We investigated selective cultivation media and previous treatments of samples suitable for detection of Legionella species from environmental water and for elimination of co-existing microbes which gave rise to an interference with the evaluation of Legionella sp. growth. Twenty thousand U of polymyxin B (PL-B)/ml and 100 micrograms of oxytetracycline (OTC)/ml seem to be useful as additives to MWY selective agar medium. Both antibiotics markedly inhibited the growth of co-existing microbes with almost no influence on the growth of Legionella sp. In the studies on the resistance of 8 strains of Legionella sp., 24 strains of co-existing microbes and 2 standard strains of Staphylococcus aureus and Escherichia coli to acid treatment (0.2 M HCl-KCl, pH 2.2, 25 degrees C, 4 minutes) and heating (50 degrees C, 20 minutes), acid treatment or heating alone showed no inhibition on the growth of almost all strains examined. However, combination with acid treatment after heating resulted in an apparent extinction of almost all microbes except for Legionella sp., Seven strains from co-existing microbes showed an apparent growth inhibition against 8 strains of Legionella sp. with different serotypes and were all identified as Pseudomonas aeurginosa, which were all eliminated by means of the combination with acid treatment after heating. From these results, it was concluded that the combined pre-treatment of water samples with acid after heating and the addition of PL-B and OTC into the selective cultivation medium is an useful method for detection of Legionella sp. from environmental water.
Anti-microbial effect of the pretreatment with various organic acid buffer solutions against co-existing microorganisms which were isolated from cooling-tower water samples along with Legionella spp. was examined. Among several buffer solutions, a 0.1 M potassium citrate-citric acid buffer solution (hereafter, citrate buffer solution, pH 2.2) hardly affected the recovery of Legionella spp., but effectively inhibited the growth of co-existing microorganisms. To evaluate the buffer action of these buffer solutions, pHs of 9 cooling-tower water samples were evaluated after addition of an equal volume of each buffer solution. When a citrate buffer solution. pH 2.2 was combined to a 200-fold concentrated solution of each cooling-tower water sample, the pH of the combined solution became 2.5 to 2.7 and maintained a stably low pH value than that (pH 3.0 to 7.4) obtained after mixture of a 0.2 M HCl-KCl buffer solution (hereafter, HCl buffer solution, pH 2.2), suggesting strong buffer action of the citrate buffer solution, pH 2.2 in the combined solutions. Furthermore, when cooling-tower water samples were pretreated with a citrate buffer solution, pH 2.2, the recovery of Legionella spp. was successful in 7 out of 9 cooling-tower water samples, suggesting 3 times higher recovery rate than that obtained by addition of a HCl buffer solution, pH 2.2 (3 out of 9 cooling-tower water samples).
The action of VA-2, the most active component of antibiotic M-92, against S. aureus is bactericidal but not bacteriolytic. The bactericidal action is markedly affected by incubation temperature, whether bacterial cells are prolific or resting.The bactericidal kinetics of VA-2 is biphasic, since addition of VA-2 caused rapid and straight decrease in viability curve and reached a plateau after several minutes. The bactericidal activity of VA-2 is blocked by 2,4-dinitrophenol.Alike to many membrane-active bacteriocins, VA-2 seems to exert its action through two stages.As reported previously" 2.1), antibacterial and anticancer antibiotic M-92 elaborated by Micromonospora verruculosa MCRL 0404 is a complex of several structurally related antibiotics having a quinoid nature. Among the six major components, VA-2 exhibited the most potent antibacterial activity, particularly against some Gram-positive bacteria (MICs: 0.000001-0.0001 ag/ml)". Such a potent activity urged us to examine the mechanism of interaction between VA-2 and bacterial cells, and also the mode of action on the target bacteria.The present paper concerns the characteristics of the interaction between VA-2 and the susceptible Staphylococcus aureus cells which suggest that interaction proceeds through two stages. In some parts of the present experiments, mitomycin C4' was used for comparison, because mitomycin C, an antitumor quinoid antibiotic, shows biological properties somewhat resembling to VA-2. Mode of action of VA-2 as a DNA synthesis inhibitor will be dealt with in the succeeding paper. Materials and MethodsAntibiotics and Chemicals VA-2 was separated and purified as described in the preceding paper'). Mitomycin C and other chemicals were obtained from commercial sources. Antibiotic solutions were prepared by dissolving the antibiotic in dimethylsulfoxide and then diluting with an appropriate amount of sterile deionized water. Microorganisms and MediaStaphylococcus aureus 209P JC-1 maintained in our laboratory was used. Nutrient broth (Difco) and Bacto-agar (Difco) were employed in the present study.Culture Conditions for Growth and Viability Tests A test strain was grown in double strength Nutrient broth (2NB) on a reciprocating shaker at 37°C. The bacterial growth was monitored by measuring absorbance at 600 nm. Bacterial cell cultures in an early exponential stage, whose absorbance at 600 nm reached to approximately 0.2, were used for the tests. Viability of cells was determined by counting the colony numbers grown on 2NB agar plates, which were prepared as follows. The cell suspensions were serially diluted with 0.9y. physiological
A new antibiotic complex, M-92 was isolated from the whole fermentation broth of Micromonospora verruculosa MCRL 0404. The whole broth was mixed with talc and filtered. The filter cake thus obtained was extracted with acidic methanol to give a crude powder of M-92 complex, which was then separated into A (acidic) and N (neutral or weakly acidic) groups. The A group components are soluble in alkaline water (pH 8.5), while the N group components are not. These components were further separated into six major components designated VA-2, BA-4, BA-5, BN-1, BN-2 and BN-3 by silica gel column chromatography.Components with the letter "V" are reddish violet and those with "B" are blue.The IR and UV spectra of these components suggest that their chromophores may be the same or very closely related to one another.The molecular formula of BN-3 was determined to be C28H23NO8 by mass spectrometry.The results of various spectroscopies on BN-3 suggest that M-92 components consisted of chromophores in which juglone (5-hydroxynaphthoquinone) is conjugated with naphthazarin (5,8-dihydroxynaphthoquinone).In screening for new antibiotics produced by Micromonospora, we found that a new strain for which the name Micronionospora verruculosa sp. nov.1) was proposed, produced a new antibiotic complex named M-92 which was exceedingly active against some Gram-positive and Gram-negative bacteria.M-92 complex was extracted with acidic methanol from the mycelial cake. Crude complex was then separated into six major components designated VA-2, BA-4, BA-5, BN-1, BN-2 and BN-3 by silica gel H column chromatography. They were acidic or neutral in nature and blue or violet in color. This paper deals with the isolation and purification of the M-92 components and their physicochemical properties. Biological properties of these components will be reported in the succeeding paper2). The structural elucidation of these components is now under way. Isolation and PurificationIsolation of a crude powder of the M-92 complex was achieved as shown in Chart 1. Thus, 24 g of a crude powder of the M-92 complex was obtained from 1,000 liters of fermentation broth.According to the procedures shown in Chart 2, the chloroform solution of the M-92 complex was separated into A group (acidic substances) which is soluble in alkaline water (Na2CO3, pH 8.5) and N group (weakly acidic or neutral substances) which is insoluble. The A group components in the chloroform layer were washed with water and then evaporated to dryness in vacuo. Thus, 6.5 g of a crude powder of the A group components and 10.4 g of a crude powder of the N group components were obtained from 20 g of crude M-92 complex.The isolation and purification of the A group components were carried out by silica gel H [Merck, impregnated with 0.5 M Mcllvain buffer (pH 5)] column chromatography as shown in Chart 3. Then,
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.