Cross reactivity of Clostridium glycolicum with the latex particle slide agglutination reagent for Clostridium difficile identification

Three strains of unidentified clostridia and 15 known strains of Clostridium glycolicum were examined to investigate cross reactions with the latex particle agglutination reagent used to identify Clostridium difficile. The unknown strains were identified as Cl. glycolicum and cross reacting agglutination occurred in 15/15 (100%) stock Cl. glycolicum strains. Characteristics such as volatile fatty acid profile, propylene glycol fermentation, u.v. fluorescence and the production of p -cresol are required to distinguish between the two species.     

A comparison of the requirements for various carbon and nitrogen sources and vitamins in some Frankia isolates

Summary It was established that anAlnus glutinosa isolate (LDAgp 1) is able to utilize mono- and disaccharides and shows a limited growth ability on arabinose and starch. This contrasts with an isolate fromAlnus viridis (AvcI 1) andComptonia peregrina (CpI1), which apparently lack glycolytic pathway activity. These latter isolates can utilize some tricarboxylic acids in contrast to LDAgp1. Volatile fatty acids or their salts, such as propionic acid and acetate, were utilized by all three isolates. Besides a general ability to utilize inorganic nitrogen sources, some amino acids and urea, selected isolates showed a limitedability to utilize adenine and uracil. A simple, synthetic medium based on propionic acid as the energy source was developed. On this medium some isolates showed growth stimulation in the presence of biotin. The metabolic aspects of the utilization of carbon and nitrogen sources, as well as some ecological consequences are discussed.     

Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion

De novo pyrimidine synthesis was studied in mouse liver, intestine, and kidney by intraperitoneal infusion of 15NH4Cl and analysis of 15N incorporation into uracil nucleotide pools. When the dose of a 1-h infusion of 15NH4Cl was increased from 50 mumol to 250 mumol the fraction of the total uracil nucleotide pool formed by de novo synthesis increased 4.0-fold in liver to 8.4% and 2.3-fold in intestine to 13.7%. The increase in intestine was independent of the increase in liver as evidenced by the lack of correlation between the increase observed in the intestine and liver of the same animal and the different distributions of label in the uracil ring nitrogens. A 2.4-fold increase in newly formed uracil nucleotides was observed in kidney when the infusion dose was raised from 150 mumol to 250 mumol. The increase in kidney was correlated with the increase in liver in the same animal and the distribution of label in the uracil ring nitrogens was similar to the distribution in liver. These results suggest that the increase in newly formed uracil nucleotides in intestine is due to increased de novo synthesis of pyrimidines in the intestine, while the increase in the kidney is due to increased salvage synthesis of uracil nucleotides from uridine synthesized in the liver and output to the circulation.     

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.     

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 .     

Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.     

Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains.

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.     

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)).     

Auxotypes of Neisseria gonorrhoeae isolated from localized and disseminated infections in Montreal.

A survey recently made in the United States on the regional distribution of auxotypes of Neisseria gonorrhoeae suggested that isolates from different geographic areas often differ in auxotype. A subsequent auxotyping study in Montreal of 901 isolates of N. gonorrhoeae, 15 from patients with disseminated gonococcal infection, proved interesting in many regards. Gonococcal genetic medium, modified by the addition of other amino acids, was used. Most (93%) of the strains isolated from patients with localized infection belonged to one of the following three phenotypes: arginine-, hypoxanthine- and uracil-dependent (44%); prototrophic (33%); and proline-dependent (16%). Of the 15 strains responsible for disseminated infection 14 required arginine, hypoxanthine and uracil for growth.     

Active pyrimidine absorption by chicken colon

Pyrimidine absorption by chicken large intestine was investigated employing the everted sac and flux chamber techniques. 3H-labelled uracil was used as substrate. The small intestine and the colon unlike the caecum, transported uracil from the mucosal to the serosal surface against a concentration gradient in the everted sac experiments. Furthermore, there was a net transport of uracil from the mucosal to the serosal side of the colon and jejunum in the flux chamber experiments. Uracil transport by the everted colon sacs against a concentration gradient was inhibited when the purine hypoxanthine was present in the incubation medium. Uracil transport by the everted colon sacs was also inhibited under anaerobic conditions and when 2,4-dinitrophenol was present in the incubation medium. Replacing the Na+ ions of the incubation medium by Li+ ions also caused an inhibition of uracil transport. It is concluded from these results that uracil (and probably other pyrimidines) are absorbed from the chicken colon by a Na+ ion-dependent active transport process having also an affinity for purines.     

Ribotyping of Clostridium perfringens from industrially produced ground meat

AIMS: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial species identification and subtyping. METHODS AND RESULTS: The current study has used ribotyping to examine 111 Cl. perfringens isolates from industrially produced ground meat in order to collect a basis for a contamination survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical ribotype patterns in three different runs, carried out 3-4 months apart from each other. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The discriminatory index for EcoRI ribotyping of the Cl. perfringens isolates was 0 x 99. Results showed that ribotyping is suitable for subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for profound epidemiologic studies of Cl. perfringens-contamination in food production and processing.     

灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定

【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots

An anaerobic, H(2)-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H(2)-CO(2), formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O(2) in the headspace of static, horizontally incubated culture tubes; the concentration of O(2) decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O(2). In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H(2) became significant end products when RD-1 was grown on glucose in the presence of O(2). Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H(2) were produced. When the concentration of O(2) in the headspace exceeded 1% (vol/vol), supplemental H(2) was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288(T), an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288(T) demonstrated that it had negligible H(2)- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288(T). A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288(T) confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O(2), (iii) oxic conditions favor the production of ethanol, lactate, and H(2) by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O(2) might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O(2).     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Uridine catabolism in Kupffer cells, endothelial cells, and hepatocytes

Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-14C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 15 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells. Kupffer cells and endothelial cells did not produce radioactive CO2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into CO2, beta-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.     

Uracil salvage pathway in PC12 cells

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.     

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.