Strains Of Clostridium Perfringens Research Articles

Collagenase gene ( colA) is located in the 3′-flanking region of the perfringolysin O ( pfoA) locus in Clostridium perfringens

The 3′-flanking region of the β-galactosidase gene ( pbg), which is located downstream of the perfringolysin O gene ( pfoA), and the 5′-flanking region of the collagenase gene ( colA) of Clostridium perfringens strains NCTC8237 and 13, respectively, were analyzed. Southern analysis revealed that the colA gene is located 6.5 kb downstream of the pbg gene in the chromosome of C. perfringens. Sequence analysis showed that between the pbg and colA genes were the arcABDC and ahrC genes, whose putative products were quite similar to enzymes of the arginine deiminase pathway of Pseudomonas aeruginosa and the arginine repressor/activator of Bacillus subtilis, respectively. It is concluded that the genomic structure of the pfoA-colA region consists of pfoR-pfoA-ORF54- pbg-arcABDC-ahrC-colA.

Lack of Correlation between the Enterotoxigenicity of Clostridium perfringens Isolates and Propionic Acid Formation or Amylase Activity at 46°C

The absence in the supernatant fluid of either extracellular amylase in cultures grown at 46°C or propionic acid has been proposed for the presumptive identification of enterotoxin-positive strains of Clostridium perfringens. However we found that propionic acid was formed by several enterotoxin-positive, as well as enterotoxin-negative, strains of this organism. In addition 4 of 13 ent+ stains produced extracellular amylase during growth at 46°C. These results indicate that neither trait can be used as a reliable indicator of the enterotoxigenicity of isolates of this organism.

Raffinose-Bifidobacterium (RB) agar, a new selective medium for bifidobacteria

A new selective medium for the detection of bifidobacteria was developed. The medium owes its selectivity to the presence of propionate (15 g/l) and lithium chloride (3 g/l) as inhibitory agents, and raffinose (7.5 g/l) as a selective carbon source. In addition, casein (5 g/l) is used as a protein source, which results in a zone of precipitation around the colonies of bifidobacteria. The medium, Raffinose-Bifidobacterium (RB) agar, is free of antibiotics and easy to prepare. Bifidobacteria growing on RB agar show a yellow colony with a yellow halo and a precipitation zone around the colony. All human and dairy bifidobacteria grew well on RB agar, except for some Bifidobacterium bifidum strains. Some uncommon or animal species, Bifidobacterium gallicum, Bifidobacterium asteroides, Bifidobacterium animalis, Bifidobacterium pullorum, and some Bifidobacterium bifidum either did not grow or did not show the characteristic reactions. No non-bifidobacterial strains used in dairy products grow with the distinctive characteristics. Some faecal strains showed good growth and acid production, resulting in characteristic reactions. These strains were Actinomyces species, a Clostridium perfringens strain and some lactobacilli of animal origin. Comparison with other media indicated that RB agar is more selective than other media used.

Chemiluminescent enzyme immunoassay for detection of PCR-amplified enterotoxin A from Clostridium perfringens

A PCR protocol was developed for the rapid and specific detection of Clostridium perfringens strains harboring the enterotoxin A gene in artificially contaminated ground beef. A biotinylated primer pair was designed for amplification of a 750 bp fragment of the C. perfringens enterotoxin A gene. A combination of 4 h enrichment incubation and nucleic acid extraction, followed by 2 h of PCR amplification allowed detection at levels below 10 CFU of freshly grown cells in raw and cooked beef samples. PCR amplified products were confirmed by a Southern hybridization assay using a digoxigenin-labeled internal probe, and two hybridization ELISA protocols (PCR-ELISA) applying a streptavidin capture step for the hybridized PCR products. Both enzyme immunoassays utilized chemiluminescent detection with Lumiphos 530 ™ as substrate, after hybridization to an internal digoxigenin-labeled probe or a 5′ conjugated alkaline phosphatase-labeled probe. The PCR-ELISA resulted in faster confirmation of the PCR products while providing a level of sensitivity comparable to Southern hybridization, and has potential for development into an automated method.

Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne.

The intron-encoded endonuclease I-CeuI from Chlamydomonas eugametos was shown to cleave the circular chromosomes of all Clostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins, beta, epsilon, and tau, in addition to the enterotoxin and lambda-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins, theta and mu, were missing.

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750

Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus. Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated ifM r from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 ( dvn750) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M rmr = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750.

Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus. Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated M(r) from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 (dvn750) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M(r) = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

Generation Times and Modeling of Enterotoxin-Positive and Enterotoxin-Negative Strains of Clostridium perfringens in Laboratory Media and Ground Beef

The growth of two enterotoxin-positive (ent+) and two enterotoxin-negative (ent−) strains of Clostridium perfringens were examined in two laboratory media, fluid thioglycollate and fluid thioglycollate with beef, and in autoclaved ground beef stored at 37, 41, 43, 46, and 48°C. There was no association between enterotoxigenicity and optimum growth temperature, which varied depending on strain and medium. Experimentally obtained values for generation times at 43°C were as low as 8.7 and 6.3 min in thioglycollate both for ent+ and ent- strains respectively. In autoclaved ground beef the shortest generation times, 7.1 min and 9.2 min, were obtained at 41 and 46°C for two ent+ strains. For ent− strains in autoclaved ground beef a generation time of 6.6 min was observed at 43°C. Temperature optima obtained experimentally were the same as those obtained by the Gompertz model. The rapid growth of the ent+ strains between 41 and 46°C underlines previous recommendations for proper holding and cooling of protein foods such as meat and poultry known to support the growth of this organism.

The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne.

The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Clostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the HindIII repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS1151. In these cases, the HindIII repeat was not linked to the cpe gene although this was generally preceded by ORF3.

Detection of Enterotoxigenic Clostridium perfringens in Raw Beef by Polymerase Chain Reaction

A polymerase chain reaction (PCR) procedure was developed for direct detection of Clostridium perfringens strains with potential for food poisoning in raw beef samples. An oligonucleotide primer pair was used to amplify a 364 base pair sequence internal to the C. perfringens enterotoxin gene. One milliliter portions of the meat homogenates were inoculated into cooked meat medium (CMM) or reduced Fluid Thioglycollate (FTG) medium and incubated at 37°C. Portions sampled at 2, 4, 6, 8 and 24 h of enrichment were assayed for detection of the enterotoxin sequence by PCR. Amplification of the 364 bp sequence could be detected in 6 h by agarose gel electrophoresis and as early as 2 h by hybridization to a 150 bp digoxigenin (DIG)-labeled probe. To increase the sensitivity of the detection assay a commercial chromosomal deoxyribonucleic acid (DNA) extraction assay was compared with a nested PCR approach. Both methods allowed detection of less than 1 log10 colony forming units (CFU)/g of C. perfringens strains harboring the enterotoxin gene, with no interference with the background microflora present in the raw ground beef.

Typing of Clostridium perfringens by in vitro amplification of toxin genes.

The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents.

Multilocus enzyme typing of human and animal strains ofClostridium perfringens

Multilocus enzyme electrophoresis was developed to evaluate the genetic diversity of 71 human strains and 17 animal strains of Clostridium perfringens. Crude protein extracts, obtained by sonication of washed bacteria, were analyzed by polyacrylamide-agarose gel electrophoresis to characterize electrophoretic mobility variants of seven enzymes (esterase, glutamate dehydrogenase, glutamic-oxaloacetic transaminase, nucleoside phosphorylase, phosphoglucose isomerase, phosphoglucomutase, threonine dehydrogenase). Genetic diversity of the enzyme loci ranged from 0.340 to 0.813. Sixty-nine electrophoretic types were described among the 88 strains tested and the index of discrimination was 0.994. All strains were typable, and epidemiological relationships between isolates could be established. This method showed a fair correlation with esterase electrophoretic typing based on hydrolytic and electrophoretic polymorphism of esterases. This work demonstrates that multilocus enzyme polymorphism is a reliable and discriminant marker of genetic diversity of strains of C. perfringens.

Improved rapid method for production and purification of Clostridium perfringens type A enterotoxin

Clostridium perfringens strain FD-1041 was grown in Duncan-Strong medium containing raffinose for 5 h at 43°C. Enterotoxin in the cell extract was purified 5.5-fold by a modified ammonium sulfate-gel filtration method using Sephacryl S-200. The enterotoxin could be purified to homogeneity with 50% recovery in less than 48 h.

EVALUATION OF METHYLXANTHINES AND RELATED COMPOUNDS TO ENHANCE CLOSTRIDIUM PERFRINGENS SPORULATION USING A MODIFIED DUNCAN AND STRONG MEDIUM1

Methylxanthines and related compounds were added to Duncan‐Strong (DS) medium in an effort to improve spore yields and enterotoxin production in three strains of Clostridium perfringens. Supplementation with 50–200 μg of caffeine/ml (0.26–1.03 mM) increased heat‐resistant spore yields for strain NCTC 8238 from 4.32 to 6.03 log10 spores/ml after 24 h of incubation. With strains NCTC 8239 and NCTC 10240, 50–200 μg/ml caffeine (0.26–1.03 mM) or theophylline (0.28–1.11 mM) were equally effective, and increased spore yields from 4.18–4.89 to 6.00–7.13 log10 spores/ml. When sporulation and enterotoxin production were evaluated in DS medium containing either starch or raffinose and supplemented with 100 μg/ml caffeine, heat‐resistant spores were first detected at 6 h, and enterotoxin at 3 h of incubation. Raffinose not only yielded higher spore numbers than starch, but also shortened the time to toxin detection and/or increased toxin production by two out of three strains. Strain NCTC 8238 produced enterotoxin (2 ng/ml) with raffinose within 3 h, while strain NCTC 10240 produced 2 ng/ml enterotoxin with starch and higher levels (4 ng/ml) with raffinose at 3 h. In contrast, strain NCTC 8239 produced enterotoxin (2 ng/ml) at 3 h only with starch.

Antibiotic Susceptibility of Anaerobic Bacteria in Europe

Twenty-two laboratories in 15 European countries determined the MICs of 12 antibiotics for 1,289 isolates of the B. fragilis group by a microdilution method. There was no resistance to metronidazole (breakpoint, 8 mg/L), and only one isolate was found to be resistant to chloramphenicol (breakpoint, 8 mg/L). Resistance was uncommon to imipenem (0.3% at concentrations > 4 mg/L), amoxicillin/clavulanate (1% at concentrations > 8 mg/L), cefoxitin (3% at concentrations > 32 mg/L), mezlocillin (6% at concentrations > 64 mg/L), and clindamycin (9% at concentrations > 4 mg/L). The majority of the isolates were resistant to ampicillin (93% at concentrations > 4 mg/L), ciprofloxacin (56% at concentrations > 4 mg/L), and tetracycline (64% at concentrations > 4 mg/L). Bacteroides fragilis was the most susceptible species of the group, and the most striking regional differences in susceptibility were found in association with clindamycin and tetracycline; more resistance was noted in isolates from the southern part of Europe (Greece, Italy, Portugal, and Spain). It has been reported that Fusobacterium species and Clostridium species occasionally produce beta-lactamases. A few metronidazole-resistant Clostridium perfringens strains but no metronidazole-resistant fusobacteria have been isolated.

Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens.

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.

A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells

Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum. A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types. The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Resistance of anaerobic bacteria to antimicrobial agents in Spain

As a consequence of antibiotic consumption, the entire microbial ecosystem attached to man is evolving towards resistance. In Spain, penicillin resistance (MIC > 0.5 mg/l) is found in about 10% of Peptostreptococcus, Clostridium perfringens and Eubacterium, and in 50% of Veillonella. Cefoxitin resistance ( > 2 mg/l) is present in 10 to 20% of Peptostreptococcus and Clostridium, and in 50% of Eubacterium; 21% of Bacteroides (fragilis group) strains are resistant to 16 mg/l. A very low rate of imipenem resistance (> or = 128 mg/l) is found among Bacteroides (1%), but for 5% of the isolates MICs of imipenem are 2 to 4 mg/l. Metronidazole resistance ( > 8 mg/l) is found in 5 to 10% of Peptostreptococcus, Clostridium and Veillonella, and in less than 1% of the Bacteroides fragilis group. Erythromycin resistance ( > 2 mg/l) is present in over two-thirds of the Peptostreptococcus, Veillonella and Fusobacterium isolates, and in 27% of the Clostridium perfringens strains. Clindamycin resistance ( > 4 mg/l) is found in 10 to 20% of the Peptostreptococcus, Clostridium and Eubacterium isolates, and in 20% of the Bacteroides of the fragilis group, this rate being higher (30%) among faecal isolates.

Stimulation of the onset of sporulation of Clostridium perfringens type A by netropsin and distamycin.

The basic peptide antibiotics, netropsin and distamycin, previously shown to inhibit sporulation of Bacillus subtilis, stimulated low levels of sporulation of Clostridium perfringens strain NCTC 8798 at concentrations of 1.0 and 0.1 microgram/ml respectively. Most sporulating cells produced in the presence of the antibiotics were defective. These were blocked at Stage III of sporulation, and many possessed forespores exterior to the sporangium. The same antibiotics could also inhibit the caffeine-induced stimulation of sporulation of this strain.

Kinetics of spore coat protein synthesis byClostridium perfringens type A

Spore coat proteins of several strains ofClostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent−) strain of this organism. The results indicated that spore coat proteins from many strains ofC. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulationspecific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393–410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.