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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.

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.

(1960). The Paper Disc Method for Determination of Bacterial Sensitivity to Antibiotics Studies on the Accuracy of the Technique. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 12, No. 4, pp. 408-413.

SummaryA procedure has been developed whereby paper chromatograms of antiviral agents can be developed by applying them to virus infected, agar overlay, chick embryo cell tissue cultures. Plaque-free zones are produced, thus indicating the area on paper chromatogram where the active material can be located. Using antiviral substances antibiotic W-112 and isatin-monothiosemicarbazone, W. Nile and vaccinia viruses, and 4 chromato-graphic solvent systems, it was possible to localize the biological activity of these antiviral agents. As little as 1 μg of antibiotic W-112 produced large plaque-free zones when paper chromatogram was applied to the overlay culture 1 hr after virus infection. When 100 μg of same material was used, large plaque-free areas resulted even when the paper was applied 46 hr after virus infection. Isatin-monothiosemicarbazone at 50 μg level not only produced a plaque-free zone but also produced a yellow spot on paper chromatogram that coincided exactly with the zone of antiviral activity.

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.

The test paper here described makes it relatively easy to choose the antibiotic best suited for treatment of a given bacterial infection. Each type of test paper bears both a culture medium and an antibiotic and is supplied, dry and sterile, in a transparent envelope. For a given test, the paper is wetted and inoculated and the envelope with its contents is incubated. It is not necessary to wait for the appearance of visible colonies because the metabolic activity of the bacteria is detected by an indicator (triphenyltetrazolium chloride) which is colorless until reduced to a bright red dye by bacterial enzymes. The reliability of the new test was established by a comparative study with other methods in 100 clinical cases. The test paper is easily stored, and the entire unit can be incinerated after use. It is therefore useful in general office practice and should help to make sure that each patient receives the most effective antibiotic.

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.

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.

SummaryA procedure has been developed whereby agar diffusion technics used in bacteriology can be applied to antiviral agents. Chick embryo cell monolayers were grown in Pyrex baking dishes, infected with virus and overlaid with agar. Filter paper discs impregnated with the test materials were deposited on the hardened agar surface. After proper incubation the virus produced plaques in the cell sheet, except in the area around a disc containing an antiviral agent. Since size of the plaque-free zone was proportioned to concentration of the antiviral agent present in the disc, bioassays were simply performed in exactly the same manner as done with antibacterial antibiotics.

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 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.

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.

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.

A graphic method of predicting antibiotic yields in continuous flow reactors is presented and discussed using the novobiocin fermentation as a model process. Extension to other antibiotic fermentations and steroid bioconversions is emphasized. In the case of the novobiocin fermentation it was concluded that a combination of one growth stage and one or two antibiotic production stages would be the most economic reactor system to conduct such a fermentation on a continuous basis.

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.

Sclerothricin is a new strongly basic antibiotic obtained from the culture filtrate of Streptomyces sclerogranulatus SHIMAZU et YONEHARA nov. sp. The antibiotic is active against gram-positive and gram-negative bacteria and some fungi. Sclerothricin (C16H30O8N6) seems to belong to the streptothricin group. It has two N-methyl, a free carboxyl and two N-terminal groups. No carbamoyl group is present.

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.

Four patients are reported in whom perforation of the colon followed exchange transfusion for haemolytic disease of the newborn. This association seems to be more than coincidental, and possibly the perforation is due to a vascular accident occurring as a mechanical result of the exchange transfusion. The insidious onset of colonic perforation may be recognized early by the passage of blood per rectum. There is no place for conservative treatment, and once the diagnosis has been made treatment must include broad-spectrum antibiotics and laparotomy.

We have isolated a temperature-sensitive mutant of S. cerevisiae which has proven useful for the isolation of antibiotic-sensitive strains.

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.