>

Twenty-four antimicrobial drugs were examined for rapidity of onset and magnitude of bactericidal activity against selected strains of Clostridium perfringens. Ceftriaxone, imipenem, metronidazole, mezlocillin, penicillin G, piperacillin, and teicoplanin reduced colony counts by at least 3 log10 units within 2-4 h after exposure. Clindamycin, fluoroquinolones, josamycin, and tetracycline caused delayed kill (greater than or equal to 99.9% reduction of viable counts at 4-22 h after exposure). Chloramphenicol and rifampin lacked bactericidal activity against 2 of 4 strains, whereas erythromycin, fusidic acid, and fosfomycin (with added glucose-6-phosphate) were merely inhibitory for all 4 strains. Imipenem and penicillin G were combined with 9 and 12 antimicrobial drugs, respectively. Essentially all drug combinations yielded indifferent effects; only penicillin G plus doxycycline resulted in an antagonistic effect against C. perfringens.

Lysogenic phages ofClosrtidium perfringens: mapping of the chromosomal attachment sites

The sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of Clostridium perfringens strain CPN50 using two techniques based on pulsed field gel electrophoresis. Phage phi 29 was mapped to the 1 Mb region of the 3.6 Mb genome, near nanH which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 Mb.

Synthetic DNA probes for detection of enterotoxigenic Clostridium perfringens strains isolated from outbreaks of food poisoning

Four synthetic oligonucleotides encoding different parts of the Clostridium perfringens enterotoxin gene were used to test the enterotoxigenicity of C. perfringens strains isolated from confirmed outbreaks of food poisoning. Of the 245 strains isolated from food and feces originating from 186 separate outbreaks, 145 (59%) gave hybridization reactions with each of the four DNA probes used, while 104 strains did not hybridize with any of the probes. There was no correlation between serotype and the presence of the enterotoxin gene, although the C. perfringens enterotoxin gene was rarely detected among nontypable strains (17%). Results show that DNA hybridization is a suitable method for the identification of C. perfringens strains which have the potential to produce enterotoxin.

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.

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.

Analysis of plasmid profiling as a method for rapid differentiation of food‐associated Clostridium perfringens strains

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

Antagonism exerted by an association of a Bacteroides thetaiotaomicron strain and a Fusobacterium necrogenes strain against Clostridium perfringens in gnotobiotic mice and in fecal suspensions incubated in vitro

Antagonism between an association of Bacteroides thetaiotaomicron and Fusobacterium necrogenes strains and two strains of Clostridium perfringens was evidenced both in vivo in gnotobiotic mice and ex vivo in fecal suspensions incubated for 22 h at 37 degrees C. Several features of this antagonism were similar in and ex vivo. (i) An obligate and continuous synergy between B. thetaiotaomicron and F. necrogenes was required; (ii) the two C. perfringens strains did not respond to the same extent to this antagonism; and (iii) expression of the antagonism was host and diet dependent. Neither diffusible nor soluble inhibitory substances were detectable in feces of gnotobiotic mice, nor could depletion of nutrients be identified as causing antagonism in both in and ex vivo experiments. Our findings support the hypothesis that a reversible bacteriostasis induced by the inhibitory strains acting together continuously, and hindering the target strain from utilizing available nutrients, was responsible for this antagonism.

The fluorescence of Clostridium perfringens by biosynthesis of porphyrins and demetallation of haem

All 100 strains of Clostridium perfringens studied showed porphyrin fluorescence due to synthesis on a special medium. A majority (84%) of the same strains were capable of porphyrin fluorescence by the removal of iron from haem.

Increased Sporulation of Clostridium perfringens in a Medium Prepared with the Prereduced Anaerobically Sterilized Technique or with Carbon Dioxide or Carbonate

Sporulation of ten strains of Clostridium perfringens was determined in Duncan-Strong sporulation medium prepared and stored in the presence of atmospheric oxygen (DS) or prepared as a prereduced anaerobically sterilized medium (DSPR). Numbers of heat-resistant spores were higher in DSPR compared to DS for 4 strains in fresh media and for 7 strains in stored media. For strains 8679 and 8799, spore numbers were as much as 5 logs greater in DSPR than in DS with a concomitant higher culture pH and fewer vegetative cell numbers. The effects of the addition of an antioxidant, degraders of hydrogen peroxide (H2O2), or scavengers of oxygen radicals to DS or H2O2 to DSPR do not suggest a role for toxic oxygen species in inhibiting sporulation of these two strains in DS. Increases in spore numbers and culture pH were similar in DSPR and in DS to which carbon dioxide (CO2) or sodium carbonate had been added. Modifications of sporulation media which increased the levels of CO2 or increased the culture pH improved sporulation of strains 8679 and 8799, respectively. These results suggest that improved sporulation in DSPR resulted from an improved buffering effect on culture pH or a direct effect of CO2 introduced during preparation of the prereduced medium.