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Paper chromatography is very helpful in the identification and comparison of antibiotics, but very few results from the application of this technique have been published so far. The awakening of interest in the search for antibiotics which are active against fungi pathogenic to plants and animals makes the investigator aware of the scant information that is available to help characterize such agents. A knowledge of the Rf values of the known antifungal agents would help advance the search for such therapeutic materials. Often these values, in conjunction with the biological inhibitory activities, are the only criteria by which one can determine whether the unknown agent appears to be a new material. When the Rf data are obtained for shake cultures, one can eliminate many previously described antibiotic-producing organisms from further study, thus allowing more research to be applied to antibiotics which are new and different. The studies reported in this paper should serve as a guide to the identification of some of the known antifungal antibiotics.

A simple quantitative chromatographic method is described for the separation of epitetracycline and tetracycline in pharmaceutical preparations. The method is applicable qualitatively to human serum and quantitatively to the biological determination of tetracycline in urine. Whatman's modified cellulose phosphate cation-exchange paper is developed by the descending technique using 0·1 per cent w/v ammonium chloride solution. The spots are located as yellow fluorescent spots under ultra-violet light and ammonia vapour, eluted in 10 per cent w/v ammonium chloride, and the extinction determined at 356 mμ. The method is not suitable for the separation of chlortetracycline or oxytetracycline from tetracycline.

Antibiotic discs were prepared, using several several batches of papers meeting Food and Drug Administration specifications. The analysis of 1,152 zones of inhibition produced showed no performance differences among these batches. Other discs were prepared using papers of different grades. These produced large differences in performance. It is obvious, therefore, that the use of a specified disc paper is necessary for standardizing the performances of the products of various manufacturers and that reproducible results can be attained with the grade of paper specified.

MANNOSIDOSTREPTOMYCIN in some streptomycin preparations is conveniently detected by the paper chromatographic procedure of Winston and Eigen1. In the presence of high concentrations of salts such as in ion-exchange column eluates, or in fermentation broths, this procedure is not applicable: attempts to desalt such solutions, by mixed-bed ion-exchange, electrolytic de-ionization or solvent extraction, have proved unsuccessful. We have found it possible to separate streptomycin, mannosidostreptomycin and allied substances by paper electrophoresis, having developed (independently) a technique similar to that used for new antibiotic broths by Hosoya et al. 2, and King and Doery3.

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ACCORDING to Miyazaki, Omachi and Kamata1, antibiotics can be grouped according to their salting-out chromatograms. Pure antibiotics and materials containing antibiotics were examined by the aid of ascending paper chromatography. As irrigation solvents in each case and with each antibiotic, distilled water and rising concentrations of ammonium chloride (0.5, 1, 2, 3, 5, 10, 20 per cent and saturated) were used. Location of the spots of the antibiotics, that is, the R F values, were ascertained bio-autographically.

EXTRACTION methods are usually used for the isolation and purification of antibiotics soluble in organic solvents which are immiscible with water. This method can be applied if the partition coefficient of the antibiotic between the solvent and water is sufficiently large. The partition coefficient often depends on the pH. value, particularly for acidic or basic compounds.

Microbiological assay of individual antibiotics in mixtures of antibiotics depends on the use of selective inactivation and/or of test bacteria with differential susceptibility. Controlled experiments revealed that streptomycin in concentrations of 20 and 40 μg/ml did not influence a disk diffusion assay of penicillin with Sarcina lutea (ATCC 9341) as the test organism. In the case of penicillin concentrations less than or equal to 1 IU/ml, addition of 80 μg of streptomycin per ml influenced the penicillin assay significantly. Clinical use of streptomycin resulting in levels above 40 μg/ml usually did not occur; therefore penicillin could be assayed as though streptomycin were not present. We observed additionally that S. lutea was unable to grow on agar plates prepared with semicarbazide hydrochloride.

1. 6-Hydroxykynurenic acid (4,6-dihydroxyquinoline-2-carboxylic acid, 6-HKA) was isolated in crystalline form from both green and cured tobacco leaves. 2. A method for the determination of 6-HKA by paper chromatography and fluorimetry is described. 3. The content of 6-HKA in the flowers, stem and roots of the tobacco plant was much lower than that in the leaf. 4. The 6-HKA content increased throughout leaf development and senescence. 5. 6-HKA was detected in the leaves of plants representing 11 out of 27 families sampled. 6. 6-HKA was found to be devoid of antibacterial and antifungal activity, and was inactive in the Avena-coleoptile and cress-seed-germination tests. 7. The presence of 6-HKA is taken as evidence in plants of the tryptophan-catabolic pathway already known in mammals and micro-organisms.

Streptomyces rishiriensis produces at least five closely related antibiotics. Strain selection yielded a culture producing only the most active component, coumermycin A. Hydrolysis of this antibiotic by barium hydroxide yielded both 5-methyl-pyrrole-2-carboxylic acid and pyrrole-2-carboxylic acid, which could be separated by paper chromatography. Coumermycin A was thus shown to be two fractions, designated A 1 and A 2 depending upon the nature of the pyrrole carboxylic acid portion. The addition of cobalt to the fermentation medium at a level as low as 0.01 μg/ml shifted the fermentation exclusively to the production of coumermycin A 1 . Other ions were ineffective, except nickel, whose activity could be explained by the presence of contaminating cobalt.

Eighteen species of Penicillium , one of Oidium and one of Paecilomyces were found to effect a stereospecific conversion of cis -propenylphosphonate to fosfomycin which was identified by paper chromatography and gas-liquid chromatography (GLC) of the trimethylsilyl esters. Penicillium spinulosum carried out the epoxidation only after the glucose substrate had been utilized. Glucose controlled the epoxidation since its residual concentrations in the broth severely depresses the reaction. At optimum levels of glucose, an epoxidation efficiency approaching 90% of olefin charged (0.2 g/liter) was obtained after 10 days of incubation. The olefin concentration could be increased to 0.5 g/liter when glucose was replaced by glycerol, whereby a 90% conversion to fosfomycin was attainable in 6 days. The high conversion efficiency, a good agreement between the GLC assay and bioactivity, are indicative of the levorotatory nature of the product.

To elucidate the amino acid sequence of glumamycin, glumamycin and DNP-glumamycin have been partially hydrolyzed with hydrochloric acid, and various peptides have been isolated by column chromatography on ionexchange resin or talc-celite, paper chromatography and paper ionophoresis. From the structures of the peptides, two possible structures (A and B) for glumamycin have been presumed.

Cell extracts from Bacillus brevis (A.T.C.C. 10068), grown with various media, incorporated certain 14C-labelled amino acids that are normally components of tyrothricin into material that was extracted by ethanol from the precipitate formed by adding acid. When this material was separated by paper and silica-gel thin-layer chromatography and paper electrophoresis 14C was located in those regions that also contained gramicidin and tyrocidine. From a study of the properties of the system responsible for the incorporation it was deduced that non-tyrothricin materials were present. It was shown that the methods normally used to characterize tyrothricin do not adequately distinguish between tyrothricin and non-tyrothricin materials. However, a method for separating these materials was devised. This involved elution with ethanol from columns of acid alumina followed by gel filtration on Sephadex LH-20 with dimethylformamide–water solvent. The behaviour of gramicidin and tyrocidine on the Sephadex LH-20 column was examined, and it was concluded that the separation was not caused simply by gel filtration of unassociated molecules. Also, tyrocidine molecules with different amino acid compositions seemed to have different affinities for the Sephadex LH-20 column.

Acidolin was isolated from skimmilk cultured for 48 hours with Lactobacillus acidophilus (CHR. HANSEN's Laboratory strain 2181). It was extracted from the skimmilk with methanol and acetone and was further concentrated and purified by Sephadex G-25 gel filtration, high voltage electrophoresis, and thin-layer chromatography on silica gel. Ultraviolet, infrared, nuclear magnetic resonance, and mass spectra results are presented for the antibiotic. Acidolin has a low molecular weight (-200), is acidic in nature, possesses a yellow-brown color, and is highly hygroscopic and thermostable. Acidolin exhibits antimicrobial activity against enteropathogenic organisms and sporeformers and only limited activity against lactic-acid bacteria. It is non-toxic to tissue culture cells (H-Ep-2) and is more active against vaccinia than polio virus.

Addition of aromatic acids to the culture medium of Streptomyces caelestis results in the production of antibiotics different from the celestosaminides normally produced by the organism. The new antibiotic produced when 4-aminosalicylic acid was added to the culture medium was isolated and characterized as desalicetin 2'-(4-aminosalicylate). Although biological evaluation of this antibiotic is not complete, its in vitro antibacterial spectrum is identical to that of celesticetin.

SUMMARY: The acetone-extractable antibacterial components of Isochrysis galbana have been resolved by paper-chromatography and bioautography. Two pigmented products with similar antibacterial properties were obtained which were tentatively identified from their absorption spectra as chlorophyll a derivatives, probably pheophytin a and an atypical chlorophyllide a.

Flavomycoin is a polyene antibiotic isolated from the mycelium of Streptomyces roseoflavus ARAI 1951 var. jenensis nov. var. JA 5068 as yellow-green crystals. It shows inhibitive activities against yeasts and fungi as well as protozoa. It is optically active, exhibits an ultraviolet spectrum without fine structure with maxima at 363 nm and 263 nm. Analytical studies indicated that the molecular formula of the antibiotic is C41H68O10 and the molecular weight 721. These results were confirmed by degradation to the parent hydrocarbon and by catalytic hydrogenation. As flavomycoin is different from the related substances mycoticin and flavofungin, it must be considered as a new antibiotic.

In this paper the term " meningitis " is used in a clinical sense, whether the inflammatory condition is of bacterial or non bacterial origin and whether or not it involves the brain. It covers all inflammatory disease that can be included within the terms " meningitis," " meningo-encephalitis," or " ence phalitis," with the exclusion of tuberculous meningitis and poliomyelitis. The initial signs of meningitis are usually those which indicate meningeal inflammation, but clinical signs of involve ment of the brain may follow in some cases. In fatal cases evidence of cerebral damage can usually be found at necropsy. Moreover, electroencephalographic studies often show disturb ance of cerebral function even without any clinical manifestations.

Scopafungin (U-29,479) is a crystalline, nonpolyenic antimicrobial agent obtained from the culture broth of Streptomyces hygroscopicus var. enhygrus var. nova UC-2397. Scopafungin inhibits, in vitro, a variety of pathogenic fungi, yeasts, and gram-positive bacteria.

When dl -isoleucine was added to an ammonium nitrate-maltose medium during cultivation of Streptomyces sp. 732, quinomycin B formation was selectively enhanced (from 3 to 70% of the quinomycin mixture) and quinomycin C synthesis was inhibited completely. In addition, two new quinomycins, designated quinomycins D and E, were produced in the presence of isoleucine. These compounds were found to contain N -methylalloisoleucine. In experiments with isoleucine enantiomorphs, it was determined that the order of effectiveness for quinomycin B synthesis was dl -isoleucine > d -isoleucine > l -isoleucine. The extent to which quinomycin B synthesis is enhanced depends upon the concentration and the time of addition of isoleucine to the medium. The effect of dl -isoleucine was reduced to some extent by the addition of l -valine. It is conceivable that amino acids which are precursors of the N -methylamino acids in quinomycin can regulate quinomycin formation.

Two metabolites, active against gram-positive bacteria, have been isolated from a strain of Actinomycetales. The chemical structure of one of the two, named tetrenolin, has been previously described1). The present paper deals with the description of the producing organism, the isolation and properties of the two metabolites.

Lomofungin is a new antimicrobial agent obtained from the culture broth of Streptomyces lomondensis sp. n. UC-5022. Lomofungin is an acidic, olive-yellow, crystalline compound which inhibits, in vitro, a variety of pathogenic fungi, yeasts, and gram-positive and gram-negative bacteria.

A system for the automatic capture and retrieval of information contained in routine bacteriology reports is described. The system depends on the preparation of reports on an electric typewriter producing punched paper tape as a byproduct. Periodically the information contained on the tape is analysed by computer. The potential value of the system for the analysis of the results of antibiotic sensitivity testing and for the study of the epidemiology of hospital infections is briefly discussed.

Pure cultures of Physarum gyrosum were grown on agar plates with autoclaved Escherichia coli suspensions as the growth medium. Portions of such agar, after growth of the slime mold, contained diffusible materials that inhibited the growth of Bacillus subtilis, B. cereus, E. coli, Staphylococcus aureus , and Pseudomonas aeruginosa . Paper chromatography of extracts of such cultures revealed at least three different active fractions. Preliminary fractionations increased the specific activity by one order of magnitude, probably in part by removal of inactive material and in part by separating active components. The fractionations also demonstrated the multiplicity of the antibiotic activity. Fractions variously obtained always retarded the growth of the bacterial species listed above.

A new antibiotic, cryomycin, was isolated from the culture filtrate of a facultatively psychrophilic streptomycete. As a result of taxonomic studies, it was considered a new subspecies for which the name Streptomyces griseus subsp. psychrophilus, YOSHIDA, TANI and OGATA, is proposed. The type strain is AKU 2881. This organism grows at 0-37°C and produces cryomycin at 0-18°C. Cryomycin is a peptide antibiotic containing a rather large amount of glycine in its molecule. It darkens at 214-217°C with decomposition. This antibiotic is highly active against Gram-positive bacteria in vitro. The LD50 in mice by intravenous injection is 150 mg/kg.

The addition of elemental sulfur to the fermentation of Streptomyces sioyaensis in a soybean meal medium resulted in a three- to fourfold increase of siomycin. Further experiments on the effect of elemental sulfur during fermentation suggest that one of the key steps stimulating siomycin synthesis is the utilization of thiosulfate, which accumulates in the medium as the result of the oxidation of elemental sulfur.

Kitasatoa kauaiensis, a new species of the genus Kitasatoa, belonging to the family Streptoplanaceae, produces 3 kinds of antibiotics in a culture broth. These antibiotics were identified as chloramphenicol, bottromycin and fradicin based on their physico-chemical and biological properties. A new differential bioassay method for chloramphenicol and bottromycin produced in the same culture broth was established.

Two new compounds, pyracrimycins A and B, have been isolated from a Streptomyces species named Streptomyces eridani Coronelli et al. n. sp. The morphological and physiological characteristics of this strain are described in the present paper together with the production and properties of the two metabolites. Pyracrimycin B is biologically inactive whereas pyracrimycin A is active in vitro against both Gram-positive and Gram-negative bacteria.

THE application of paper electrophoresis to the study of new antibiotics1 has been in use at these laboratories for some time. Our technique consists of applying spots of culture fluid, or a concentrate of it, to paper strips soaked in buffer, and subjecting the strips to electrophoresis by the technique of Kunkel and Tiselius2. Spots of chloromycetin are applied to the paper on each side of the spot of unknown antibiotic as a control for electro-osmosis. After migration, the position of the antibiotic is revealed by the microbiological technique as usual in paper chromatography of antibiotics3. Thus it is possible to determine whether unknown antibiotics in culture fluids are acidic, basic or amphoteric, and to obtain an approximate measure of their isoelectric point as a guide to subsequent purification procedures. The migration of an unknown antibiotic 179, relative to chloromycetin, to the cathode at pH 3 and pH 8, with lessened mobility at pH 8, indicates a basic substance with an isoelectric point greater than pH 8, as illustrated in the accompanying photograph.

Streptomyces sioyaensis , which produces the antibiotic siomycin, oxidizes elemental sulfur when added to the culture medium and accumulates thiosulfate in the fermented broth. The accumulated thiosulfate was isolated as the ammonium salt and was identified by melting point, IR spectrum, and paper chromatography. A variety of other streptomycetes also oxidized elemental sulfur and accumulated thiosulfate.

A new antibiotic, designated LL-A491, was isolated by butanol extraction and crystallization from beers of an unidentified streptomycete, Lederle culture A491. LL-A491 was primarily active against gram-positive bacteria and was related to monazomycin. A tentative molecular formula of C 72�2 H 144�8 O 25�1 N for the antibiotic, based on analyses of the crystalline hydrochloride, picrate, and picrolonate salts, is considerably larger than that proposed for monazomycin, from which LL-A491 was not differentiated by paper chromatography. Analysis of the amorphous polyacetate ester suggested 15 to 16 acetylable groups. Upon hydrolysis, LL-A491 liberated ammonia and a reducing sugar which appeared to be mannose. LL-A491 was dextrorotatory, [α] D 25 + 14�, and exhibited only end absorption in the ultraviolet region.

Antibiotics that inhibit the biosynthesis of the cell wall, such as vancomycin, penicillin, d -cycloserine, and bacitracin, stimulate the incorporation of lysine into lipids that are extractable with n -butanol-6 M pyridinium acetate. Approximately 93% of this lysine is in lysylphosphatidylglycerol (LPG). The remaining lysine is incorporated in another as yet uncharacterized lipid. Because the lysine in the latter lipid is released by mild alkaline hydrolysis, it is not the C 55 -isoprenyl-pyrophospho- N -acetyl-muramyl pentapeptide. Vancomycin and penicillin stimulate the incorporation of lysine into both LPG and the minor lipid fractions, whereas treatment with d -cycloserine results in an increase only in LPG. Antibiotics that inhibit protein synthesis do not influence the incorporation of lysine into the lipid fractions. Analysis of the extracted lipids indicate that the incorporation of radioactive lysine into LPG is due to an enhancement in synthesis of LPG from phospholipids in the cytoplasmic membrane.

A prospective study of 358 medical and 234 postoperative patients with clinical evidence of secondary chest infection showed that previous administration of antimicrobial agents greatly reduced the chance of obtaining a clear-cut laboratory report. In patients with radiographical evidence of pneumonia this led to a fourfold decrease in the overall rate of isolation of potential pathogens. Furthermore, 81 diverse “coliforms” were isolated from 258 medical and surgical patients who had received previous antimicrobial therapy while only four coliforms (all Escherichia coli) were isolated from 334 untreated patients. Thus the general hospital environment on its own seemed to have a negligible influence in promoting the growth of coliform flora in sputum. Any unique effect of underlying disease in this regard was masked by that of previous therapy. Finally, the results raised the possibility that previous antimicrobial therapy might have increased the risk of secondary pneumonia in hospital patients.

The normal sequence of cell separation in Saccharomyces cerevisiae begins with the formation of a primary septum, presumably consisting of chitin, on which secondary septa are later deposited. In the presence of the antibiotic polyoxin D, a potent inhibitor of chitin synthetase, pairs of abnormal cells of two different types were observed by phase-contrast microscopy: the “exploded pair,” consisting of two lysed cells from which the cytoplasm had been extruded at the cell junction, and the “refringent pair,” consisting of two highly refractile cells joined by a thin bridge. Thus, in both cases the septal region appears to be affected. Observations with the electron microscope showed that the primary chitin septum was not formed in either of these cell types, and as a consequence secondary septa of varying thicknesses were laid down in an abnormal pattern. With [ 3 H]glucose as carbon source the incorporation of tritium into the chitin of abnormal cells was inhibited about 90%, whereas the labeling of mannan was normal and that of glucan somewhat reduced. The effective concentrations of polyoxin D (0.1 to 1 mg/ml) were much greater than those required to inhibit chitin synthesis in vitro. Dimethylsulfoxide and amphotericin B, both known to increase cell permeability, enhanced the action of the antibiotic.

Different groups of patients in a general practice have been under close observation by defined methods to assess the extent of their adherence to drug schedules. Patients taking antibiotics, admitted to a short drug trial and prescribed long-term treatments, proved in most cases to be compliant. Antenatal patients taking a once-daily dosage of prophylactic iron were more co-operative than those taking divided dosages. Socially isolated patients and those of low social class were particularly likely to neglect their drugs. It is suggested that the significance and extent of the problem have been largely ignored and that all drug trials undertaken on outpatients should incorporate methods of detecting defaulters.

Anticapsin is a new fermentation product which inhibits formation of the hyaluronic acid capsule of Streptococcus pyogenes . Production of this metabolite in a complex medium by S. griseoplanus is enhanced by high levels of carbohydrate. A number of carbon sources support biosynthesis but sucrose is most effective, the optimum level being 150 g/liter. Neither glucose nor fructose, alone or in combination, serves as an equivalent substitute for sucrose. The addition of dibasic potassium phosphate to the medium further increases anticapsin production. Dissolved oxygen levels are important for synthesis and stability of the metabolite. Anticapsular activity diminishes rapidly in previously aerated broths which are held under static conditions. This decrease does not occur in pasteurized broths or unpasteurized filtrates.