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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.

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.

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.

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.

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.

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.

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.

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.

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.

The classification of antibacterial drugs into bactericidal and bacteriostatic groups is of clinical value. Bactericidal drugs are to be preferred when possible as they are more rapidly effective, may be synergistic when used in combination against bacteria difficult to eliminate, and are less likely to leave residual “persistent” organisms. Antagonism between chemotherapeutic agents is of little clinical importance. The modes of action of antibacterial drugs are reviewed. The penicillins, cephalosporins, and cycloserine interfere with bacterial cell wall synthesis. Polymyxin, colistin, and nystatin affect the bacterial cell membrane. Protein manufacture by the bacterial ribosome is interfered with by the tetracyclines, chloramphenicol, erythromycin, and lincomycin, and the initiation of protein molecules by streptomycin. Streptomycin, kanamycin, and neomycin also cause the ribosome to manufacture warped proteins. Nalidixic acid, griseofulvin, and novobiocin upset the D.N.A. replication of the bacterial chromosome. Many of the factors adverse to the success of chemotherapy are unimportant when powerful drugs are given in large doses to relatively fit patients infected with highly sensitive bacteria. But these factors become important when patients, particularly debilitated patients, are infected acutely or chronically with some of the more obstinate bacteria. Some grasp of these principles is therefore both intellectually satisfying and clinically useful.

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.

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.

Antibiotic-resistant enterobacteria, the vast majority of which carried transmissible R factors, were isolated from 81 out of a total of 100 healthy infants. There was surprisingly little correlation between previous antibacterial therapy and the resistant enterobacterial flora.

The use of many different antibiotics to treat chest infection has led us to test the sensitivity of 68 strains of Haemophilus influenzae to 15 different compounds. These included established compounds such as ampicillin and tetracycline and newer agents such as cephalosporins and clindamycin. The minimum inhibitory concentrations of the compounds for H. influenzae were then compared with blood levels attained after the usual dose regimens. There has been a significant increase in tetracycline resistance in the last few years, but all strains were sensitive to ampicillin, chloramphenicol, sulphamethoxazole, and trimethoprim, Several antibiotics were found to be microbiologically unsuitable for treating H. influenzae infections.

Out of 200 infections due to Bacteroides fragilis occurring over a period of three years 133 were related to the intestinal tract, 55 to the genitourinary tract, and the remainder were in bedsores and ulcers; 56% occurred in patients undergoing major intestinal surgery.B. fragilis was isolated in pure culture from 56% of the infections. In mixed culture it was most commonly associated with Klebsiella and Enterobacter species. Other anaerobic bacteria were isolated in 9% of the mixed cultures.Altogether 131 (65.5%) of the patients recovered without antibiotic therapy or further surgery, but 59 (29.5%) developed complications and 10 (5%) died. The commonest complication was abscess formation, and the incidence was highest with infections associated with malignancy (44%) and lowest with obstetric infections (5%). The mortality was 5% overall but in the presence of bacteraemia it rose to 33%.Only 43 patients received appropriate chemotherapy. Clindamycin was the most effective antibiotic, having a recovery rate of 78%, but this rate was little better than in untreated patients (65%). The role of prophylactic antibiotic therapy in preventing bacteroides infection remains to be studied.The incidence of the isolation of bacteroides from wound infections after major intestinal surgery rose from 13% in 1970 to 81% in 1973. This increase was due to both the accurate collection and care of specimens while in transit to the laboratory and the use of selective media for the isolation of bacteroides in laboratory culture. The importance of these precautions is emphasized.

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.

Tobramycin and amikacin are new aminoglycoside antibiotics which resemble gentamicin and kanamycin respectively in pharmacokinetic, toxicological, and some microbiological respects. Minimum inhibitory concentrations for these four antibiotics have been determined against 393 bacterial strains (13 species from eight genera) isolated from clinical materialKanamycin was the least active, 89% of the Pseudomonas aeruginosa strains being resistant. Resistance was also found in 21% of the Klebsiella aerogenes, 16% of the indoleproducing Proteus species, 4% of the E. coli and 3% of the Enterobacter spp. tested. Providenca stuartii strains were often resistant to gentamicin and tobramycin.Apart from these exceptions, however, all the organisms tested were sensitive to all four compounds, gentamicin and tobramycin usually showing the highest intrinsic activity.Amikacin was the most active compound against a selection of strains chosen because of their resistance to one or more of the aminoglycosides. Thus, amikacin represents a significant advance over kanamycin and seems to be superior to gentamicin in terms of its in-vitro properties. This is especially the case in those unusual circumstances when gentamicin-resistant strains are a problem. We suggest that amikacin warrants further investigation since it may prove to be a valuable alternative to gentamicin in the treatment of life-threatening infections.

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.

Within a period of only seven months three patients in one hospital under treatment with antibiotics by intravenous infusion developed secondary bloodstream infection originating from the site of insertion of the cannula. Two of these infections were fatal. Precautions suggested include strict asepsis, the skin being sterilized with iodine in spirit solution; antibiotic spray; and changing the cannula site.