Persistent high numbers of Clostridium perfringens in the intestines of Japanese aged adults.

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10(7) to 10(9), while some others ranged from 10(3) to 10(6) per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10(9) C. perfringens per ml of feces, no trace of a-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of a-toxin although it allowed the organism to grow sufficiently.     

Rapid Technique for the Enumeration of Clostridium perfringens

A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar.     

Cross-complementation of Clostridium perfringens PLC and Clostridium septicum α-toxin mutants reveals PLC is sufficient to mediate gas gangrene

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different α-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens α-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum α-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP⁻/rtPCR⁺ colonies were identified as C. perfringens, whereas 3 mCP⁺/rtPCR⁻ colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.     

Clostridium perfringens α-toxin up-regulates plasma membrane CD11b expression on murine neutrophils by changing intracellular localization

Gas gangrene caused by Clostridium perfringens type A infection is a highly lethal infection of soft tissue characterized by rapid spread of tissue necrosis. This tissue destruction is related to profound attenuation of blood flow accompanied by formation of platelet-leukocyte aggregates in the blood vessels. Several studies have identified α-toxin, which has both sphingomyelinase and phospholipase C activities, as a major virulence factor in the aggregate formation via activation of the platelet gpIIbIIIa. Here, we show that α-toxin greatly and rapidly increases plasma membrane localization of CD11b, which binds to the platelet gpIIbIIIa via fibrinogen, in mouse neutrophils. Interestingly, short-term treatment of α-toxin has little effect on gene expression profiles in neutrophils, and the toxin does not change the total protein expression levels of CD11b in whole cell lysates. The following analysis demonstrated that CD11b localizes to intracellular vesicles in intact cells, but the localization changed to the cytoplasmic membrane in α-toxin-treated cells. These results suggest that CD11b is recruited to the cytoplasmic membrane by α-toxin. Previously, we reported that α-toxin promotes the formation of ceramide by its sphingomyelinase activity in mouse neutrophils. Interestingly, a synthetic cell-permeable ceramide analog, C₂-ceramide, increases plasma membrane localization of CD11b, suggesting that ceramide production by α-toxin recruits CD11b to the cytoplasmic membrane to promote platelet-leukocyte aggregation. Together, our results illustrate that the increase of cell membrane CD11b expression by α-toxin might be crucial for the pathogenesis of C. perfringens to promote formation of platelet-leukocyte aggregates, leading to rapid tissue necrosis due to ischemia.     

Characterization of a Toxin-Deficient Clostridium perfringens Strain,KZ1340

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an α, θ-, and κ-toxin non-producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (θ-toxin) gene, but the plc (α-toxin) and colA (κ-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin-deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.     

A simple method for the isolation and determination of Clostridium perfringens

Summary A method for the isolation and determination of small numbers of vegetative cells or spores of Clostridium perfringen has been developed based on enrichment under anaerobic conditions in a fluid thioglycollate medium without dextrose, containing 400 g of D-cycloserine/ml at 46°C for 18 h (PEM). It allows virtually complete recovery of vegetative cells of all strains of Clostridium perfringens tested, whereas facultative anaerobes present in food are inhibited. Undamaged Clostridium perfringens spores can also be detected by this procedure. After enrichment, isolation of Clostriduum perfringens is carried out on iron sulphite agar at 46°C for 18 h. Typical black colonies are picked and confirmed by the following tests: neutralization of the -toxin by a specific diagnostic antiserum and absence of indole, motility, and ability to liquify gelatin.     

Sensitivity of Direct Plating for Detection of High Levels of <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 in Bovine Fecal Samples

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10⁹ colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10⁻¹ to 10⁻⁴) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37°C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37°C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 μg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 × 10⁴ cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.     

Improved Medium for Enumeration of Clostridium perfringens

An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.     

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C. difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty-four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 10^4.1/g in young adults and 10^4.6/g in elderly adults, with a range of 10^2.0 to 10^6.9/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.     

NMR assignment of backbone and side chain resonances for a dockerin-containing C-terminal fragment of the putative μ-toxin from Clostridium perfringens

The μ-toxin of Clostridium perfringens, termed CpGH84A, is modular hydrolytic enzyme that contributes to the pathogenicity of this organism. Backbone and side chain ¹H, ¹³C, and ¹⁵N resonance assignments have been determined for the C-terminal 15.5 kDa FIVAR-Doc modular pair of CpGH84A.     

Adhesive Property of <Emphasis Type="Italic">Bifidobacterium lactis</Emphasis> LKM512 and Predominant Bacteria of Intestinal Microflora to Human Intestinal Mucin

The adhesive property to the intestinal mucin of Bifidobacterium lactis LKM512, B. longum, B. breve, B. bifidum, B. adolescentis, B. infantis, Bacteroides vulgatus, Bacteroides distasonis, Eubacterium aerofaciens, Clostridium perfringens, Escherichia coli, and Lactobacillus acidophilus were examined. Adhesive rate of LKM512 to the mucin was significantly (p < 0.05, 0.01, or 0.001) stronger than the other strains from 2 to 100 time. Though the adhesive property of many strains was almost same to the mucin of 20-year-old and 50-year-old generations, in case of 4-month-old was different. Adhesive inhibitory effect of C. perfringens to the mucin by LKM512 was examined. Under the condition that LKM512 was 10⁸/ml and that C. perfringens was 10⁶/ml, adhesion of C. perfringens to the mucin was inhibited at 99.6%, when LKM512 adhered in advance. There was the strong inhibition of adhesion at 74.0%, when C. perfringens adhered to mucin in advance. Thus, LKM512 can inhibit the adhesion of harmful bacteria to the intestinal mucin, the possibility of using as a probiotic strain has to be verified.     

Comparison of Media for the Enumeration of Clostridium perfringens

For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 10(4) organisms per g, but with 10(6) organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested.     

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

Management of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 10⁸ conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 10², 3.2 × 10⁴, 3.2 × 10⁶ and 3.2 × 10⁸ conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 10⁸ conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments

Aims: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. Methods: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species‐specific PCR, along with full‐length sequencing of the 16S rRNA gene for a subset of isolates. Results: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species‐specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. Conclusions: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99·5% similarity). Significance and Impact of the Study: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.     

Sperm motility and fertilizing ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca fluviatilis)

The objective of this study was to freeze sperm of sex‐reversed females (neomales) of perch and to test their fertilization ability. Sperm used was testicular (TSN), collected from females that have been inverted by means of externally administered 17‐alpha methyltestosterone. Sperm collected from intact males (SSNM) of the same origin were used as control. Prior to freezing, both TSN and SSNM were diluted into 300 mm glucose solution at the ratio of 1 : 6 and DMSO was used as cryoprotectant (10% final concentration). Crypreservation was performed in 0.5 ml straws placed into a polystyrene box, three cm above the liquid nitrogen level for 10 min and thereafter transferred fully into liquid nitrogen. Samples were thawed in 40°C water bath for 8 s and used for the fertilization experiments. Spermatozoa concentration of fresh TSN and SSNM were estimated with 45.3 × 10⁹ and 37.8 × 10⁹ spermatozoa ml^?1, respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 μm s^?1 and 12.8%) compared to the SSNM (203.2 μm s^?1 and 94.7%) at 10 s after sperm activation. However, no differences were observed in terms of hatching rates between fresh TSN and SSNM (42.5 vs 49.3%) at fertilization densities of 12 × 10⁵ spermatozoa per egg. Frozen/thawed SSNM exhibited similar hatching rates at 12 × 10⁵ and 2.4 × 10⁵ spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen/thawed TSN were about 7.3% with 12 × 10⁵ spermatozoa per egg and did not show any difference at 2.4 × 10⁵ spermatozoa per egg (6.6%). Stripped sperm of normal perch can be successfully frozen. Squeezing of the testes is not a good method for collection of testicular sperm resulting into low velocity, motility and hatching rate. To understand the influences of neomales on sperm quality on reproductive success further studies should be performed addressing a full assay of motility and fertility criteria when using stripped sperm from normal males and neomales. Additionally, the results indicate that many of sex reversed perch neomales are not able to release sperm and that for further studies some well spermiating neomales must to be selected.