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Publisher Summary This chapter discusses the principles of paper chromatography of antibiotics. It includes all steps of analysis beginning with the preparation of samples and papers, development techniques, detection methods, and means of documentation. Paper chromatography of the best-known groups of antibiotics is also presented in the chapter. General theoretical aspects of paper chromatography are not dealt with except for those that directly emerged from studies of antibiotics. The chapter concludes with a discussion of paper chromatographic classification and systematic analysis of antibiotics.

An attempt to establish a systematic chromatographic analysis of antibiotics has been made. Sixty-two known antibiotics were distributed into five classes and into fourteen subclasses, according to their RF values in four principal solvent systems. Principles of the classification of antibiotics are given. For more detailed comparison and characterization of antibiotics further additional solvent systems were used. Additional solvent systems A, B, C, and D were used for class I, systems E, F, G, and H for classes II and III, systems I, J, K, and L were used for classes IV and V.

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.

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.

Antibiotic 136, a mixture of five similar basic antibiotics, has been partially resolved by chromatography on activated carbon. Component A appeared identical with streptothricin by paper chromatography, elemental analysis, infrared spectrum and animal toxicity. Fraction ABCD could not be distinguished by paper chromatography from pleocidin. Fraction CDE showed chemical properties similar to the mixture streptolin AB. Among the acid hydrolysis products of Antibiotic 136 chromatographic fractions, l -β-lysine, α- d -gulosamine, streptolidine, and N-guan-streptoldyl- d -gulosaminide have been identified. Component D difers from components A, B, and C by containing an imidazoline structural unit differing from streptolidine. The general properties of the mixture Antibiotic 136 indicate that its components are members of the streptothricin complex of antibiotics.

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.

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.

Six healthy men were given a fruit- and vegetable-free diet, and one man was given a pure sucrose-water diet and antibiotics for 4 days. Vanylglycol in urine was detected by paper chromatography and thin-layer chroma tography after enzymatic hydrolysis with glusulase (glucuronidase + arylsul fatase). Acid hydrolysis destroyed vanylglycol. Treatment with antibiotics and a sucrose diet did not change the vanylglycol excretion, while the mono amineoxidase inhibitor, alphamethyldopa, and reserpine decreased the excre tion. Disulfiram treatment increased the vanylglycol excretion.

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.

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.

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.

Column chromatography on a strongly basic ion-exchange resin, with water as eluent, fractionated the gentamicin antibiotic complex; each fraction, representing different mixtures of antibiotics, further separated upon thin-layer chromatography. Sixteen antibiotics were clearly differentiated. Paper chromatography of the N-acetyl derivatives supplemted thin-layer chromatography, detecting many additional constituents of the complex. A number of important, basic, water-soluble antibiotics were chromatographically compared with the major gentamicin antibiotics.

Identity tests for antibiotic sensitivity disks have been developed. Suitable tests previously reported in the literature have been modified and adapted to the micro-quantities involved in these disks. Once a system of testing had been established, it was screened for interference from all other disk antibiotics. Penicillins are identified generally by an iodometric method and specifically by thin-layer chromatography (TLC). The polypeptide antibiotics are differentiated by TLC in combination with nitration, reduction, and diazotization to further distinguish colistin from polymyxin. Lincomycin, oleandomycin, and kanamycin are identified by TLC systems, the tetracyclines by paper chromatography. Under varying conditions of solvents and wavelengths, streptomycin, erythromycin, nystatin, vancomycin, novobiocin, cephalothin, and viomycin are identified spectrophotometrically. Color tests have been devised for chloramphenicol, dihydrostreptomycin, and neomycin.

The inhibitory action of an antibiotic isolated fromB. subtilis is studied onC. albicans, C. clausenii, C. langeronii, C. stellatoidea, andC. visvanathii, as a possible means forC. albicans rapid identification. Growth inhibition ofC. albicans by the antibiotic was observed when the yeast was preincubated on malt-agar slants, at 30° C, during 24 hours, and tested afterwards for antibiosis on Sabouraud-agar plates. No growth inhibition could be detected withC. clausenii, C. langeronii, C. stellatoidea, andC. viswanathii, under the same experimental conditions.

Isolation, purification and physicochemical properties of the antibiotic complex YA-56, a family of phleomycin-bleomycin group, are described. The antibiotic YA-56 complex was found to be composed mainly of two active components named YA-56 X and Y. These components were clearly differentiated from the reported phleomycins and bleomycins by their physicochemical properties. However, by direct comparison, YA-56 X was found to be identical with zorbamycin reported recently. YA-56 Y was recognized to be a new antibiotic.

The fate of the orally effective antibiotic D-cephaloglycin and its much less active L-enantiomer has been studied in the rat. D-Cephaloglycin-14C is metabolized by two pathways, (1) hydrolysis of the amide linkage to form D-2-phenylglycine-14C and (2) deacetylation to form deacetyl-D-cephaloglycin-14C. D-Cephaloglycin is absorbed from the gastro-intestinal tract at least in part as the intact antibiotic, L-Cephaloglycin-14C is rapidly metabolized to L-2-phenylglycine-14C and its metabolites. In contrast to the results with D-cephaloglycin-14C, no intact L-cephaloglycin-14C or deacetyl-L-cephaloglycin was excreted in the urine of rats.

Dianemycin is an antibiotic previously reported to affect cation transport and to be effective in treating coccidiosis in chickens. It is isolated from a fermentation broth of Streptomyces hygroscopicus. The compound was found to be primarily active against gram-positive organisms. The chemical properties of dianemycin indicate a close relationship to monensin and other acidic cation complexing compounds.

A new antibiotic, gluconimycin, was isolated from Streptomyces AS 9. The systematic position of the organism is discussed. Gluconimycin has a polypeptide nature. It contains iron and gluconic acid in its molecule. Thus it has been classified as a member of sideromycins. Gluconimycin is considered from the fast moving type when chromatographed by butanolacetic acid-water. The antibiotic is active against Gram+ve, Gram-ve bacteria and some fungi. The antibiotic exerts high toxicity when injected in mice.

Partial hydrolysis of streptothricin was most readily carried out in dilute acid solution. The hydrolysate had no antimicrobial activity, and its toxicity was six times that of streptothricin. From this inactive substance the original active antibiotic was regenerated with dicyclohexylcarbodiimide.

Of a total of 739 actinomycetes freshly isolated from soil samples from China, 515 (69·7%) were antibiotically active. Their antibiotic spectrum was determined against 14 test micro-organisms, including bacteria, yeasts and fungi, and 386 of them were characterized by paper chromatography using agar blocks, by behaviour on agar plates incorporating ion-exchange resins and by u.v. absorption spectrum (to detect polyene substances). Of these strains, 195 (50·5%) produced a mixture of antibiotics of both polyene and non-polyene type. On the basis of these results the active strains have been classified in various ways.

Purpuromycin (C26H18O13) is a new antibiotic isolated from the culture broth of Actinoplanes ianthinogenes proposed as a new species on the basis of its morphological, cultural and physiological characteristics. The antibiotic, obtained as purple crystals, belongs to the naphthoquinone group of antibiotics and resulted to be related to rubromycins. The isolation and purification procedures and the chemico-physical properties of the product are reported. Purpuromycin is active in vitro against Gram-positive bacteria at 0.005-0.02μg/ml, at 1-20μg/ml on Gram-negative bacteria and at 0.2-0.5 μg/ml on fungi.

A strain of Myxococcus xanthus, referred to as M. xanthus TA, has been isolated which produces an antibiotic capable of inhibiting growth of certain Gram-positive and Gram-negative bacteria. Antibiotic production was significantly inhibited when the concentration of protein hydrolysate in the production medium exceeded 1%. The antibiotic was purified over 1000 times to apparent homogeneity by silicic acid chromatography and by a variety of preparative alumina thin-layer chromatographic procedures. The purified antibiotic was partially characterized by its chromatographic behavior in six solvent systems, stability to acid, alkali and heat, infrared, ultraviolet, and mass spectra. The antibiotic has a λmax of 242 nm in methanol.

A new amino acid antibiotic, amiclenomycin is produced by Streptomyces lavendulae subsp. amiclenomycini OKAMI. The chemical structure of amiclenomycin is L-2-amino-4-(4'-amino-2', 5'-cyclohexadienyl)-butyric acid. The antibiotic inhibits growth of mycobacteria and this activity is reversed by biotin and desthiobiotin. α-Methyl-desthiobiotin is coproduced along with amiclenomycin.

Strains of Escherichia coli are resistant to the antibiotic kasugamycin due to the partial non-methylation of 16S ribosomal RNA. An RNA methylase activity, absent from resistant strains, is shown here to methylate in vitro the 16S RNA of resistant as well as sensitive strains.

tetaine, a low molecular weight broad spectrum antibiotic of aminoacid derivative type and of low toxicity, is an inhibitor of cell wall synthesis in Escherichia coli K-12 3000 HFr strain. Tetaine inhibits the incorporation of 3H DAP and 3H L-Ala to murein of E. coli. In the presence of tetaine nucleotide precursors of murein synthesis were radioactive mainly due to the 3H uridine and not 14C alanine, what indicates the lack of incorporation of alanine to UDP-Mur-NAc. It is postulated that tetaine inhibits the synthesis of murein nucleotide precursors at the level of incorporation of first alanine moiety.

A strain ofStreptomyces viridochromogenes produced a new crystalline antibiotic, Avilamycin, related to but not identical with Curamycin and Exfoliatin. Avilamycin, C63H94O35Cl2, gave on solvolytic degradation the following products: dichloroisoeverninic acid, 2-deoxy-d-rhamnose, 2,6-di-O-methylmannose, 4-O-methylfucose,l-lyxose and 3,5-diacetoxy-γ-caprolactone.

The sideromycin antibiotic A 22,765 is taken up from the medium by Staphylococcus aureus SG 511 against a concentration gradient. In contrast to this, A 22,765-resistant cells display an extremely low degree of permeability to the molecule. Ferrioxamine B penetrates the cells in only very minute quantities, but nevertheless antagonises permeation by A 22,765. This permeation-antagonising effect of ferrioxamine B is discussed.

Three new antibiotics, neoantimycin, neaureothin and ochramycin have been isolated from Streptoverticillium orinoci. The systematic position of the organism is discussed. Neoantimycin is related to the antimycin antibiotics group; neoaureothin shows some similarity with aureothin. Ochramycin is an amphoteric compound containing in its molecule an octaene chromophore. The first two antibiotics show a slight antifungal activity only. Ochramycin is active against gram +ve, but not against gram -ve bacteria; its activity against yeasts and fungi is very slight. Toxicity data are reported; no therapeutical effectiveness has been observed in experimental infections in mice.

An analytical procedure was devised which can distinguish members of the streptothricin family of antibiotics. It is based upon an analysis of the hydrolysis products of the antibiotics using a Technicon amino acid autoanalyzer under special conditions. The various fragments including the different streptolidine-amino sugar compounds were well resolved. A basic water-soluble antibiotic discovered in our laboratories and named LL-BL136 was compared to other members of this group by this technique. It was not differentiated from the antibiotic SF-701 reported by Tsuruoka. The autoanalyzer results along with other physicochemical data permitted a structure proposal for this antibiotic, which is the N -methyl-desformimino derivative of antibiotic LL-AC541.

Ferrous sulphate administered together with tetracycline and three of its derivatives-oxytetracycline, methacycline, and doxycycline-was found seriously to impair the absorption of these antibiotics. Thus even small doses of iron taken simultaneously should be avoided during tetracycline treatment.

A new antibiotic O-2867 inhibiting the growth of Piricularia oryzae, a plant pathogenic fungus of rice, was isolated from a Streptomyces sp. Antibiotic O-2867 is water-soluble, amphoteric and consists of 2 components, O- 2867-α and β. O-2867-α was obtained as an amorphous powder and O-2867-β as colorless needles. Both antibiotics are active against P. oryzae in vitro but are not effective in pot tests using rice plants infected with P. oryzae.

PRISTINAMYCIN is a new antibiotic obtained from Streptomyces pristinae spiralis, a Streptomyces isolated from a soil sample taken at San Carlos (Cordoba) in Argentina1. It is made up of several components and belongs to the family of synergistic antibiotics which includes streptogramin, staphylomycin and ostreogrycin. Chromatographic analysis has shown that pristinamycin is a mixture of two groups of components, the proportions of which vary within certain fixed limits

Summary. Transformability of a large number of Streptomyces strains was examined with respect to antibiotic production. Of 8 antibiotically inactive strains studied, 3 could be transformed to antibiotic production by treatment with the deoxyribonucleic acid of antibiotically active strains. When both the donor and the recipient strains were antibiotically active, 3 of 32 pairs of strains examined yielded positive transformations. The recipient strains acquired the antimicrobial spectra of the donor antibiotics; in some cases the chemical properties, including Rf values in several solvent systems, of the antibiotic substances elaborated by some of the donor and transformant strains were found to be similar. Although an antibiotically inactive mutant of S. viridifaciens could be transformed to antibiotic production by treatment with the DNA of S. aureofaciens NCIB 8234, all attempts at transformation in the Streptomyces strains producing the tetracycline group of antibiotics failed. A candicidin producing strain of S. griseus NCIB 8891 acquired antibacterial property when transformed with the DNA of S. griseus MA 8. A strain of Streptomyces sp. A(22) 3 transformed 4 other strains of Streptomyces.

Edein D wurde aus dem komplexen Antibioticum Edein mit Hilfe von Verteilungs-Chromatographie abgetrennt. Als Bausteine von Edein D wurden 3-Amino-3-phenyl-propionsaure, 3-Amino-2-hydroxypropionsaure, 2,3-Diaminpropionsaure, 2,6-Diamin-7-hydroxy-azelainsaure, Glycin und Spermidin ermittelt.

Ein neues antifungales Antibioticum,Streptomyces sp. Ac2(435) wurde mit breitem Spektrum isoliert. Die aktive Substanz ist ein amorphes, nicht-kristallines weisses Pulver, das in Wasser massig loslich ist. Die Substanz ist lichtempfindlich und bei 100°C inaktiv. Vergleichsproben mit bekannten Substanzen ergaben eine Tetraenverbindung.

The myxomycete Physarum gyrosum was cultured in its plasmodial stage on agar plates containing 0.025 M phosphate buffer at pH 6.5, 2% bakers' yeast, and 0.2% glucose and was supplemented with live Escherichia coli . Extracts of these plasmodia contained several antibiotic substances. Antibiotic materials were partially purified by dialysis of the agar medium-mold mixture, evaporation of the dialyzate, and butanol extraction of the residue. Further purification in two paper and two thin-layer chromatographic systems gave one product which was pure in six thin-layer chromatographic systems. Antibiotic activity against some gram-positive and gram-negative bacteria and yeasts was demonstrated with partially purified extracts and a paper-chromatographically separated fraction. One pure antibiotic was effective against Bacillus cereus .