Strains Of Clostridium Perfringens Research Articles

Gas-Liquid Chromatographic Determination of Propionic Acid Production Differentiates between the Food-Poisoning Strains and Toxigenic-Type Strains of Clostridium perfringens

Fatty acids of 32 strains belonging to five types of Clostridium perfringens were extracted from the supernatant liquid of cultures in various different media prepared with or without the addition of sugar and examined by application of the gas-liquid chromatographic technique. The major products in nearly all media were acetic, propionic and butyric acid among the volatile fatty acids and only lactic acid among the nonvolatile fatty acids. Quantitative comparison of the resulting chromatograms with regard to the presence and relative amounts of propionic acid as one of the large major peaks revealed clear differences between the toxigenic types and the food-poisoning strains of this organism. In cooked meat medium containing 2% fructose, all the toxigenic-type strains produced propionic acid, but the food-poisoning strains tested entirely lacked production of this acid. This differentiation characteristic may be useful in routine laboratory bacteriology.

Recovery of Clostridium perfringens from Food Samples Using an Oxygen-Reducing Membrane Fraction

Three strains of Clostridium perfringens were inoculated into different food products and their recovery rates on tryptose sulfite cycloserine (TSC) agar, with and without an oxygen-reducing membrane fraction (ORMF), were compared. Organisms were generally recovered in greater numbers using TSC+ORMF and aerobic incubation than with TSC and anaerobic incubation. Organisms inoculated into beef stew were subjected to heat and cold stress for various periods. In all instances, presence of ORMF in TSC and aerobic incubation resulted in greater recovery of viable C. perfringens than did TSC and anaerobic incubation. Of 35 uninoculated raw meat samples evaluated, C. perfringens was recovered from 22 using TSC+ORMF compared to 18 using TSC alone.

A Sporadic Case of Diarrhoea due to EnterotoxigenicClostridium perfringens

Enterotoxigenic strains of Clostridium perfringens have been recently implicated in some cases of antibiotic associated diarrhoea, especially in hospitalised elderly patients. We present a case of diarrhoea associated with enterotoxigenic C. perfringens in a young adult not implicated in an episode of food poisoning and who did not receive antimicrobial therapy. Keywords: Diarrhoea; Clostridium perfringens ; enterotoxin.

Nonimmune binding of Ig to Clostridium perfringens. Preferential binding of IgM and aggregated IgG.

In an attempt to find a bacterial IgM receptor, a large number of bacterial strains of different species were screened for the ability to bind human IgM. Certain strains of the anaerobic bacterium Clostridium perfringens were found to bind a major fraction of polyclonal IgM. One bacterial strain showed a particularly high binding capacity and was studied in more detail. This strain is also able to bind a minor fraction of polyclonal IgA and IgG. Inhibition experiments indicate that the different Ig classes bind to one and the same R structure. The ability of the strain to bind polyclonal Ig is correlated to the number of subunits in the Ig. This correlation can most simply be explained by increasing avidity with increasing number of subunits. In agreement with this hypotheses, experiments with aggregated IgG show that binding ability increases with aggregate size. Experiments with Ig fragments indicate that the binding structure in Ig is located in the F(ab')2 region. The ability of this bacterial strain to bind a majority of IgM molecules as well as aggregated IgG is potentially useful in immunologic work and represents a new type of Ig binding to bacteria.

A Sporadic Case of Diarrhoea due to EnterotoxigenicClostridium perfringens

Enterotoxigenic strains of Clostridium perfringens have been recently implicated in some cases of antibiotic associated diarrhoea, especially in hospitalised elderly patients. We present a case of diarrhoea associated with enterotoxigenic C. perfringens in a young adult not implicated in an episode of food poisoning and who did not receive antimicrobial therapy.

Sensitivity of Foodborne Bacteria (Spoilage and Pathogenic) to a Methanol-Acetone Extract of Milk Fermented by Streptococcus thermophilus

Effects of an inhibitory methanol-acetone (MA) extract of Streptococcus thermophilus-fermented milk was tested on growth of Salmonella enteritidis, two strains of Staphylococcus aureus (types A and E), two strains of Clostridium perfringens (types A and C) and Pseudomonas fluorescens. Each organism was tested at three levels of the extract, e.g., 250, 500 and 1000 ppm. Results indicated that the degree of sensitivity among the test organisms varied. C. perfringens (C) was the most sensitive, with a mean % inhibition (average % inhibition over the three MA extract concentrations) of 73.3, while S. enteritidis was the least sensitive (mean % inhibition = 51.8) to the extract. The differences between the mean % inhibition of P. fluorescens (65.4), S. aureus (A) (64.8), and C. perfringens (A) (62.2) were not significant (P>0.05); however, the sensitivity of these three organisms to the extract was significantly less (P<0.05) than C. perfringens (C) but significantly greater (P<0.05) than S. aureus (E) and S. enteritidis.

Growth curves of clostridium perfringens in schaedler and brain heart infusion broths

Four Clostridium perfringens strains recovered from clinical specimens, four strains obtained from faecal specimens of healthy adults, and control strain ATCC 13124 were inoculated into Schaedler and Brain Heart Infusion broths, respectively, and incubated anaerobically at 35°C for 24 h. The exponential growth phase usually lasted from 2 to 6 h following inoculation; the generation times averaged 23 min at 2 to 4 h and 27 min between 4 and 6 h of incubation. The early stationary growth phase (generation time between 6 and 8 h = 89 min) was reached at 8 h following inoculation. All strains displayed essentially identical in vitro growth rates, even though they differed in murine virulence.

Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases.

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.

Enterotoxigenicity and Tetracycline Resistance of Clostridium perfringens Isolated from Chicken

Fifty chickens prepared for sale and 15 samples each of minced and non-minced chicken for use as raw material for processed food were collected and strains of Clostridium perfringens were isolated from them. The number of isolates was 153 and the isolation frequencies of the organism from these 3 kinds of samples were 34%, 100%, and 87%, respectively. Among these 153 isolates, there were 32 strains that were extraordinarily resistant to heat; 30 of the 32 strains were resistant to 100°C for 10 minutes, and the other 2, to 100°C for 60 minutes. However, no enterotoxigenicity was found with these 32 strains, though 1 strain was found to be enterotoxigenic by reversed passive hemagglutination tests.The MICs of tetracycline for 114 selected strains were examined by the plate dilution method. Strains resistant to 12.5μg/ml of tetracycline amounted to 84 of the 114 strains, and 53 of these 84 strains were found to be highly resistant to the drug with MICs of more than 25μg/ml. Tetracycline resistance-transfer experiments were carried out using 53 highly resistant strains as donors and 2 recipient strains which are maintained in our laboratory. Transferable tetracycline R-plasmids were detected in 41 of the 53 highly resistant strains.Tetracycline R-plasmids were extracted from these transcipients and treated with the restriction endonuclease, EcoRI. Several different restriction profiles were observed among the plasmids.

Enterotoxigenicity of heat-resistant and heat-sensitive strains of Clostridium perfringens.

Fourteen Clostridium perfringens strains including 5 heat-resistant and 9 heat-sensitive strains were tested for enterotoxigenicity comparatively using reversed passive latex-aggultination and rabbit ileal loop tests. The results obtained by both the immunological and biological methods were in good agreement. All of the 5 heat-resistant strains were enterotoxin positive, while 3 of the 9 heat-sensitive strains were enterotoxin positive. All these heat-sensitive strains, whether enterotoxigenic or nonenterotoxigenic, produced spores in vitro as well as in vivo at approximately the same frequency as did the heat-resistant strains, indicating that absence of enterotoxin was not due to inability of these strains to produce spores.

Improved Media for Sporulation and Enterotoxin Production by Clostridium perfringens

Sporulation of 24 strains of Clostridium perfringens isolated from stools of food poisoning patients and normal controls was improved by adding sodium carbonate to Duncan-Strong (DS) sporulation medium and replacing starch with raffinose in Taniguti's AEA medium. Viable counts of heat-tolerant spores (75°C for 20 min) were 2 to 186 times greater in modified AEA medium and 2 to 169 times greater in modified DS than in DS medium. Reversed passive latex agglutination assays revealed a corresponding increase in enterotoxin titers in supernatant fluids of the 12 enterotoxigenic strains grown in modified AEA medium and in modified DS medium.

The effect of pH, sodium chloride, sodium nitrite and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media

The effects of salt concentration (1–12% w/v) in combination with unheated sodium nitrite (0–400 μg/ml) on growth of mixed strains of Clostridium perfringens and of faecal streptococci at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. At pH 6.2, following storage at 15°C, 1% salt and 50 μg/ml nitrite inhibited growth of C. perfringens. At 20°C and pH 6.2, 200 μg/ml nitrite plus 3% salt, or 50 μg/ml plus 4% salt were required to inhibit growth. Growth of C. perfringens was prevented by levels of curing salts used commercially providing the pH was 6.2 or below. At pH 6.8 or above at least 4% salt and 50 μg/ml nitrite was required to prevent growth at 20°C. The faecal streptococci grew in medium containing 6% salt and 400 μg/ml nitrite irrespective of pH or storage temperature. In 8% salt growth was prevented by storing at or below 17.5°C or, if pH was 6.2 or lower, by adding 200 μg/ml nitrite irrespective of storage temperature. Growth of faecal streptococci was not controlled by concentrations of curing salts which would be acceptable in meat products.

Toxin-types of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria

Two-hundred and forty-five strains of Clostridium perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments (paddock soils) in Kano and Kaduna States of Nigeria were studied. The isolates were examined by the toxin-antitoxin neutralisation tests performed intradermally in depilated albino guinea-pigs. One-hundred and twenty-seven (53.1%) of the isolates were type A, 17 (7.1%) were type B, 14 (4.9%) were type C, 44 (18.4%) were type D and 19 (7.9%) were type E. Eighteen (7.5%) were not typable while six were lost during storage. The significance of this distribution and the potential danger to animal health, especially as regards enterotoxaemias, cannot be over-emphasized.

Biochemical differentiation between enterotoxigenic heat-sensitive and heat-resistant Clostridium perfringens strains.

Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat-selection methods and of two NCTC strains were studied. Two different biochemical patterns closely related to the heat resistance of the strains were found. The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat-resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat-sensitive spores. Sorbitol fermentation was variable among the strains of this last group. The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2. Only the strains placed into group 2 synthesized toxins of sufficient levels for typing. In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1. The heating of these two strains did not modify their characteristics. We conclude that enterotoxigenic C. perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil. These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity. The heat-selection methods used for isolation of C. perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.

Beta-glucuronidase-producing bacteria in bile from the common bile duct in patients treated with endoscopic papillotomy for gallstone disease.

This paper reports the occurrence of beta-glucuronidase-producing bacteria in the bile in gallstone patients treated with endoscopic papillotomy (EPT). The study included 36 patients--18 women and 18 men, aged 43-87 years, with a median of 72.5 years. Bile sampling was done with an endoscopic technique. All bacterial strains were tested for beta-glucuronidase activity with a rapid chromogenic tablet test, using 4-nitrophenyl-beta-D-glucuronic acid as substrate. Bacterial growth was found in the bile in 35 patients. Of 103 strains isolated, 30 produced beta-glucuronidase. Twenty-five of the patients had at least one beta-glucuronidase-producing strain in the bile. All 26 strains of Escherichia coli were producing the enzyme. Both strains in the Bacteroides fragilis group and one out of two strains of Clostridium perfringens were producing beta-glucuronidase. The activity of the bacterial beta-glucuronidase was found within the pH range of the bile in these patients. A relationship between the presence of beta-glucuronidase-producing bacteria in the bile and pigment gallstone is suggested.

Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens.

Enterotoxigenic strains of Clostridium perfringens have recently been implicated in some cases of antibiotic-associated diarrhoea. We present here the results of an epidemiological study of this disease. Five cases of diarrhoea caused by C. perfringens serotype 41 occurred during a 9-week period, and then during a 6-week period there were three cases due to serotype 27 and two due to serotype 24; in all but one case two geriatric wards were involved. In total there were 16 cases in 22 months. All cases were identified by the detection of C. perfringens enterotoxin in the faeces. The mean number of C. perfringens in these cases was 10(8.8) cfu/g of faeces. Of 37 patients who had negative test results for C. perfringens enterotoxin, 18 had positive cultures for C. perfringens, with mean faecal counts of 10(5.3) cfu/g, and nine of these patients had diarrhoea. Thirteen different serotypes were isolated from these 18 patients, including type 41 from seven patients and type 27 from one. Hand carriage of the offending serotype was demonstrated in three of four infected patients, none of four controls and two of 14 ward staff. C. perfringens of serotypes causing disease was isolated from 59% of environmental areas where there was active disease, 27% of areas where there had been disease which had since resolved and 9% of areas where there was no history of disease. The findings imply that cross infection may occur.

Inhibition of Clostridium perfringens by Naphthoquinones

ABSTRACTThe minimum inhibitory concentrations (MICs) of several naphthoquinones (NQ) were determined in the presence or absence of nitrite (NO2−) against various strains of Clostridium perfringens. In fluid thioglycollate medium, MICs ranged from 70–100 ppm for 2‐methyl‐1,4‐NQ (menadione), 200–280 ppm for 1,4‐NQ, 180–250 ppm for 1,2‐NQ, &gt;500 ppm for several water‐soluble derivatives and 100–300 ppm for NO2−. Using a type B strain in homogenized meat medium, MICs were 670 ppm for menadione, 620 ppm for 1,4‐NQ and 770 ppm for NO2−. Nitrite, menadione and 1,4‐NQ exhibited comparable and additive rather than synergistic inhibition. Some NQ compounds may have potential as partial nitrite substitutes subject to safety evaluation.

The occurrence of antibiotic resistance in Clostridium perfringens from pigs.

Porcine faecal specimens were collected from piggeries in south western Australia. Clostridium perfringens strains were isolated, identified, and examined by disc susceptibility tests for their resistance to several antibiotics. The resultant data were correlated with the known exposure of the animals to antimicrobial agents in the feed or water. The results showed that the percentage of C. perfringens isolates resistant to tetracycline or the macrolide-lincosamide antibiotics was significantly higher from weaners fed one of a number of combinations of antimicrobial agents than was the percentage of resistant strains isolated from the one piggery that did not use antimicrobials. No differences in the levels of chloramphenicol or penicillin resistance were observed. Genetic analyses showed that 3 of the resistant strains carried conjugative R-plasmids which carried the tetracycline resistance determinants.

The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains.

Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively. Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tórtora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests. By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained. The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice. Tórtora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.

Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3

Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.