Isolation and Characterization of Uracil-Degrading Clostridia from Soil

Five strains of uracil-degrading clostridia were isolated from soil using an enrichment medium containing uracil as the principal energy source. All the isolates resembled Clostridium glycolicum , although they lacked the characteristic ability to ferment ethylene glycol.
Both representative isolates and a reference strain of Cl. glycolicum degraded uracil and produced β-alanine when grown in a semi-defined medium. Thus, the uracil-degrading activity was similar to that described previously for Cl. uracilicum.     

Clostridium bartlettii sp. nov., isolated from human faeces

Seven obligately anaerobic, Gram-positive, rod-shaped, spore-forming organisms isolated from human faecal specimens were characterized using phenotypic and molecular taxonomic methods. Strains of the unidentified bacterium used carbohydrates as fermentable substrates, producing acetic acid, isovaleric acid and phenylacetic acid (PAA) as the major products of glucose metabolism, and possessed a G +C content of approximately 29.8 mol%. Comparative 16S rRNA gene sequencing showed that the 7 strains were genetically highly related to each other (displaying >99.5% sequence similarity) and represent a previously unknown sub-line within the Clostridium Cluster XI. The closest described species to the novel bacterium is Clostridium glycolicum, although a 16S rRNA sequence divergence of 4% demonstrates that they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and C. glycolicum (30.6% similarity between the proposed type strain of the novel species, WAL 16138, and C. glycolicum ATCC 14880(T)). Based on morphologic, phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium be classified as C. bartlettii sp. nov. The type strain of C. bartlettii is WAL 16138(T) (= ATCCBAA-827(T)=CCUG48940(T)).     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Comparison of latex agglutination and ELIS A for the detection of Clostridium perfringens type A enterotoxin in faeces

One hundred and thirty one faecal specimens from cases of suspected Clostridium perfringens food poisoning were examined by both a reverse passive latex agglutination test and a standard ELISA test for the presence of Cl. perfringens enterotoxin. The latex agglutination test proved as sensitive and specific as the ELISA, and required less time at the bench without the need for specialized equipment.     

Thiaminase Activity Amongst Strains of Clostridium sporogenes

All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum

The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man. Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion. The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h. The aim of this study was to examine the efficiency of the identification of Cl. botulinum using the Rapid ID32 A. Forty-two strains of Cl. botulinum , one strain each of botulinum toxin-producing Cl. butyricum and Cl. baratii , and four strains of Cl. sporogenes , were tested. One strain of Group I Cl. botulinum gave a presumptive identification of Group II Cl. botulinum , six strains of Cl. botulinum were identified as 50–98% Cl. botulinum in some tests, and 17 strains of Cl. botulinum were identified as <50% Cl. botulinum. Thirteen strains of Cl. botulinum were identified as >99% Cl. sporogenes or 86% Cl. histolyticum , and five strains gave a combination of these results. All strains of Cl. sporogenes were correctly identified. These results show that some strains of Cl. botulinum may not be correctly identified using the Rapid ID32 A.     

Phylogenetic characterization of several para- and meta-PCB dechlorinating Clostridium species: 16s rDNA sequence analyses

The genus Clostridium has more than 127 species, grouped according to their morphology and functions. Nine Clostridium species were identified based on their ability to dechlorinate meta- and para-PCB (polychlorinated biphenyl) contaminated sediments. The phylogenetic relatedness of these PCB-degrading Clostridium species was studied using ribosomal RNA genes. The diversity of small-subunit rRNA genes associated with the domain bacteria was examined using defined operational taxonomic units (OTUs) in samples from PCB contaminated sediments from Lake Medinah, New York. The RFLP (restriction fragment length polymorphism) of the OTVs was measured. OTUs B (105 clones), A (33 clones) and C (45 clones) accounted for 75% of all the 16S rDNA clones expressing anaerobic para- and meta-PCB dechlorinating activity. In this report we describe complete 16S rDNA sequences of OTU-A and OTU-B, and partial rDNA sequences of OTUs C-J. The OTU-B and OTU-I form a phylogenetically related cluster, closely affiliated with Clostridium hydroxybenzoicum strains. OTUs A, C, D, G, H and J also belong to the genus Clostridium, but they represent separate species. OTU-E, a close affiliate to Bacteroides forsynthus, is a meta-PCB dechlorinator. The Cl. hydroxybenzoicum strains (OTU-B) are primarily para-PCB dechlorinators and are the most common. Some less prevalent OTUs (- E, -G, -H and -I), are also mostly para-PCB dechlorinators. Other Clostridium species such as Cl. beijerinckii (OTU-A), Cl. intestinalis (OTU-D) and Cl. thermolacticum (OTU-J) are primarily meta-PCB dechlorinators. Cl. paraputrificum (OTU-C) and Cl. cellulosi (OTU-F), were less prevalent in the total consortium, but they could dechlorinate both para- and meta-PCB. Although a few less prevalent Clostridium species can degrade both para- and meta-PCBs, this study confirms that para- and meta-PCB dechlorinating species are generally phylogenetically different.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

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.     

Further development of a bacteriocin typing system for Clostridium perfringens

The specificity of typing Clostridium perfringens with bacteriocins was improved by adding new bacteriocins and deleting others from the original typing set of ten. A total of 516 new isolates of Cl. perfringens were screened for bacteriocin production and, of these, 162 strains (31%) were found to be producers. The sensitivity patterns obtained by testing 40 bacteriocins against 200 isolates of Cl. perfringens were recorded and the data subjected to a computer analysis. A total of 18 bacteriocins capable of dividing the 200 isolates into 98 typing patterns was selected. The repro-ducibility of the new system was tested by performing three sequential typings of 60 strains of Cl. perfringens. No variation was found in 73% of the strains, while a further 16% of the strains demonstrated a change in sensitivity to only one bacteriocin. Common serological types of Cl. perfringens were divisible into subtypes based upon both their ability to produce bacteriocins and their sensitivity to bacteriocins, suggesting a useful role for bacteriocin typing in conjunction with an already well-established tool for typing Cl. perfringens.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

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 .     

Some Observations on OS Variants of Clostridium botulinum Type E

Three strains of OS variants of Clostridium botulinum type E (Dolman, 1957) have been studied using additional biochemical tests and fluorescent antisera. The relationship of these organisms, and other nontoxic clostridia resembling Cl. botulinum , to typical toxic strains is discussed.     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

The Incidence of Organisms of the Genus Clostridium in Vacuum Packed Fish in the United Kingdom

Of a total of 224 samples of commercial vacuum packed fish products examined for the presence of Clostridium spp., 151 gave positive cultures. The predominant species was Cl. welchii; Cl. botulinum was not isolated. Spore suspensions of 29 strains of Cl. welchii were tested for heat resistance; none survived treatment for 5 min at 100°.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N-methyl-N'-nitro-N-nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N -methyl- N '-nitro- N -nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

The effect of sodium chloride and temperature on the rate and extent of growth of Clostridium botulinum type A in pasteurized pork slurry

A selective medium was used to enumerate Clostridium botulinum growing in the presence of natural spoilage organisms in a model cured pork slurry. The growth responses of a mixed spore inoculum of six strains of Cl. botulinum type A were studied at 15 degrees, 20 degrees and 27 degrees C with 1.5, 2.5, 3.5 or 4.5% (w/v) salt added (aw range 0.961-0.990). Gompertz and logistic curves, which have a sigmoid shape, were fitted to the data and lag times, growth rates, generation times and time to maximum growth rates were derived. Variation in germination rates of the spores occasionally gave a falsely extended lag time resulting in an exceptionally high estimate for growth rate. Products containing 4.5% (w/v) NaCl would be capable of supporting growth of proteolytic strains of Cl. botulinum, even at 15 degrees C, although the lag period would be extended. In products where absence of Cl. botulinum cannot be assured additional preservative measures are essential. The information obtained provides a framework to investigate the effects of a wider range of additives or variables on the growth responses of Cl. botulinum.