Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum.

A rapid plasmid isolation procedure for Clostridium perfringens and C. absonum is described. The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation. The method can be scaled up, without difficulty, for large-scale plasmid preparation.     

Nitrosoguanidine-induced attenuated Clostridium perfringens type A mutant in gas gangrene]

A fully virulent classical type A strain of Clostridium perfringens was treated during its logarithmic growth phase with 100 mug/ml of N-méthyl-N'-nitro-N-nitrosoguanidine, the bacteria being exposed to the mutagen for 30 min at 37 degrees C in a phosphate buffer adjusted to pH 6.2; after treatment the suspension was streaked on sheep blood agar plates, and colonies that showed an alteration in the theta-hemolysis pattern were selected for isolation. The virulence of two mutants, thus altered in their theta-hemolysis, was studied. One, designated LNG 5, was still capable of killing most of the inoculated guinea pigs in less than 24 h with all the clinical, macroscopic, and bacteriological signs of gas gangrene; however, histological sections showed that tissue damage was not as marked as with the wild strain. On the contrary, the second mutant, labelled LNG 11, was completely avirulent as far as gas gangrene was concerned; indeed, the injection of fluid cultures containing 1 times 10(8) - 10(9)/ml viable bacteria, was not followed by any clinical, bacteriological, or histological signs of gas gangrene. However, strain LNG 11 did give rise to a firm swelling of the inoculated thigh with a corresponding acute inflammatory response of the connective tissue, although the muscle fiber was unaltered. Eventually, this local reaction was followed by necrosis of the skin accompanied by an acute or subacute inflammation with fibroblastic proliferation. These superficial lesions healed spontaneously. They could not be reproduced with crude filtrate alone or with washed bacilli. Strain LNG 11 was therefore considered to be soletly an attenuated strain since, although avirulent as far as gas gangrene is concerned. it is still capable of producing low levels of toxic material. This appears to be the first time that such a strain of C. perfringens type A has been obtained by nitrosoguanidine treatment.     

Clostridium tertium isolated from gas gangrene wound; misidentified as Lactobacillus spp initially due to aerotolerant feature

Clostridium tertium has been increasingly reported as a human pathogen. This organism is an aerotolerant Gram-positive rod that is often mistaken for other organisms, such as Lactobacillus or Bacillus species. We describe a case of a patient with a history of intravenous drug use presenting to UCLA-Olive View Medical Center with gas gangrene of both upper extremities. The organism was initially misidentified as a Lactobacillus species on aerobic culture plates. However, terminal spore formation was detected in this isolate on a sub-cultured anaerobic culture plate and this isolate was confirmed as C. tertium biochemically and genetically by 16S rDNA sequencing. Additional DNA cloning libraries made from the formalin-fixed specimen revealed Peptoniphilus species and an uncultured Clostridium clone, but not C. tertium. C. tertium might be a causative organism of gas-producing myonecrosis but such an association has never been described. Clinicians should be aware of the phenomenon of aerotolerance of some anaerobes and need to clarify the identification of organisms if the clinical picture does not fit the isolated organism.     

The role of neutrophils and monocytic cells in controlling the initiation of Clostridium perfringens gas gangrene

Clostridium perfringens is a common cause of the fatal disease gas gangrene (myonecrosis). Established gas gangrene is notable for a profound absence of neutrophils and monocytic cells (phagocytes), and it has been suggested that the bactericidal activities of these cells play an insignificant role in controlling the progression of the infection. However, large inocula of bacteria are needed to establish an infection in experimental animals, suggesting phagocytes may play a role in inhibiting the initiation of gangrene. Examination of tissue sections of mice infected with a lethal (1 x 10(9)) or sublethal (1 x 10(6)) inoculum of C. perfringens revealed that phagocyte infiltration in the first 3 h postinfection was inhibited with a lethal dose but not with a sublethal dose, indicating that exclusion of phagocytes begins very early in the infection cycle. Experiments in which mice were depleted of either circulating monocytes or neutrophils before infection with C. perfringens showed that monocytes play a role in inhibiting the onset of gas gangrene at intermediate inocula but, although neutrophils can slow the onset of the infection, they are not protective. These results suggest that treatments designed to increase monocyte infiltration and activate macrophages may lead to increased resistance to the initiation of gas gangrene.     

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.     

Further studies on the bile salt induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum

Optimal induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase in 100-ml cultures grown to stationary phase was achieved by the addition of metabolizable bile salt inducers: chenodeoxycholate, 7-ketolithocholate or cholate at 2.5-3 h after inoculation. Bile salt addition prior to or after this period markedly reduced the enzyme levels induced. However, when the non-metabolizable inducers deoxycholate and 12-ketolithocholate were similarly added, no significant differences in enzyme levels were observed between addition at 2.5-3 h or at earlier times. The ability of both metabolizable and non-metabolizable bile salts to induce the enzymes fell markedly when additions were made later than approximately 3.5 h. Kinetic studies using 1-l cultures suggest that in a larger culture a somewhat earlier inducer addition period is optimal. When ranked according to the level of enzymes induced the order in decreasing induction power was: chenodeoxycholate, 7-ketolithocholate, deoxycholate, 12-ketolithocholate and cholate. Mixtures of cholate and suboptimal concentrations of deoxycholate induced the culture better than the sum of the two concentrations individually. The end product, ursodeoxycholate, was very effective in blocking the induction by chenodeoxycholate or deoxycholate. Ursocholate (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoate) was less effective. Cultures when grown for 3 h with various bile salts or none, then centrifuged and recultured for a further 3 h in fresh medium containing chenodeoxycholate, all yielded identical enzyme levels within experimental error. We conclude that exposure of the organism to bile salt inducer in the last 3 h of culture was important, while the history of the culture prior to this time was unimportant in the induction process.     

Conversion of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium absonum in culture and by immobilized cells

Abstract Volatile fatty acids (VFA) have interesting biological effects on eukaryotic cells, inducing alterations of cell shape, morphological differentiation, changes in the composition of the cytoplasmic membrane and growth inhibition. This paper describes the effects of VFA on granulocyte chemotaxis. The influences observed, usually inhibitory, were shown to depend on the concentration tested and the incubation atmosphere (anaerobic or 5% CO₂).
Because these metabolites are produced by several anaerobic bacteria both in vitro and at infected sites, one can speculate that they might act as potential 'leukotoxins' contributing to the pathogenicity of anaerobic bacteria.
We suggest that the well-known ability of anaerobes to inhibit their own phagocytosis and intracellular killing, as well as that of facultative anaerobes, might be at least partially due to the impairment of granulocyte functions induced by VFA.     

Purification and Characterisation of Xylanolytic Enzymes of a Cellulase-free Thermophilic strain of Clostridium absonum CFR-702

Two endo-β-1-4-xylanases (EC 3.2.1.8), xylanase-I and xylanase-II, were purified fromClostridium absonum CFR-702 by ammonium sulphate precipitation and chromatographed on DEAE-Cellulose and phenyl-Sepharose. The enzymes in sodium dodecyl sulphate polyacrylamide gels resolved as proteins corresponding to molecular mass 150 and 95 kDa for xylanase-I and xylanase-II, respectively. The optimum pH and temperature ranges for the enzyme activities on birchwood xylan were between 6.5 and 7.5 and 75°C for xyl-I and 7.5 and 80°C for xyl-II. Xyl-I was stable up to 60°C whereas xyl-II was stable at 50°C. Both the enzymes liberated xylobiose, xylotriose and xylotetraose from birchwood xylan. Xyl-I and xyl-II with birchwood xylan had K_mvalues of 1.1 and 1.4%, and V_maxvalues of 454.54 and 363.63 μmol/min/mg protein respectively.