Strains Of Clostridium Perfringens Type Research Articles

Enterotoxin formation by Clostridium perfringens type A in a defined medium.

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.

GROWTH OF Clostridium perfringens STRAINS ON α‐GALACTOSIDES

ABSTRACTFive strains of Clostridium perfringens type A varied greatly in their ability to utilize and grow on the α‐galactosides raffinose, stachyose, and melibiose when these sugars were added (1%) to a casein‐digest medium. Stachyose and raffinose were utilized rapidly by NCTC strain 8798, and at moderate rate by NCTC strains 8238 and 10240. Strain KA3 utilized raffinose at moderate rates, but stachyose not at all. Strain FD‐1 utilized raffinose only to a slight extent, and stachyose not at ail. NCTC strains 8238 and 10240 sporulated heavily during growth on raffinose and stachyose. The implications of these findings on previous work linking C. perfringens to legume‐induced human flatulence is discussed.

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.

Changes in the Population of Clostridium perfringens Type A Frozen in a Meat Medium

Survival of eight strains of Clostridium perfringens type A frozen in a meat medium at −23 C and held under aerobic conditions for 1,7, and 14 days was 17, 12, and 6.2%, respectively. Holding under anaerobic conditions for 1, 7, and 14 days gave survival percentages of 51, 26, and 10%, respectively. Survival was not affected by cooling rates of 0.5, 1.5, and 20 C per min. Addition of 10% sucrose to the meat medium did not affect survival. The meat medium with glycerol concentrations of 1.0 and 2.0M gave survival percentages of 13, 63, 144, and 58, respectively, when frozen at −23 C and held for 22 to 24 h.

Temperate phages of Clostridium perfringens type C

Four phages isolated from carrier strains of Clostridium perfringens type C belong to two classes. The three phages of class I, c1, c3, and c4, and homoimmune and serologically closely related. The phage of class II, c5, is heteroimmune to the class I phages and not related to them serologically. Transduction experiments with several of the phages were negative. Mutants of the indicator strain with surface alterations occurred spontaneously in stock cultures. Electron micrographs show the phages of each class to be distinct yet similar, having polyhedral heads of about the same diameter 55 nm, and long, flexible tails without sheaths or collars. Phages c4 and c5 were characterized for their lysogenic properties. Phage c4 was inducible with mitomycin C. Both c4 and c5 were temperate viruses by the test of stability of their respective lysogens to phage-specific antisera.

Heterogeneity of Enterotoxin‐Like Protein Extracted from Spores of Clostridium perfringens Type A

Enterotoxin-like protein was extracted from spores of three enterotoxin-positive and three enterotoxin-negative strains of Clostridium perfringens type A by urea/mercaptoethanol, alkaline mercaptoethanol and alkaline dithiothreitol. Disc immunoelectrophoresis demonstrated that three distinct enterotoxin-like proteins could be extracted. In 7% acrylamide gels, type I, type II, and type III enterotoxinlike proteins had relative mobilities of 0.52, 0.63, and 0.73 respectively. In contrast to disc immunoelectrophoresis, immunoelectrophoresis in agar gel demonstrated identical electrophoretic properties for the various entertoxin-like proteins. Immunoelectrofocusing experiments gave isoelectric points of 4.43, 4.43, 4.36, and 4.52 for purified entertoxin and type I, type II, and type III enterotoxin-like proteins respectively. Ferguson plots (i.e., log relative mobility versus acrylamide concentration) yielded nonparallel lines which intersected at a nonsieving concentration of acrylamide indicating that the various species of enterotoxin-like protein differed in size. Estimation of the molecular weight of purified enterotoxin and the three species of enterotoxin-like protein was done by comparing the slopes obtained in Ferguson plots with those obtained using proteins of a known molecular weight. Molecular weights of 38000, 36500, 23000, and 15400 were obtained for purified enterotoxin, type I, type II, and type III enterotoxin-like protein respectively. Collectively, the evidence indicates that fractionation of the different species of enterotoxin-like protein was due primarily to differences in their size, and that different forms of enterotoxin-like protein can be extracted from spores of different strains of C. perfringens type A.

Distribution of Enterotoxin-producing Strains of Clostridium perfringens Type A in Human Beings, Foods and Soils

On the isolation frequency of enterotoxin-producing strains of Cl. perfringens, a significant difference was existed between the isolates from food poisonings and those from natural environments, that is, enterotoxinogenic strains were found in 77.3% of the isolates from food poisonings caused by the organism and in 0.8% of those from natural environments.Percentage of enterotoxin-producing strains in Cl. perfringens isolates from natural environments was as follows; 0.8% of the strains from human feces, 0.8% of the strains from foods, 7.7% of the strains from swabs in cooking establishments, 0% of the strains from river water and 0% of the strains from soils.Spore formation was found in 100% of enterotoxin-positive strains and in 67.5% of enterotoxin-negative strains.

A paracrystalline inclusion formed during sporulation of enterotoxin-producing strains of Clostridium perfringens type A.

A large paracrystalline inclusion is formed by certain strains of Clostridium perfringens type A during spore morphogenesis. In most cell thin sections, the inclusion appeared rod-shaped when sectioned at an angle perpendicular to its longer axis, and circular or oval-shaped when sectioned at an angle parallel to its longer axis. Measurements performed on electron micrographs of inclusions sectioned to reveal the rod shape indicated a fairly consistent thickness (width) of 192 +/- 23 nm. The length of the inclusions varied considerably with a maximum of approximately 2,120 nm being observed. Ultrastructurally, the inclusion was composed of closely packed, periodically spaced, parallel layers. Usually a single inclusion was randomly located in the cytoplasm of the cell. Two inclusions per cell were rarely observed. The inclusion was formed only by ent(+) strains of C. perfringens. Mutants of the ent(+) strain NCTC 8798 that were altered in their sporulating and enterotoxin-producing capacities and revertants of these mutants were tested for inclusion formation. The results indicate that, as with the ent(+) trait, a direct relationship exists between inclusion formation and spore formation. The synthesis of enterotoxin, formation of a morphologically distinct inclusion, and the initial deposition of discontinuous coat fragments around the forespore appear to be events closely related in time during spore morphogenesis.

Antigenic homogeneity of enterotoxin from different agglutinating serotypes of Clostridium perfringens.

Fifteen strains of Clostridium perfringens type A which included Hobbs' agglutinating serotypes 1 to 13 and two unknown serotypes were studied with respect to the serological nature and the quantity of enterotoxin produced. Ten of the 13 serotypes, and the two unknown ones, produced enterotoxin while three strains (serotypes 4, 5, and 7) failed to do so. Enterotoxins from the producing strains showed a line of identity in immunodiffusion tests using specific antiserum to enterotoxin, and they exhibited similar erythemal and enteropathogenic effects. Enterotoxin was produced only when these strains sporulated, but spore producing, nonenterotoxigenic strains were encountered.

The role of enterotoxin in Clostridium perfringens type A enteritis.

Vegetative cells of three strains of Clostridium perfringens type A, free of erythemal activity, were suspended in fresh medium and injected into ligated intestinal loops of lambs. Examination of the loop contents after 6.5 h showed significant accumulation of fluid, multiplication and sporulation of C. perfringens, and erythemal activity in both the supernatant fluids and the sediments.The erythemal factor produced in vivo was identical with the erythemal factor of sporulated cells of C. perfringens grown in vitro, and again caused accumulation of fluid when transferred into ligated intestinal loops of recipient lambs. Immune rabbit serum prepared against extracts from sporulated cells of C. perfringens, and absorbed with extracts from vegetative cells of the same strain, completely neutralized the enterotoxic and erythemal activities of the in vivo-produced factor.It is concluded that the erythemal factor is the causative agent in C. perfringens type A enteritis. The term "Clostridium perfringens enterotoxin" is proposed to characterize the erythemal factor.

Immunization of sheep against experimental Clostridium perfringens type A enteritis.

Lambs were injected parenterally with formalized cell extract, or bacterin, prepared from sporulating cultures of an enteropathogenic strain of Clostridium perfringens type A. This procedure induced an antibody in the serum which neutralized the erythemal and enteropathogenic activities of the cell extract. Immunodiffusion of the serum against the toxic cell extract produced a single, specific band associated with enteropathogenic activity. Protective immunity was not evident when the immunized lambs were challenged by the intraduodenal and the ligated intestinal loop methods with toxic cell extract or with whole cells of C. perfringens. Non-immunized lambs, challenged nine times by the intraduodenal method over a period of 70 days, had no measurable antibody and remained susceptible to experimental enteritis.

Experimental gas gangrene with food-poisoning Clostridium perfringens type A.

Classical and food-poisoning strains of Clostridium perfringens type A were tested for their capacity to produce gas gangrene in guinea pigs.The virulence of food-poisoning strains producing heat-sensitive spores and showing beta hemolysis on sheep-blood agar was comparable to that of the classical strains. The most virulent strains of both groups produced fatal infection with only three to five vegetative cells. Of 13 food-poisoning, heat-sensitive strains showing no beta hemolysis, only three were lethal when a minimum of 4 × 104 to 4 × 108 cells was injected. None of the food-poisoning, heat-resistant strains produced fatal infection with cell numbers up to 4 × 108. The groups of strains showed a correlation between virulence and formation of alpha toxin in liquid culture.It is concluded that a number of heat-sensitive, beta-hemolytic strains of C. perfringens may cause gas gangrene as well as food poisoning, and that the current subdivision of C. perfringens type A strains into classical and food-poisoning groups is no longer tenable.

Experimental enteritis with food poisoning and classical strains of Clostridium perfringens type A in lambs.

Experimental enteritis with food poisoning and classical strains of Clostridium perfringens type A in lambs.

Variations in Recovery of Clostridium perfringens on Commercial Sulfite‐Polymyxin‐Sulfadiazine (SPS) Agar

SUMMARY— Liquid cultures of several strains of Clostridium perfringens type A were diluted with phosphate buffer and enumerated on laboratory‐prepared sulfite‐polymyxin‐sulfa‐diazine (SPS) agar, four lots of commercial SPS agar, and on non‐selective media. Recoveries on laboratory‐prepared SPS agar and on one lot of commercial SPS agar were the same as on non‐selective media. Recoveries on three commercial lots of SPS agar were significantly lower.Replacement of phosphate buffer with 0.1% peptone allowed quantitative recoveries of C. perfringens on three out of four lots of commercial SPS agar, irrespective of whether the cells were grown in liquid cultures or on meat and meat products.

Effects of animal alimentary passage on the heat resistance of Clostridium perfringens.

The resistance to heat, as measured by D values and phantom thermal death time curves, was observed to increase for one of three strains of Clostridium perfringens type A subsequent to animal passage. Animal passage was accomplished by the force-feeding of germ-free mice with bacterial suspensions of the organism, followed by the force-feeding of additional gnotobiotic mice with the contaminated feces. For the one strain in which an increase in heat resistance was noted, the result could not be attributed to mouse feces per se, since the presence of sterile germ-free mouse feces in a suspending medium did not protect C. perfringens spores from elevated temperature destruction.

EFFECT OF CONTROLLED PH ON TOXINOGENESIS BY CLOSTRIDIUM PERFRINGENS TYPE D.

A strain of Clostridium perfringens type D was grown in a complex medium with or without pH control. Rate and extent of growth was the same without pH control and at controlled pH levels between 6.0 and 7.4, but was reduced at pH 7.6. With controlled pH at optimum levels the yields of alpha, epsilon, kappa, and theta toxins were 2, 6, 2, and 8 times higher than without pH control. These yields were 25 MLD, 70 × 103 MLD, 400 kappa units, and 60 hemolytic units per milliliter respectively. Optimum pH was 7.0 to 7.2 for alpha, 6.7 to 7.4 for epsilon and kappa, and 7.0 to 7.4 for theta toxin. Lambda and mu toxins were not produced, and only traces of nu antigen were formed.Fourteen additional strains of C. perfringens type D were grown with pH controlled at 7.0 or without pH control and assayed for production of epsilon, kappa, and theta toxins. Epsilon was formed by 14, kappa by 10, and theta by 13 strains. With pH controlled at 7.0, 14 strains produced more epsilon toxin, 4 strains more kappa toxin, and 10 strains more theta toxin than without pH control. It is concluded that increased toxinogenesis at controlled pH is a general phenomenon in cultures of C. perfringens type D.

Effects of Animal Alimentary Passage on the Heat Resistance of Clostridium perfringens1

The resistance to heat, as measured by D values and phantom thermal death time curves, was observed to increase for one of three strains of Clostridium perfringens type A subsequent to animal passage. Animal passage was accomplished by the force-feeding of germ-free mice with bacterial suspensions of the organism, followed by the force-feeding of additional gnotobiotic mice with the contaminated feces. For the one strain in which an increase in heat resistance was noted, the result could not be attributed to mouse feces per se, since the presence of sterile germ-free mouse feces in a suspending medium did not protect C. perfringens spores from elevated temperature destruction.