Inhibition of Clostridium botulinum type C by Bacteria Isolated from Mud

Clostridium botulinum type C was not detected in 54 samples of mud even after seeding them with a small number of spores of this organism. From 35 of these mud samples. 108 strains of bacteria were isolated which inhibited the growth of Cl. botulinum type C. strain FH 6513, but did not denature preformed toxin. These strains fell into three groups: Bacillus spp. (73%); Gram positive non-sporing rods (11%); and Gram positive cocci (16%). Seven strains of Bacillus spp. were further investigated and found to produce from two to five peptide antibiotics.     

Toxigenic Profile of Clostridium perfringens Strains Isolated from Natural Ingredient Laboratory Animal Diets

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium that ubiquitously inhabits a wide variety of natural environments including the gastrointestinal tract of humans and animals. C. perfringens is an opportunistic enteropathogen capable of producing at least 20 different toxins in various combinations. Strains of C. perfringens are currently categorized into 7 toxinotypes (A, B, C, D, E, F, and G) based on the presence or absence of 6 typing-toxins (α, β, epsilon, iota, enterotoxin, and netB). Each toxinotype is associated with specific histotoxic and enteric diseases. Spontaneous enteritis due to C. perfringens has been reported in laboratory animals; however, the source of the bacteria was unknown. The Quality Assurance Laboratory (QAL) at the National Institute of Environmental Health Sciences (NIEHS) routinely screens incoming animal feeds for aerobic, enteric pathogens, such as Salmonella spp. and E. coli. Recently, QAL incorporated anaerobic screening of incoming animal feeds. To date, the lab has isolated numerous Clostridium species, including C. perfringens, from 23 lots of natural ingredient laboratory animal diets. Published reports of C. perfringens isolation from laboratory animal feeds could not be found in the literature. Therefore, we performed a toxin profile screen of our isolated strains of C. perfringens using PCR to determine which toxinotypes were present in the laboratory animal diets. Our results showed that most C. perfringens strains we isolated from the laboratory animal feed were toxinotype A with most strains also possessing the theta toxin. Two of the C. perfringens strains also possessed the β toxin. Our results demonstrated the presence of C. perfringens in nonsterile, natural ingredient feeds for laboratory animals which could serve as a source of this opportunistic pathogen.     

Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Avirulent Clostridium perfringens Strains Obtained by Euflavine Treatment

Clostridium perfringens was incubated in the presence of euflavine (EU); the resistant mutants which were thus isolated had highly reduced capacity to release alpha-toxin. This fact was confirmed by lecithinase determinations and by immunoelectrophoresis. Injected into guinea pigs and into 6- to 7-day-old chicks, these mutants were completely avirulent. A study of their properties indicated biochemical differences between wild types and mutants.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌α,β,ε,τ毒素基因cpa、cpb,etx及iA序列合成了针对4种毒素基因的4对特异引物,建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法.结果本所保存的A,B,c,D,E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带,而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性;将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段.并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定,并与毒素中和试验鉴定结果进行了比较,结果表明两种方法具有较高的符合率.本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌a, b, e, t 毒素基因cpa、cpb、etx 及iA序列合成了针对4种毒素基因的4对特异引物, 建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法。结果本所保存的A, B, C, D, E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带, 而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性; 将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段。并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定, 并与毒素中和试验鉴定结果进行了比较, 结果表明两种方法具有较高的符合率。本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义。     

Analysis of the Phospholipase C Gene of Clostridium perfringens KZ1340 Isolated from Antarctic Soil

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.     

Inhibition of Clostridium perfringens by a Novel Strain of Bacillus subtilis Isolated from the Gastrointestinal Tracts of Healthy Chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.     

Inhibition of macromolecular synthesis by caffeine in Clostridium perfringens

Caffeine (2 mg/mL) inhibited the incorporation of [14C]adenine into actively growing cells of Clostridium perfringens NCTC 8679 in a dose-dependent manner. Also reduced by caffeine was incorporation of [14C]thymidine and 14C-labeled amino acids. No effect on guanine, uracil, adenosine, guanosine, or uridine was detected. Actual incorporation of [14C]caffeine or [14C]thymine in control cultures did not occur.     

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.     

Characterization of Clostridium perfringens (welchii) Isolated from Market Poultry

Strains of Clostridium perfringens capable of producing heat-resistant spores, characteristic of the food-poisoning types, were not recovered in a random survey of feces and livers of market poultry. Favorable growth response with a known food-poisoning strain indicated that the media and methods employed were adequate. Spores produced in vitro from this strain survived at 100 C for several hours. Animal feeding experiments with this strain showed that heat-resistant spores (surviving for 1 hr at 100 C) could be readily demonstrated 24 hr after oral instillation of vegetative cells in mouse feces, but not in chicken feces. One experiment suggests that this strain might adapt to the environment of the intestinal tract of chickens, but not all of the spores recovered were as heat resistant as those of the parent culture.     

Radiation Resistance of Spores of Some Clostridium perfringens Strains

Clostridium perfringens spores (eight strains) were irradiated in a model system with 60Co gamma rays at -30 C. The quantal response data obtained were analyzed with extreme value statistics. It was found (at the 95% confidence level) that all eight strains followed the same rate of death and that this rate was probably (at the 95% level) not exponential. The statistics did not exclude, however, a normal, lognormal, Weibull, or related rate of spore kill. A more definitive study would be required to distinguish between the latter distributions.     

Distribution of Neuraminidase among Food-poisoning Strains of Clostridium perfringens

A survey was made to determine the distribution of the enzyme neuraminidase among 76 strains of Clostridium perfringens. Representative strains from each toxigenic type (A to F) and atypical C. perfringens type A food-poisoning strains of both American and English (Hobbs types) origin were tested. Both the American food-poisoning and nonfood-poisoning associated cultures consisted of both neuraminidase-positive and -negative strains. Furthermore, American strains which could not be differentiated from the original Hobbs cultures consisted of both neuraminidase-positive and -negative representatives. In contrast, the English (Hobbs) strains uniformly failed to produce an active intracellular or extracellular neuraminidase. No enzyme activity was detected in these strains when cultures were grown in different growth media, when grown in the presence of substrate (neuraminlactose), or upon extended incubation of enzyme preparations with substrate. With the exception of a type F strain, representative strains of the other toxigenic types (A to F) produced neuraminidase; 85% of the typical type A strains contained the enzyme.     

Genetic Characterization of Type A Enterotoxigenic Clostridium perfringens Strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.     

Characterization of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity.