Rapid and Sensitive Detection of Bacteria by Gas Chromatography

A gas chromatograph fitted with electron capture and flame ionization detectors was employed for the rapid detection of bacteria by analysis for their metabolic products. The presence of Proteus vulgaris, Streptococcus faecalis, S. liquefaciens, Escherichia coli B, Bacillus cereus, and B. popilliae was detected in 2 to 4 hr in media inoculated with less than 10(4) cells per ml, whereas a 7- to 12-hr growth period was required for the detection of products formed in cultures of Serratia marcescens, Aerobacter aerogenes, E. coli K-12, Staphylococcus aureus, and Salmonella typhimurium. Metabolites elaborated by the equivalent of less than a single cell of B. cereus, S. faecalis, P. vulgaris, or E. coli B were sensed by the electron capture detector. The flame ionization detector was generally not as sensitive. Volatile metabolites were identified, and their concentrations were determined.     

Metabolism of Tryptophan by Pseudomonas aureofaciens: III. Production of Substituted Pyrrolnitrins from Tryptophan Analogues 1

Exogenous tryptophan is metabolized by Pseudomonas aureofaciens to yield pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorophenyl)-pyrrole], an antifungal agent. The ability of this culture to metabolize tryptophan analogues in a similar manner was investigated by addition of the appropriate compound to the fermentation. Tryptophan precursors and metabolites or nonphenyl-substituted tryptophans had little effect on pyrrolnitrin biosynthesis, but simple derivatives of indole inhibited the production of pyrrolnitrin. Tryptophans substituted at the 4 position decreased pyrrolnitrin production and were converted into the corresponding substituted indoles. Tryptophans substituted at the 5, 6, and 7 position with fluorine or at the 5 and 7 position with methyl yielded new pyrrolnitrin derivatives. Substitution of larger groups (such as chloro, bromo, trifluoromethyl, and methoxy) at these positions led to the formation of the intermediate, amino pyrrolnitrin [3-chloro-4-(2'-amino-3'-chlorophenyl)-pyrrole], with the appropriate new substituent. The trifluoromethyl group at the 6 position of tryptophan prevented chlorination at the 3 position of pyrrolnitrin.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Mechanism of Action of the Antifungal Antibiotic Pyrrolnitrin

Pyrrolnitrin at 10 mug/ml inhibited the growth of Saccharomyces cerevisiae, Penicillium atrovenetum, and P. oxalicum. The primary site of action of pyrrolnitrin on S. cerevisiae was the terminal electron transport system between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. At growth inhibitory concentrations, pyrrolnitrin inhibited endogenous and exogenous respiration immediately after its addition to the system. In mitochondrial preparations, the antibiotic inhibited succinate oxidase, NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and succinate-coenzyme Q(6) reductase. In addition, pyrrolnitrin inhibited the antimycin-insensitive reduction of dichlorophenolindophenol and of the tetrazolium dye 2,2'-di-p-nitrophenyl-(3,3'-dimethoxy-4,4'-bi-phenylene)5,5'-diphenylditetrazolium. The reduction of another tetrazolium dye, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, that was antimycin-sensitive, was also inhibited by pyrrolnitrin. The antibiotic had no effect on the activity of cytochrome oxidase, and it did not appear to bind with flavine adenine dinucleotide, the coenzyme of succinic dehydrogenase. In whole cells of S. cerevisiae, pyrrolnitrin inhibited the incorporation of (14)C-glucose into nucleic acids and proteins. It also inhibited the incorporation of (14)C-uracil, (3)H-thymidine, and (14)C-amino acids into ribonucleic acid, deoxyribonucleic acid, and protein, respectively. The in vitro protein synthesis in Rhizoctonia solani and Escherichia coli was not affected by pyrrolnitrin. Pyrrolnitrin also inhibited the uptake of radioactive tracers, but there was no general damage to the cell membranes that would result in an increased leakage of cell metabolites. Apparently, pyrrolnitrin inhibits fungal growth by inhibiting the respiratory electron transport system.     

Glass capillary columns applied to prostaglandins measurement: a useful tool for gas-chromatography mass spectrometry analysis

The difficulties to analyse prostaglandins (PG) by gas-liquid chromatography are mainly due to the lack of sensitivity of the gas-chromatograph itself (higher than 200 ng) and to the poor resolution of the packed columns. Therefore the use of glass capillary columns which has been applied with success for other biological compounds was tempting. We describe a comparison of the preparation of the columns and their use for PG analysis of standards and of human semen. A complete resolution of PG-1 from PG-2 series was achieved. The sensitivity was multiplied 100 fold with a flame ionisation detector when compared to packed columns and was equal to the one obtained with electron capture detectors without the inconveniences of this technique. The successful coupling of glass capillary columns to a mass spectrometer leads to promising results and allows profile studies of primary PG and their metabolites as seen with human semen.     

Quantitative determination of histamine metabolites by capillary gas chromatography

A method using capillary gas chromatography is described for the determination of histamine and eight of its basic and acid metabolites in a single biological sample of serum, urine, or gastric juice. Ion-exchange chromatography and extraction with organic solvents are used for isolation and purification, and gas chromatography for identification and quantitation. The heptafluorobutyryl derivatives of histamine and some basic metabolites are compatible with nitrogen-phosphorus and electron capture detection modes and offer an excellent sensitivity (detection limit 0.1 pmol with electron capture). The acid metabolites are quantitated after esterification. The linearity range, the sensitivity, a partial study of reproducibility and typical chromatograms show that the method is adaptable to a variety of applications.     

Metabolism of Tryptophans by Pseudomonas aureofaciens: VI. Production of Pyrrolnitrin by Selected Pseudomonas Species

Twenty-nine strains of Pseudomonas, classified as P. fluorescens biotype D or E or as P. multivorans, were examined for the production of pyrrolnitrin, an antifungal agent synthesized in P. aureofaciens. Eight strains were shown to produce pyrrolnitrin in shake-flask fermentation. Four cultures were from the multivorans taxon, and the remaining four were members of the fluorescens group. The antifungal agent produced in these strains was isolated and shown to be pyrrolnitrin by comparison with an authentic sample. The strains differed markedly with respect to the amount of pyrrolnitrin produced and in their utilization of exogenous tryptophan. Secondary metabolites, not related to pyrrolnitrin, were also examined and compared with those synthesized in P. aureofaciens. Marked differences were noted in both phenazine pigments and phenolic metabolites. The results of the study suggest that the production of pyrrolnitrin may be widespread in selected taxonomic groups of Pseudomonas.     

Gas—liquid chromatographic method for the measurement of plasma levels of d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine acid maleate (SCH-12679) and its major metabolites in aggressive mental retardates

A sensitive gas—liquid chromatographic technique for the quantitative analysis of SCH-12679 (d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine acid maleate) and its major metabolites in plasma of aggressive mental retardates receiving therapeutic doses of the medicament has been developed. The lower limits of detection are 20 ng/ml for SCH-12679, 0.5 ng/ml for 3-desmethyl SCH-12679 and 0.4 ng/ml for 7-desmethyl plus 8-desmethyl SCH-12679. SCH-12679 is estimated with a flame ionization detector. Its metabolites are quantitated using an electron-capture detector after conversion of the compounds to their heptafluorobutyryl derivatives by reaction with the appropriate anhydride. Data on plasma levels of unchanged SCH-12679, 3-desmethyl SCH-12679 and a combination of 7-desmethyl and 8-desmethyl SCH-12679 in fifteen patients treated with the medicament are presented.     

Work in progress: Analysis of the prostaglandins E by glass capillary gas chromatography with electron capture detection

Using a wide bore capillary column coated with D.E.G.S. in combination with a micro volume ⁶³Ni electron capture detector a simple and rapid method with high sensitivity was developed for the analysis of E prostaglandins.For electron capture detection the naturally occurring PGE's have to be transformed to the PGB configuration, esterified and silylated. This method with sensitivity in the low picogram range, tested with prostaglandin standards, will be used for the analysis of prostaglandins and prostaglandin metabolites in biological fluids.     

Gas chromatography and detection of microquantities of gibberellins and indoleacetic acid as their fluorinated derivatives

Gas chromatographic analysis of halogenated derivatives of the plant hormones IAA and gibberellins is described, employing an electron capture detector. Heptafluorobutyryl (HFB) and trifluoroacetyl (TFA) derivatives of the gibberellin and IAA methyl esters have excellent chromatographic properties and allow quantitative and qualitative analysis on the nanogram to picogram level. The methods have been used in analysis of apple seed hormones.     

Application of the flame photometric sulfur detector to the gas-chromatographic analysis of trimethylsilylated methylthiohydantoins of amino acids

Application of the flame photometric detector was made to the gasliquid chromatographic analysis of trimethylsilylated methylthiohydantoins. Principal advantage of the detector proved to be its specificity. The detector was sensitive to the trimethylsilyl derivatives of each of nineteen methylthiohydantoins studied, and extraneous peaks which were occasionally observed with the flame ionization detector were completely absent. Linear calibration curves (log peak area vs log amount chromatographed) were obtained for twelve of the methylthiohydantoins studied, and a rough quantitative estimate of the amount present could be made for the remaining seven.     

A sensitive determination of α-keto acids by gas-liquid chromatography and its application to the assay of l-glutamate dehydrogenase and aminotransferases

A sensitive and selective determination of α-keto acids was established by the use of a gas chromatograph equipped with an electron capture detector. α-Keto acids (pyruvic, oxaloacetic, α-ketobutyric, and α-ketoglutaric acids) were reacted with pentafluorophenylhydrazine, and the derivatives were extracted with ethyl ether, reacted with diazomethane, and were subjected to gas-liquid chromatography with an electron capture detector. In the course of the reaction, oxaloacetic acid was decarboxylated, and yielded pyruvic acid. In the case of pyruvic (oxaloacetic) and α-ketobutyric acids two peaks corresponding to the syn and anti forms of the hydrazone appeared, and in the case of α-ketoglutaric acid, two peaks corresponding to the hydrazone and the cyclization compound produced from the hydrazone. The sum of the two peaks was taken for the determination. The present method was applicable to the assay of l-glutamate dehydrogenase, aspartate: 2-oxoglutarate, and l-alanine: 2-oxoglutarate aminotransferases.     

Determination of urinary amino acids by means of glass capillary gas—liquid chromatography with alkali-flame ionisation detection and flame ionisation detection

The determination of amino acids as their N(O)-heptafluorobutyryl-isobutyl ester derivatives by glass capillary gas—liquid chromatography has been studied. Separations of amino acids obtained from insulin hydrolysate and human urine analysed with a flame ionisation detector, an alkali-flame ionisation detector and an electron-capture detector are shown. The quantitative results of urinary amino acids of one deep-sea diver are presented.     

Pyrrolnitrin from Burkholderia cepacia: antibiotic activity against fungi and novel activities against streptomycetes

A bacterial strain identified as Burkholderia cepacia NB-1 was isolated from water ponds in the botanical garden in Tübingen, Germany, and was found to produce a broad spectrum phenylpyrrole antimicrobial substance active against filamentous fungi, yeasts and Gram-positive bacteria. In batch culture containing glycerol and L- glutamic acid, the isolate NB-1 produced the antibiotic optimally late in the growth phase and accumulated a main portion in their cells. Isolation and purification of the antibiotic from Burkholderia (Pseudomonas) cepacia NB-1 by acetone extraction, gel filtration on Sephadex LH-20 and preparative HPLC yielded 0·54 mg l⁻¹ of a pure substance. Spectroscopic data (HPLC, MS and NMR) confirmed that the compound was pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chloro-phenyl) pyrrole]. Pyrrolnitrin has an inhibitory effect on the electron transport system, as demonstrated by isolated mitochondria from Neurospora crassa 74 A. This inhibition was relieved by N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), indicating that pyrrolnitrin blocked the electron transfer between the dehydrogenases and the cytochrome components of the respiratory chain. Among Gram-positive bacteria, pyrrolnitrin was most active against certain Streptomyces species, especially S. antibioticus , which has not previously been described in the literature. In the presence of pyrrolnitrin, aerial mycelium and spore formation of Strep. antibioticus was suppressed, although growth continued via substrate mycelium. The new findings of inhibition of streptomycetes and their secondary metabolism by pyrrolnitrin may contribute to the fact that Pseudomonas species predominate in soil and compete even with antibiotic-producing Streptomyces.     

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).     

Changes in the Levels of Abscisic Acid and Its Metabolites in Excised Leaf Blades of Xanthium strumarium during and after Water Stress

The time course of abscisic acid (ABA) accumulation during water stress and of degradation following rehydration was investigated by analyzing the levels of ABA and its metabolites phaseic acid (PA) and alkalihydrolyzable conjugated ABA in excised leaf blades of Xanthium strumarium. Initial purification was by reverse-phase, preparative, high performance liquid chromatography (HPLC) which did not require prior partitioning. ABA and PA were purified further by analytical HPLC with a μBondapak-NH₂ column, and quantified by GLC with an electron capture detector.     

Production and composition of phenylpyrrole metabolites prodcued by Pseudomonas cepacia

Summary In shaken cultures, a strain of Pseudomonas cepacia isolated from apple leaves produced pyrrolnitrin and four other phenylpyrrole antibiotics. The concentrations of these metabolites were determined at intervals for 7 days in three different media at two initial pH levels. Optical density measurements revealed maximum cell concentrations after 24 h in nutrient broth, after 48 h in King's B medium, and after 96 h in minimum salts solution. The effects caused by initiating fermentations at pH 5.8 rather than 7.0 were in most cases not dramatic, although in some instances, especially in minimum salts broth, higher concentrations of metabolites were produced with the lower initial pH. Concentrations of the phenylpyrrole antibiotics were greatly affected by choice of culture medium and incubation time. Concentrations of the two nitrophenyl metabolites, pyrrolnitrin and 2-chloropyrrolnitrin, rose throughout the 7-day incubation and were more than 20 times greater in minimum salts medium than in either King's B medium or nutrient broth. The maximum concentrations of each of the three aminophenyl metabolites (dichloroamino, trichloroamino and monochloroamino) occurred in different media, the monochloro compound in nutrient broth, the dichloro compound in Kings B medium and the trichloro compound in minimum salts medium. The time dependence of the concentrations of the five metabolites supports the proposed biosynthesis of these pyrroles from tryptophan by successive chlorinations followed by oxidation of the amino group at the end of the pathway.     

Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic β-cells

The effects on pancreatic β-cell viability and function of three microbial secondary metabolites pyrrolnitrin, phenazine and patulin were investigated, using the rat clonal pancreatic β-cell line, INS-1. Cells were exposed to 10-fold serial dilutions (range 0-10 μg mL(-1)) of the purified compounds for 2, 24 and 72 h. After 2 h exposure, only patulin (10 μg mL(-1)) was cytotoxic. All compounds showed significant cytotoxicity after 24 h. None of the compounds altered insulin secretion with 2 and 20 mM glucose after 2 h. However, after 24 h treatment, phenazine and pyrrolnitrin (10 and 100 ng mL(-1)) potentiated insulin production and glucose-stimulated insulin secretion, whereas patulin had no effect. Exposure (24 h) to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 ng mL(-1)) caused similar increases in the Ca(2+) content of INS-1 cells. The outward membrane current was inhibited after 24 h exposure to either phenazine (100 ng mL(-1)) or pyrrolnitrin (10 or 100 ng mL(-1)). This study presents novel data suggesting that high concentrations of pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and thus possibly diabetogenic, whereas at lower concentrations these agents are nontoxic and may be insulinotropic. The possible role of such agents in the development of cystic fibrosis-related diabetes is discussed.     

Electron spin resonance investigations of mitochondrial electron transport in Neurospora crassa. Characterization of paramagnetic intermediates in a standard strain.

1. Submitochondrial particles from Neurospora strain inl-89601 have been analyzed by electron spin resonance spectroscopy (ESR). Numerous signals due to iron-sulfur proteins are observed at low temperatures. Analysis of these ESR signals at various temperatures allows the assignment of resonances to iron-sulfur centers 1-5 that have been described in other organisms. There are no discrepancies between the signals seen in Neurospora and those described in other organisms and it is likely that Neurospora mitochondria contain the same iron-sulfur centers that are observed elsewhere. 2. NADPH and NADH act to reduce the iron-sulfur centers of respiratory complex I. 3. The drug pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorphenyl)pyrrole] is an effective inhibitor of both NADH-supported and succinate-supported electron transport in Neurospora. 4. Analysis of pyrrolnitrin inhibition curves, respiration studies, ESR spectra, and the steady-state level of reduction of cytochrome b in the presence and absence of the drug shows that pyrrolnitrin acts to inhibit electron transport in Neurospora mitochondria at multiple sites in the region between ubiquinone and cytochrome b.