Antibacterial Polyketides from Antarctica Sponge-Derived Fungus Penicillium sp. HDN151272.

Three new polyketides, ketidocillinones A-C (1-3), were discovered from the extract of an Antarctica sponge-derived fungus Penicillium sp. HDN151272. All the structures were deduced by spectroscopic data, including NMR and HRESIMS. The absolute configuration of compound 3 was established by using ECD calculation. Compounds 1-3 can be slowly oxidized to quinone form when exposed to air. Ketidocillinones B and C (2 and 3) exhibited potent antibacterial activity against Pseudomonas aeurigenosa, Mycobacterium phlei, and MRCNS (methicillin-resistant coagulase-negative staphylococci) with MIC values ranging from 1.56 to 25.00 µg/mL.

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria.

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE: Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.

Doubly prenylated tryptamines: cytotoxicity, antimicrobial activity and cyclisation to the marine natural product flustramine A.

The marine natural product flustramine A was synthesised via oxidative cyclisation of Nb-methylated 1-prenyl-2-tert-prenyl-6-bromotryptamine and subsequent reduction of the resulting amidinium salt. Only the tert-prenyl group migrated, whereas the 1-prenyl group remained in place. Interestingly, the 2-tert-prenylated precursor revealed to be the biologically most active of our entire series of 21 compounds. Required for cytotoxicity and antimicrobial activity was the presence of a non-cyclised tryptamine side chain carrying a free secondary amine, whereas the presence of a 6-bromo substituent did not enhance cytotoxicity. In a panel of 42 human tumor cell lines, most sensitive were the lung and mammary cancer cell lines LXFA629L (IC50 1.9 µM) and MAXF401NL (IC50 2.4 µM), respectively. In a serial dilution assay, satisfying IC50 values of 5.9 µM against Micrococcus luteus and 7.7 µM each against Mycobacterium phlei were determined for Nb-methyl-1-prenyl-2-tert-prenyl-6-bromotryptamine.

Aromatic polyketides from a sponge-derived fungus Metarhizium anisopliae mxh-99 and their antitubercular activities.

In our screening for antitubercular agents, five naphtho-γ-pyrones including two new naphtho-γ-pyrones glycosides, indigotides G and H (1 and 2), and two diphenyl ethers were isolated from the extract of a sponge-derived fungus Metarhizium anisopliae mxh-99. Their structures were established on the basis of chemical and spectroscopic evidence. The antitubercular activities of all the compounds were evaluated against Mycobacterium phlei. The known isochaetochromin B2 (6) and ustilaginoidin D (7) exhibited the highest activity with MICs 50.0 µg/mL.

HLE-inhibitory alkaloids with a polyketide skeleton from the marine-derived fungus Coniothyrium cereale.

The marine endophytic fungus Coniothyrium cereale produces the structurally unusual polyketide-type alkaloids (-)-cereolactam (1) and (-)-cereoaldomine (3), incorporating a lactam and an imine functionality, respectively, as well as the related metabolite (-)-trypethelone (2). Compounds 1 and 3 showed selective inhibition of human leukocyte elastase with IC50 values of 9.28 and 3.01 µM, respectively. Compound 2 was found to be inhibitory toward Mycobacterium phlei, Staphylococcus aureus, and Escherichia coli and also cytotoxic against mouse fibroblast cells (IC50=7.5 µM).

Identification and manipulation of the caprazamycin gene cluster lead to new simplified liponucleoside antibiotics and give insights into the biosynthetic pathway.

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5'-(beta-O-aminoribosyl)-glycyluridine and comprises a unique N-methyldiazepanone ring. The biosynthetic gene cluster has been identified, cloned, and sequenced, representing the first gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation, and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster and inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as an acyltransferase in caprazamycin biosynthesis. In vivo and in silico analysis of the caprazamycin biosynthetic gene cluster allows a first proposal of the biosynthetic pathway and provides insights into the biosynthesis of related uridyl-antibiotics.

Antibacterial activity of labdane diterpenoids from Stemodia foliosa.

As part of a continuing interest in exploring the chemistry of Brazilian medicinal plants, three new labdane diterpenoids, 6alpha-acetoxymanoyl oxide (1), 6alpha-malonyloxymanoyl oxide (2), and 6alpha-malonyloxy-n-butyl ester manoyl oxide (3), together with the known betulinic acid, lupeol, sitosterol, and stigmasterol, were isolated from the aerial parts of Stemodia foliosa. The structures of 1-3 were established on the basis of interpretation of spectroscopic data, including HRESIMS, and 1D and 2D NMR techniques. All compounds were tested against a bacteria panel consisting of Staphylococcus aureus, Bacillus cereus, B. subtilis, B. anthracis, Micrococcus luteus, Mycobacterium smegmatis, and M. phlei. Compound 2 showed moderate activity against these strains, with MIC values in the range 7-20 microg/mL.

Novel propeptin analog, propeptin-2, missing two amino acid residues from the propeptin C-terminus loses antibiotic potency.

A novel inhibitor of prolyl endopeptidase, the propeptin analog propeptin-2 (C105H130N24O(24)), missing two amino acid residues from the propeptin C-terminus was isolated from the fermentation broth of propeptin-producing Microbispora sp. SNA-115 grown using a large inoculum. It shows the same enzyme inhibition activity as propeptin against prolyl endopeptidase (Ki=1.5 microM), but its antimicrobial activity against Mycobacterium phlei is more than 100-fold lower.

Efficacy of rifabutin-loaded microspheres for treatment of Mycobacterium avium-infected macrophages and mice.

Two poly(DL-lactide-co-glycolide) microsphere formulations (A, 10% wt/wt, and B, 23% wt/wt, 1-10 microns) were evaluated for intracellular delivery of rifabutin using the J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. Within 7 days, formulation A released 100% in both cell lines and B released 53 and 67% in the J774 and MM6, respectively. Intracellular release of rifabutin with both formulations caused significant reduction of intracellularly replicating Mycobacterium avium (MAC). In MAC-infected beige mice, formulation B (50 mg, intraperitoneal days 0 and 7) completely eliminated infection by 21 days (p < 0.001), similar to a rifabutin daily oral regimen.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria.

Extracts of the roots of plants of the Geraniaceae family have been used for many years in South Africa as native herbal remedies and there is circumstantial evidence for efficacy in the treatment of pulmonary tuberculosis. We have examined dried roots of Pelargonium reniforme and P. sidoides for antibacterial activity against rapidly growing mycobacteria. Fractions with activity against Mycobacterium aurum and M. smegmatis were obtained from both plant species by bioassay-guided fractionation of n-hexane extracts and were found to contain mixtures of straight-chain fatty acids. Analysis by gas chromatography-mass spectrometry (GC-MS) of the corresponding fatty acid methyl esters revealed structures with chain lengths ranging from C12 to C26. Unsaturated compounds were analysed as the corresponding dimethyl disulfide adducts to determine double-bond positions. Active mixtures differed in the relative abundance of their components, but all contained 16:0 (palmitic), Delta9-18:1 (oleic) and Delta9,12-18:2 (linoleic acid) as the major components. When tested against M. aurum, M. smegmatis and other rapidly growing mycobacteria (M. fortuitum, M. abscessus and M. phlei), all saturated compounds except 12:0 were devoid of antimycobacterial activity, whereas unsaturated compounds showed antimycobacterial activity related to their degree of unsaturation, their chain length and the bacterial species tested. The most potent compound was linoleic acid, with MIC of 2 mg/l against M. aurum.

Nontuberculous mycobacteria in soils of La Pampa province (Argentina).

The presence of nontuberculous mycobacteria (NTM) was investigated in forty soil samples belonging to the four physiographic regions (Eastern, Central, Southern and Western) that constitute La Pampa province. The presence of NTM in 67.5% of these soil samples was determined. The density of mycobacteria ranged 25-4,500 mycobacteria g-1 dry soil (mean = 516 CFU g-1). Significant differences were found in relation to both the investigated regions (p < 0.01) and the soil pH (r = 0.44*) (P = 0.02). The mycobacteria represented less than 0.00001% of the total aerobic bacteria found in the soils. Twenty-seven isolated mycobacteria were classified according to the culture, biochemical, enzymatic characteristics and antibiotic sensitivity. Mycobacterium fortitium was the dominant mycobacterium and was detected in 63% of the positive soils. This species showed ability for living in sandy to sandy loam soils, within a wide pH range (6.5-9.7) and organic matter (4.15-83.63 g kg-1). Two other species were M. phlei (range = 50-4,500 CFU g-1) and M. kansasii (range = 50-500 CFU g-1).

Nontuberculous mycobacteria in soils of La Pampa province (Argentina)

The presence of nontuberculous mycobacteria (NTM) was investigated in forty soil samples belonging to the four physiographic regions (Eastern, Central, Southern and Western) that constitute La Pampa province. The presence of NTM in 67.5 of these soil samples was determined. The density of mycobacteria ranged 25-4,500 mycobacteria g-1 dry soil (mean = 516 CFU g-1). Significant differences were found in relation to both the investigated regions (p < 0.01) and the soil pH (r = 0.44*) (P = 0.02). The mycobacteria represented less than 0.00001 of the total aerobic bacteria found in the soils. Twenty-seven isolated mycobacteria were classified according to the culture, biochemical, enzymatic characteristics and antibiotic sensitivity. Mycobacterium fortitium was the dominant mycobacterium and was detected in 63 of the positive soils. This species showed ability for living in sandy to sandy loam soils, within a wide pH range (6.5-9.7) and organic matter (4.15-83.63 g kg-1). Two other species were M. phlei (range = 50-4,500 CFU g-1) and M. kansasii (range = 50-500 CFU g-1).(AU)

New types of liposidomycins that inhibit bacterial peptidoglycan synthesis and are produced by Streptomyces. I. Producing organism and medium components.

Liposidomycins are atypical lipid-bearing nucleoside antibiotics that inhibit bacterial peptidoglycan synthesis. A producing strain was identified as a Streptomyces sp. from its cultural characteristics and physiological properties. It produced new types of liposidomycins that lacked sulfate and/or 3-methylglutaric acid moieties present in known liposidomycins by changing medium components. Sucrose and malt extract were particularly suitable sources for specific production of the new types of liposidomycins.

New types of liposidomycins that inhibit bacterial peptidoglycan synthesis and are produced by Streptomyces. II. Isolation and structure elucidation.

Various new liposidomycins were isolated from a culture of the strain Streptomyces sp. SN-1061M by changing medium components and they were classified into four types (I-IV) based on their structures. They were purified by butanol extraction, silica gel and LH-20 column chromatographies, and high performance liquid chromatography on ODS columns. Type (I) has the original structure which has sulfate and 3-methylglutaric acid moieties. Type (II) has no 3-methylglutaric acid moiety and type (III) has no sulfate moiety. Type (IV) has neither moiety. Type (III) and (IV) compounds, which have no sulfate moiety, exhibited more potent antimicrobial activity.

[Toxic effect of hydroxylated ions of heavy metals on the cytoplasmic membrane of bacterial cells]. / Toksicheskoe deistvie gidroksilirovannykh ionov tiazhelykh metallov natsitoplazmaticheskuiu membranu bakterial'nykh kletok.

The influence of nickel, copper, cadmium, and lead ions at concentrations of 50 to 100 microM on the barrier properties of the plasma membrane (PM) and the electrophoretic mobility (EPM) of Pseudomonas fluorescens 71, Escherichia coli K-12, and Mycobacterium phlei B-1291 VKM cells was studied at pH values from 5 to 9 by electro-orientational (EO) spectroscopy and microelectrophoresis of cells. According to the data of EO spectroscopy, the increase in the toxicity of heavy metal cations to cells corresponded to transition of cations to monovalent hydroxylated forms. Hydroxylated ions were found to more easily adsorb on, or penetrate across, the PM and to bind to competent proteins. During the treatment of all three investigated microorganisms with Cu and Pb ions, and gram-negative bacteria also with Ni ions, the EPM of cells changed in a pH range corresponding to the transition of bivalent metal ions to their monovalent hydroxylated forms. Changes in the EPM induced by increasing pH correlated well with the enhanced toxicity of these metals to the PM, as evidenced by the EO spectroscopy data. At the same time, this correlation was less pronounced for cadmium sulfate toxicity to all of the microorganisms studied and for nickel chloride toxicity to M. phlei cells.

[Ciprofloxacin, clarithromycin, rifampin: in vitro bactericidal activity of double and triple drug combinations against strains of atypical mycobacteria]. / Ciprofloxacine, clarithromycine, rifampicine: activité bactéricide in vitro de doubles et triple associations sur des souches de mycobactéries atypiques.

Bactericidal activity of ciprofloxacin (CIP), clarithromycin (CLA), rifampin (RIF) alone and in combinations was studied against five atypical mycobacteria (4 isolates from patients with Acquired Immunodeficiency Syndrome and 1 collection strain) by time killing curves method. Drugs were used at their attainable serum levels: CIP (1 microgram/ml), CLA (2 micrograms/ml), RIF (16 micrograms/ml). The decrease in CFU in comparison to inoculum (10(6)-10(7) CFU/ml) was evaluated by viable counts after 1, 2, 3, 4 and 5 days of incubation at 35-37 degrees C. Drug combinations enhanced slightly the killing of drugs alone. CIP+CLA was the most effective against MAC strains whereas CIP+RIF appeared as the best combination against rapidly growing species except M. phlei which was the most resistant strain.

[Inclusion of labeled precursors of macromolecular compounds in the mycobacterial cells in the presence of DP-2 preparation]. / Vkliuchenie mechenykh predshestvennikov makromolekuliarnykh soedinenii v kletki mikobakterii v prisutstvii preparata DP-2.

The changes in the inclusion of 3H-phenylalanine, methyl-3H-thymidine, [2-14C]-thymidine and [5-3H]-uridine in the cells of pathogenic M. bovis-8, opportunistic M. fortuitum and saprophitic M. phlei, M. B-5 were comparatively investigated in introduction of 0.01 and 0.10% concentrations of the drug DP-2. It is shown that 0.10% DP-2 completely stops the radionuclides supply to the cells. It is suggested that the target of the drug may exist in the sphere of amino acid biosynthesis. The presence of DP-2 is capable of disturbing transport of substances to the cells.