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PubMed1969

Biosynthesis of desthiobiotin in cell-free extracts of Escherichia coli.

Cell-free extracts of Escherichia coli were active in catalyzing the synthesis of a biotin vitamer from 7,8-diaminopelargonic acid. The vitamer was identified as desthiobiotin on the basis of its chromatographic and electrophoretic characteristics and its biotin activities for a variety of microorganisms. The reaction was stimulated five-fold by bicarbonate, suggesting that an "active CO(2)" was incorporated into the carbonyl carbon of desthiobiotin. The enzyme was demonstrable in a wild-type (K-12) and in all biotin mutants of E. coli that were tested, with the exception of a strain which was able to grow on desthiobiotin but not on diaminopelargonic acid. Furthermore, the enzyme was repressible by biotin in all of the strains tested. These results are consistent with the hypothesis that the biosynthesis of desthiobiotin from 7,8-diaminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.

Bicarbonates↗
PubMed1983

Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography.

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.

Caproates↗
PubMed1984

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin.

A procedure was devised for routine examination of feces for Clostridium difficile with selective enrichment broth culture containing increased levels of carbohydrates and antibiotics to detect cytotoxin and volatile acids in broths inoculated with fecal samples. C. difficile was detected and identified with a rapidity comparable to that of conventional culture on selective cycloserine-cefoxitin fructose agar. Detection rates for C. difficile in inoculated broths (111/401 or 27%) were significantly higher than for culture on cycloserine-cefoxitin fructose agar (47/401 or 11%, P greater than 0.001). All fecal samples containing C. difficile and cytotoxin were correctly identified by the procedure. Isocaproic acid peak heights greater than 2 mm in selective enrichment broths inoculated with fecal samples indicated that C. difficile was present in the fecal sample examined. Of the positive specimens examined, 58% (64/111) produced peak heights greater than 10 mm. Peak heights less than 2 mm were not associated with C. difficile in the fecal sample. The investigated procedure provided a reliable alternative to the routine processing of feces for detecting C. difficile and associated cytotoxin in feces. Inoculated broths with isocaproic acid peak heights greater than 2 mm, after 24 to 48 h of incubation, and in which cytotoxin was detected, were subcultured to blood agar to obtain isolates of the organism as required. Broths which showed isocaproic acid peak heights less than 2 mm, and in which cytotoxin was not detected, were discarded as negative for C. difficile. The procedure was deemed potentially useful for epidemiological surveys of C. difficile.

Bacterial Proteins↗
PubMed1978

Detection of alcohols and volatile fatty acids by head-space gas chromatography in identification of anaerobic bacteria.

A head-space gas chromatographic technique for the analysis of volatile bacterial metabolites is described. Bacteroides fragilis, Clostridium perfringens, and Propionibacterium acnes, cultured in a glucose-containing peptone yeast extract medium, were studied. The head-space technique was compared with the injection of the complete liquid culture medium, and solvent extracts thereof, into the gas chromatograph. Volatile fatty acids could be detected by all three methods, whereas alcohols produced by C. perfringens and P. acnes were detectable only in the head-space chromatograms. Both FFAP and Porapak Q were used as gas chromatography stationary phase. Porapak Q was found more suitable than FFAP for the separation of alcohols. The head-space technique requires a minimum of preparation before the analysis and is well suited to automation.

Alcohols↗
PubMed1983

Gas-liquid chromatographic analysis of synovial fluid: volatile short-chain fatty acids in septic arthritis.

Volatile short-chain fatty acids (SCFA) in synovial fluid from 80 patients were quantified by gas-liquid chromatography (GLC). Characteristic patterns of volatile SCFA could not be associated with septic, nonseptic inflammatory, or noninflammatory groups. Mean concentrations of pentanoic and hexanoic acids were similar in all groups studied. In the septic arthritis group 3 of 4 patients with acetic acid and of 3 of 3 with 3-methyl butanoic acid had culture-proved staphylococcal infections. In patients with synovial fluid findings consistent with septic arthritis, including markedly raised leucocyte count, decreased glucose level, or detectable succinic acid, the detection of acetic and 3-methyl butanoic acids by GLC analysis may increase the suspicion that Staphylococcus aureus is the cause of the septic arthritis.

Arthritis↗
PubMed1974

Medium chain triglycerides and hepatic encephalopathy.

The oral administration of short (C(6)) and medium (C(8) and (C(10)) chain triglycerides produced no clinical or electroencephalographic changes in patients with cirrhosis of the liver. Arterial ammonia levels were also monitored in these patients and showed no significant change after medium chain triglycerides. It was concluded that medium chain triglycerides, known to be of potential value in the treatment of malabsorption in patients with cirrhosis, are not clinically contraindicated, even in patients with evidence of hepatic encephalopathy.

Ammonia↗
PubMed1997

Oxidation of short and medium chain C2-C8 fatty acids in Sprague-Dawley rat colonocytes.

BACKGROUND: The predominant colonic short chain fatty acids, acetate, propionate, and butyrate, are oxidised into CO2 in colonocytes from rat and humans in the preferred order of butyrate (C4) > propionate (C3) > acetate (C2)- hence butyrate is considered to be the principal oxidative substrate for colonocytes. AIMS: To compare colonocyte oxidation of valerate (C5), hexanoate (C6), and octanoate (C8) with that of butyrate. METHODS: Isolated rat colonocytes were incubated in the presence of a concentration range of 1-14C labelled C2-C8 fatty acids. Oxidation rates were obtained by quantifying the production of 14CO2, and Vmax (maximum velocity) and K(m) (Michaelis-Menten constant) were calculated by computer fitting of the data to a Michaelis-Menten plot. RESULTS: The K(m) value of acetate (0.56 (SEM 0.02) mmol/l) was about fourfold higher than the K(m) of butyrate (0.13 (0.01) mmol/l), whereas the K(m) values of valerate (0.19 (0.01) mmol/l), hexanoate (0.19 (0.01) mmol/l), and octanoate (0.16 (0.01) mmol/l) were of the same order of magnitude as the K(m) of butyrate. Acetate did not influence butyrate oxidation, whereas butyrate strongly inhibited the oxidation of acetate. By contrast, valerate, hexanoate, and octanoate inhibited colonocyte oxidation of butyrate equally or more than the reverse inhibitory effect of butyrate on valerate, hexanoate, and octanoate oxidation. The maximum rates of ATP production were in the order of valerate > octanoate = hexanoate > butyrate > acetate (28.47 (0.70), 21.78 (0.75), 21.33 (0.78), 16.12 (0.49), 9.09 (0.34) (mumol/min/g) respectively). CONCLUSIONS: Valerate, hexanoate, and octanoate seem to be excellent substrates for colonocyte oxidation, similar to butyrate. These results may influence the choice of fatty acid composition in enemas used for treatment of patients in whom deficient colonocyte oxidation is suspected-for example, patients with ulcerative colitis and diversion colitis.

Acetates↗
PubMed1983

Use of gas-liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools.

In order to determine if gas-liquid chromatography (GLC) on concentrated stool extracts could be substituted to cell culture assay for cytotoxicity, we prospectively studied 154 diarrhoeal stools submitted for detection of Clostridium difficile toxin. Isocaproic-positive samples were cultured on egg yolk agar supplemented with cycloserine, cefoxitin and fructose for isolation of C difficile, and on egg yolk agar plus kanamycin for isolation of other clostridium species. Of the 154 samples, 129 were GLC-negative (height of the isocaproic peak less than 1.2 cm) and were toxin-negative. Twenty-five stools yielded isocaproic acid; C difficile isolated from 13 of them, six of which were also toxin-positive. Four other isocaproic-positive samples yielded C bifermentans and C sordellii; all were toxin-negative. These results indicate that a negative GLC is an excellent screening test for excluding C difficile infection; positive results must be checked by toxin testing and culture since they are not necessarily associated with the presence of C difficile or its toxin.

Bacterial Proteins↗
PubMed1984

Detection of Clostridium difficile in faeces by direct gas liquid chromatography.

Stool specimens examined for the presence of Clostridium difficile and its cytotoxin were screened by gas liquid chromatography for the presence of volatile fatty acids and p-cresol. Twenty seven of 110 (25%) stools yielded C difficile or cytotoxin; iso-valeric acid was detected in 63/110 (57%) and iso-caproic acid in 18/110 (16%) stools. Para-cresol was found in 24/71 (34%) stools examined. Iso-valeric acid was detected in 85% of stools positive for C difficile, whereas iso-caproic acid (41%) and p-cresol (52%) were found in much lower numbers of C difficile-positive stools. It is concluded that gas chromatographic detection of volatile fatty acids or p-cresol in faeces are not satisfactory screening tests for the presence of C difficile.

Caproates↗
PubMed1985

Gas chromatographic identification of Clostridium difficile and detection of cytotoxin from a modified selective medium.

A modification of an existing selective medium for Clostridium difficile is described. Inclusion in the medium of DL nor-leucine and p-hydroxyphenylacetic acid enables identification of C difficile to be made directly from primary isolation plates by gas chromatographic detection of caproic acid and p-cresol. Plugs of agar withdrawn from the selective medium also allow the detection of cytotoxin production in vitro.

Caproates↗
PubMed1985

Evaluation of gas-liquid chromatography for the rapid diagnosis of Clostridium difficile associated disease.

Direct gas-liquid chromatography of faecal specimens with isocaproic acid as a marker was used for the rapid diagnosis of Clostridium difficile associated diarrhoeal diseases. Ninety stools were examined and results were compared with conventional culture on selective medium and cytotoxin assay in tissue culture. Using a combined analysis of isocaproic acid and butyric acid peak heights we defined three categories: positive, negative, and indeterminate. When the indeterminate group was excluded, the positive and negative predictive values of gas-liquid chromatography analysis were 86.9% and 85% respectively compared with culture and 71.4% and 95% respectively compared with cytotoxin assay.

Caproates↗
PubMed1986

Latex particle agglutination for detecting and identifying Clostridium difficile.

A total of 329 selective enrichment broth cultures were tested for detection of Clostridium difficile by latex particle agglutination (LPA), gas-liquid chromatography, and bacterial culture. Of 53 broths positive by LPA, 36 were positive by gas-liquid chromatography, and 42 were positive by bacterial culture. The sensitivity and specificity of LPA relative to bacterial culture was 95.6% and 96.3%, respectively, while the sensitivity and specificity of gas-liquid chromatography relative to bacterial culture was 84.6% and 100%, respectively. The high predictive value of a negative test (99%) should make LPA on broth cultures a good screening test for detecting C difficile. Of several other Clostridium spp tested in pure culture, strains of C sordellii and C bifermentans also gave a positive result by LPA. These results, together with the low cost and simple facilities required, suggest that the LPA test will be a useful procedure for the presumptive identification of C difficile in selective enrichment broths and for the identification of pure cultures.

Caproates↗
PubMed1998

Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate).

The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment. A 2.8-kb XhoII subfragment was found to contain the intact PHA synthase. This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function. On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity). The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R. eutropha and Pseudomonas putida. The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed. In these strains, the PHA synthase from N. corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids.

Acyltransferases↗