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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.

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 mug/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 mug of streptomycin per ml influenced the penicillin assay significantly. Clinical use of streptomycin resulting in levels above 40 mug/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.

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.

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.

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.

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.

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.

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.

Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B(1), and B(2). The B(1) and B(2) activities were studied in greater detail than A. The B(1) substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively.

Kalafungin is a new antimicrobial agent obtained from the culture broth of a soil isolate of Streptomyces tanashiensis, designated Streptomyces tanashiensis strain Kala UC-5063. Kalafungin is a chemically stable, nonpolyene agent which is ex-extremely inhibitory in vitro against a variety of pathogenic fungi, yeasts, protozoa, gram-positive bacteria, and, to a lesser extent, gram-negative bacteria.

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.

Recent literature on the antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP suggested an interaction with murein synthesis. Incubation of sensitive strains from Bacillus cereus and Staphylococcus aureus in a "wall medium" containing labeled l-alanine showed that all four antibiotics inhibited the incorporation of alanine into murein and gave rise to accumulation of radioactive uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide. Peptidoglycan was synthesized when the particulate enzyme of B. stearothermophilus was incubated with the murein precursors UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-MurNAc-pentapeptide. The newly formed polymer was less accessible for lysozyme and more strongly bound to the acceptor than the same product from the Escherichia coli particulate enzyme. After incubation in the presence of penicillin, a greater part of the peptidoglycan was lysozyme sensitive and more loosely bound to the acceptor. The antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP inhibited peptidoglycan synthesis by the B. stearothermophilus particulate enzyme. The rate of synthesis of GlcNAc-MurNAc(-pentapeptide)-P-P-phospholipid was independent from the addition of these antibiotics, but its utilization was strongly inhibited. With the present results, it is not possible to distinguish the mechanisms of action of enduracidin, moenomycin, prasinomycin, and 11.837 RP from the mechanisms of action of vancomycin and ristocetin.

Novobiocin-supersensitive (NS) mutants which could not grow on plates containing 40 mug or less of novobiocin per ml were isolated from Escherichia coli strain JE1011 (derived from E. coli K-12). Most of these NS mutants were found to have incomplete lipopolysaccharides (LPS), and they lack phosphate diester bridges in their backbone structure, with or without total loss of heptose, to which the phosphate diester is linked, and consequently lack external outer-core oligosaccharides. The phosphate diester bridges in the LPS backbone are apparently very important in forming a cell surface structure resistant to the penetration of antibiotics such as novobiocin, spiramycin, and actinomycin D. NS mutants, with incomplete LPS, lacking phosphates in their backbone structure were found to be resistant to phage T4, and those which also lacked heptose were resistant to phages T4 and T7. In contrast to the generally accepted idea that resistances to phages T3, T4, and T7 are linked genetically, no NS mutant was found to be resistant to T3. The possible structures of the receptors for T4 and T7 are discussed. The positions of novobiocin-supersensitive genes on the chromosome of several of the NS mutants defective in LPS were mapped. The genes were designated lpcA (between ara and lac) and lpcB (between 55 min and 60 min). The latter seemed to be a group of several related genes.

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.

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.

Examination of phospholipid metabolism in Haemophilus parainfluenzae with inhibitors of various cellular functions indicated that macromolecular synthesis and lipid metabolism can be dissociated at least for a short time. Two classes of inhibitors have relatively specific effects on cardiolipin (CL) metabolism. Pentachlorophenol and p-hydroxymercuribenzoate blocked CL synthesis but allowed CL hydrolysis to phosphatidic acid and phosphatidyl glycerol (PG); 3,3',4,5'-tetrachlorosalicylanilide (TCS) and carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) blocked CL hydrolysis with the stoichiometric accumulation of CL. It appeared as if TCS and m-CCCP inhibited a vital activity coupled with the hydrolysis of CL by the highly active, CL-specific phospholipase D found in this organism. Because TCS and m-CCCP are thought to act by destroying the proton gradient thereby interrupting energy-dependent transport, it is possible that a highly active portion of the cellular CL could be coupled to some phase of this process.

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 mug/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.

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, [alpha](D) (25) + 14 degrees , and exhibited only end absorption in the ultraviolet region.

The site of action of related mono- and bis-quinaldinium compounds was investigated in Staphylococcus aureus and Bacillus megaterium. The effects of these compounds on cell morphology and on protoplast formation and fragility were studied, and the distribution of C(14)-labeled quinaldinium compound in cell fractions was measured. The latter studies showed that a major part of the quaternary compound penetrates the cell, leaving a very small quantity associated with the cell wall. Similar antibacterial effects were seen with both the mono- and bis-quinaldinium compounds studied, and these effects were comparable with antibacterial properties of known cationic surface-active antibacterial agents.

In Escherichia coli K-12, the conversion of glycerol to triose phosphate is regulated by two types of control mechanism: the rate of synthesis of glycerol kinase and the feedback inhibition of its activity by fructose-1,6-diphosphate. A strain which has lost both control mechanisms by successive mutations, resulting in the constitutive synthesis of a glycerol kinase no longer sensitive to feedback inhibition, can produce a bactericidal factor from glycerol. This toxic factor has been identified by chemical and enzymological tests as methylglyoxal. Methylglyoxal can be derived from dihydroxyacetone phosphate through the action of an enzyme which is present at high constitutive levels in the extracts of the mutant as well as that of the wild-type strain. Nine spontaneous mutants resistant to 1 mm exogenous methylglyoxal have been isolated. In all cases the resistance is associated with increased levels of a glutathione-dependent enzymatic activity for the removal of methylglyoxal. Methylglyoxal-resistant mutants derived from the glycerol-sensitive parental strain also became immune to glycerol.

Two paper strips, each containing different antimicrobial agents, were placed on plates on Mueller-Hinton agar to permit antibiotic to enter the agar. A filter membrane was placed on this plate, and the microorganisms were planted on the membrane. After 6 h of incubation at 37 C, the membrane was transferred to antibiotic-free Mueller-Hinton agar containing triphenyltetrazolium hydrochloride and incubated for 18 h at 37 C. Specific growth patterns were indicative of additive (indifferent), synergistic, or antagonistic effects of the drug combination used. Trimethoprim and sulfamethoxazole proved to act synergistically against 85% of Escherichia coli, 86% of Klebsiella, and 89% of Proteus mirabilis strains tested. A few strains resistant to either drug were susceptible to their combination. The technique was useful against organisms with widely differing susceptibilities to the two antimicrobial agents tested.

Koburger, J. A. (North Carolina State College, Raleigh), M. L. Speck, and L. W. Aurand. Identification of growth stimulants for Streptococcus lactis. J. Bacteriol. 85:1051-1055. 1963-The growth of Streptococcus lactis in milk was accelerated by pancreas extract, and three distinct components that were detected by bioautography were isolated and identified. These were purified by precipitation with silver ions, and their silver salts were regenerated in acidic solution. After adsorption and elution from characoal, contaminating alpha-amino compounds were destroyed by treatment with ninhydrin. Final purification was by chromatography. Identification of the purified fractions was by paper chromatography, and by ultraviolet and infrared spectra. The active compounds were found to be inosine, hypoxanthine, and adenine.

Actinobolin was evaluated in vitro by a paper disc-agar diffusion method for inhibitory activity against mixed microbial cultures obtained from patients with periodontal disease and against pure bacterial cultures tentatively identified as strains of Bacteroides melaninogenicus, Fusobacterium fusiforme, Leptotrichia buccalis, and Veillonella parvula. Every culture tested was inhibited to some degree by actinobolin. These observations suggest that actinobolin may be effective in the treatment of periodontal disease.

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.

Antibiotic concentrations in human sera were estimated in 5 to 6 hr by a modified microbiological assay. By using Staphylococcus aureus and Streptococcus pyogenes as the assay organisms, the seeded assay plates were preincubated for 2 to 6 hr and then were stored at 4 C until used for assay. Paper discs saturated with the specimen were placed on the preincubated assay plates with reference discs saturated with known concentrations of antibiotic. After 5 to 6 hr of incubation, zones of antibacterial activity were measured and compared with a standard curve for estimation of antibiotic concentration. Results from this rapid assay method compared favorably with those from the commonly used 24-hr assay.

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.

The apparent stimulation of cephalosporin C biosynthesis by sodium thiosulfate is due to a nonbiological conversion of cephalosporin C to a new derivative, cephalosporin C(x). The new compound is more active than cephalosporin C against the assay organism, Escherichia coli W-208. Cephalosporin C(x) retains the properties of ultraviolet absorption and resistance to penicillinase, but migrates more slowly than cephalosporin C in the paper-chromatographic system used.

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.