Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water

Two media (mCP medium and Tryptose Sulphite Cycloserine (TSC) agar) were evaluated for recovery of Clostridium perfringens in environmental and part-treated drinking water. For laboratory strains of Clostridium , mCP was more selective and specific for Cl. perfringens than TSC, but was markedly less efficient for the enumeration of both vegetative cells and spores. For samples of river water and part-treated drinking water, TSC recovered significantly greater numbers of Cl. perfringens than mCP. In contrast to previous reports, there was a significant number of false presumptive positive and negative isolates on mCP. TSC is a more suitable medium for the routine monitoring of water supplies for the presence of Cl. perfringens .     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

A Study of Rapid and Simplified Confirmatory Tests for Clostridium perfringens

Strains of Clostridium perfringens and culturally similar species which also may grow on selective isolation media for this organism were examined by conventional confirmatory tests, the API ZYM system and by individual tests for phosphatase and glutamic acid decarboxylase activity.
API ZYM tests, involving 19 different enzymes, confirmed the known similarity between Cl. perfringens, Cl. absonum, Cl. paraperfringens and Cl. sardiniensis but effectively distinguished this group from Cl. bifermentans, Cl. celatum, Cl. perenne and Cl. sordellii. A similar separation was achieved by a single test for acid phosphatase which could be applied to individual colonies on a plating medium.
Because the acid phosphatase test was found to be of greater value than nitrate reduction in distinguishing Cl. perfringens , it could replace the latter in the usual series of confirmatory tests. It is suggested that strains from Cl. perfringens isolation media should be screened for acid phosphatase activity at the purification stage and only positive strains subjected to further tests.
It was found that Cl. perfringens could not be distinguished from the other species on the basis of glutamate decarboxylase activity.     

A New Medium for the Detection and Enumeration of Clostridium perfringens in Foods

S ummary . This paper describes the development and use of a selective diagnostic medium for the detection and enumeration of Clostridium perfringens in foods. The medium gives higher counts of Cl. perfringens and fewer false positives than existing media.     

Note: Occurrence and persistence of culturable clostridial spores on the leaves of horticultural plants

Clostridial spores were found in numbers from less than 1 to over 50 colony-forming units cm^-2 on mature leaves of 19 species of horticultural plants under commercial cultivation in five localities in Apulia (SE Italy). Of 1828 clostridial isolates, 87% were identified phenotypically and ascribed to Clostridium pasteurianum, Cl. sporogenes, Cl. butyricum, Cl. roseum, Cl. perfringens, Cl. felsineum and Cl. acetobutylicum , in decreasing order of frequency. When spore suspensions of Cl. pasteurianum, Cl. perfringens, Cl. roseum and Cl. sporogenes were inoculated onto the leaves of basil, leaf-beet, lettuce, rocket-salad, spinach and tomato in the greenhouse, spore counts at first invariably declined shortly after inoculation, then rose again significantly for Cl. pasteurianum and Cl. perfringens on basil and for Cl. sporogenes on tomato in summer.     

New media for detection and counting of clostridia in foods

The growth of pure cultures of Clostridium perfringens (ATCC 12922) and Cl. sporogenes (PA 3679) in five non-selective media, fluid thioglycollate medium (FTM), rapid perfringens medium (RPM), Columbia broth Malthus (CBM), reinforced clostridial medium (RCM) and lactose sulphite (LS), was monitored using conductance measurements with a Malthus analyser. Only FTM and CBM gave useful results. The correlation of log₁₀ plate counts on blood agar of the pure strain of Cl. sporogenes with detection times in FTM was highly significant ( r = 0·96, n = 73), and with detection times in CBM less so ( r = −0·909, n = 33). The correlation of log₁₀ counts on tryptose sulphite neomycin medium (TSN) of wild strains of Cl. sporogenes and Cl. perfringens with detection times with FTM in meat was also highly significant ( r = 0·933, n = 54).     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

The possibility of the germination of spores of pathogenic clostridia and bacilli in soil

A possibility of germination of clostridia (Cl. tetani and Cl. perfringens) and bacilli (Bac. anthracis, STI vaccine strain) has been studied in model experiments with native soil. Mature spores did not germinate upon contact with native soil of deferent agrochemical types. Addition of meat-pepton medium and other protein, amino acid, and sugar-containing media led only to "swelling" of spores. The data obtained support the conclusions drawn by many researches that pathogenic clostridia and bacilli do not germinate in soil.     

A sporulation medium for Clostridium perfringens

A new solidified medium for inducing sporulation of Clostridium perfringens is described. The essential components of the medium are bile, bicarbonate and quinoline. The medium induced significant sporulation in all of 100 strains of Cl. perfringens isolated at random from human faecal specimens. The majority (94%) of strains sporulated profusely.     

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10² bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

The Role of Clostridium perfringens in the Growth Response of Chicks to Dietary Penicillin

S ummary . The previous observation that Clostridium perfringens is responsible for penicillin-relieved growth depression in chicks has been reassessed. The administration of Cl. perfringens to germfree chicks, to birds harbouring a non-growth depressing gut flora and to birds reared in an environment free from Cl. perfringens was without effect on growth rate. No correlation was observed in conventional chicks between the incidence of growth depression and the presence of Cl. perfringens in the gut of the chicks one day after food was first given.     

In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.     

In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production

AIMS: Clostridium perfringens type A causes both clinical and subclinical forms of necrotic enteritis in domestic avian species. In this study the inhibitory effect of hen egg white lysozyme on the vegetative form of Cl. perfringens type A and the production of alpha-toxin in vitro was investigated. METHODS AND RESULTS: A micro-broth dilution assay was used to evaluate the minimal inhibitory concentrations (MIC) of lysozyme against three clinical isolates of Cl. perfringens type A in 96-well microtitre plates. The MIC of lysozyme against Cl. perfringens isolates was found to be 156 microg ml(-1). Scanning electron micrographs of the cells treated with 100 microg ml(-1) of lysozyme revealed extensive cell wall damage. A quantitative sandwich ELISA for alpha-toxin produced by Cl. perfringens was developed based on a commercial ELISA kit allowing only qualitative detection. Addition of 50 microg ml(-1) of lysozyme did not inhibit the growth of Cl. perfringens but significantly inhibited the toxin production. CONCLUSIONS: Lysozyme inhibited the growth of Cl. perfringens type A at 156 microg ml(-1). At sublethal levels, lysozyme was able to inhibit the alpha-toxin production. SIGNIFICANCE AND IMPACT OF STUDY: Inhibition of Cl. perfringens type A and its alpha-toxin production by hen egg white lysozyme had never previously been reported. By inhibiting this avian pathogen and its toxin production, lysozyme showed potential for use in the treatment and prevention of necrotic enteritis and other Cl. perfringens type A related animal diseases.     

Characteristics of a sporulation stimulating factor from Clostridium perfringens type A

A product in the culture supernatant fluid of Clostridium perfringens NCTC 8239 stimulated the sporulation of a test strain, NCTC 8679, of the same organism. The responsible factor, termed sporulation factor (SF), was present in seven cultures of Cl. perfringens grown in either a defined or complex medium. The SF reversed glucose-mediated catabolite repression of sporulation by this organism. Preliminary characterization of the SF demonstrated a resistance to elevated temperatures and proteases and a molecular weight of less than 500 Da. The known association of Cl. perfringens enterotoxin with sporulation highlights the importance of interactions between strains of this organism as may occur in the human intestine during foodborne illness.     

Use of a synthetic medium for cultivating pathogenic anaerobes]

It was shown that a synthetic medium suggested by the authors earlier was useful for the growth and toxin formation of Cl. tetani, Cl. botulinum and Cl. perfringens, types B and E. A study of the character of growth and toxinogensis, microscopic examination of morphology of culture cells and results of passages showed the suggested synthetic medium to be of value; a possibility of its application for studying the nutrient requirements and the role of individual components of the nutrient media in the process of growth and toxinogenesis was also demonstrated.     

Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages

AIMS: Clostridium perfringens is recommended as a suitable indicator bacterium for human enteric viruses, Giardia cysts and Cryptosporidium oocysts in finished water and in the assessment and evaluation of water treatment. Several agars and confirmation procedures were evaluated in parallel with the Australian/New Zealand Standard (AS/NZ) Method for the enumeration of Cl. perfringens from treated and untreated sewage samples. METHODS AND RESULTS: The current AS/NZ method utilizes tryptose sulfite cycloserine agar (TSC), lactose gelatin medium (LG) and nitrate motility medium (NM) at an incubation temperature of 37 degrees C. Sixty treated and untreated sewage samples were used to evaluate TSC agar, membrane Cl. perfringens agar (mCP), Perfringens agar (OPSP) and Perfringens agar with 4-methylumbelliferyl phosphate (OPSP-MUP) for enumeration of Clostridium. An incubation temperature of 44 degrees C for 24 h was used for comparison. Confirmation procedures were also evaluated using 103 isolates and included LG and NM, ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) with MUP (ONPG-MUP) and phosphatase reagent (PR). OPSP compared favourably with TSC agar. One false negative result was obtained from each of the LG/NM and ONPG-MUP procedures. No false results were obtained using the PR confirmation procedure. CONCLUSIONS: OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a simple and rapid method for isolating and enumerating Cl. perfringens from sewage samples and confirmed results can be reported more quickly due to shorter analytical turnaround times.     

Changes in the electrical activity of the cerebral cortex under the combined influence of Cl. perfringens type A toxin and products of Cl. butyricum activity]

A study of the changes of the electrocorticogram (ECoG) and the electrocardiogram (ECG) in combined action of Cl. perfringens, type A, and of the Cl. butyricum broth culture filtrate showed that desynchronization of the cortical electrical activity and its subsequent depression occurred at earlier periods than in the case of isolated administration of Cl. perfringens toxin. The general character of the changes in the cortical rhythmic activity remained the same as in intoxication caused by Cl. butyricum toxin alone. The ECoG and ECG changes occurred at shorter intervals. Cl. butyricum filtrates induced no ECoG and ECG changes. It is supposed that the effect of the products of the Cl. butyricum vital activity consisted in increase in the tissue barrier permeability and, in this connection, in a greater penetration of Cl. perfringens toxin into the tissues, including the central nervous system.