Synthesis and in vitro antimicrobial activities of 2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-ones

A number of 2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-ones have been synthesized. 3-Oxo-2-(arylhydrazono)butyric acid ethyl ester were acetylated and treated with triethyl amine and formamide in presence of 1,4-dioxane to yield N-(5-acetyl-4-ethoxy-2-oxo-2,5-dihydro-imidazol-1-yl)-N-arylacetamide, which on refluxation with urea and freshly prepared sodium ethoxide yielded the title compound. All the newly synthesized compounds have been characterized by spectroscopic and elemental analysis data. The synthesized compounds were screened against a representative panel of susceptible and resistant Gram-positive and Gram-negative bacteria using a standard antibiotic drug purinthol as control. Quantitative structure-activity relationship has also been interpreted in terms of correlation of biological activity with molecular refractive index parameters (M(R)) and Hammett substituent constant (sigma).     

Isolation and characterization of a high molecular weight antibiotic produced by a marine bacterium

Many marine bacteria demonstrate antibiotic activity against organisms of terrestrial origin. Low molecular weight antibiotics have been extracted and, in some cases, purified, but few attempts have been made to isolate high molecular weight antibiotics produced by marine bacteria. In the study reported here, a high molecular weight antibiotic was extracted from whole cells ofAlteromonas strain P18 (NCMB 1890) grown on 2216E medium. Purification included ammonium sulfate precipitation, ultracentrifugation, chromatography on DEAE cellulose, and gel filtration on Ultrogel. A rapid method for measuring specific activity of the antibiotic was developed.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

Solid-state and submerged fermentations show different gene expression profiles in cephalosporin C production by Acremonium chrysogenum

Despite the importance of Acremonium chrysogenum as the only cephalosporin C (CPC) producer, there is still a limited understanding about the molecular mechanisms regulating antibiotic biosynthesis in this fungus. Based on the previously described relationship between environmental pH and antibiotic production in numerous filamentous fungi, we studied the expression of genes related to CPC production in A. chrysogenum. We report for the first time similarities and differences, characterizing CPC production by A. chrysogenum under a variable pH environment, in submerged and solid-state fermentation. This characterization is supported by measurements of parameters, like CPC production, pH, growth, and expression levels of several genes involved, directly or indirectly, in CPC production. Interesting differences in intermediate (Pen N) and certain biosynthetic gene expression levels were observed. Our results point out some relationships between physiological features and gene expression that open important improvement perspectives for both culture systems.     

链霉菌遗传不稳定性研究进展与展望

摘要： 链霉菌中广泛存在着遗传不稳定性现象,这种遗传不稳定性影响着链霉菌的诸多表型,尤其是工业生产上的菌种退化,抗生素产量不稳定等,给工业生产和实验研究带来很大麻烦。经多年深入研究,已逐步揭示了链霉菌遗传不稳定性和染色体不稳定性的关系。链霉菌遗传不稳定性机制的揭示,可为改良生产菌种,构建高产而又稳定的基因工程菌奠定理论基础。本文综述了链霉菌遗传不稳定性的研究进展,并对后续研究进行讨论与展望。     

Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non-Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain.     

Genetic determinants of active antibiotic-producing soil streptomycetes.

Plasmids or covalently closed circular (CCC)-DNA molecules are abundant in the genus Streptomyces, and have been suggested to be involved in the genetic control of the production of many antibiotics in these organisms. In this study, 21 active antibiotic-producing Streptomyces isolates were screened for their plasmid content by an alkaline lysis method which revealed the presence of a small plasmid DNA in the positive control Streptomyces lividans ATCC 35287, containing pIJ702 plasmid (5.65 kb in size). However, no low molecular weight plasmids were observed in the tested antibiotic-producing Streptomyces strains suggesting that antibiotic production in these strains is likely chromosomally encoded DNA. Treatment of 2 Streptomyces strains with 10 mM ethidium bromide (EB) resulted in the failure to produce aerial mycelia and antibiotic activity.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.     

The peptide pheromone-inducible conjugation system of Enterococcus faecalis plasmid pCF10: cell-cell signalling, gene transfer, complexity and evolution

Expression of a large set of gene products required for conjugative transfer of the antibiotic resistance plasmid pCF10 is controlled by cell-cell communication between plasmid-free recipient cells and plasmid-carrying donor cells using a peptide mating pheromone cCF10. Most of the recent experimental analysis of this system has focused on the molecular events involved in initiation of the pheromone response in the donor cells, and on the mechanisms by which the donor cells control self-induction by endogenously produced pheromone. Recently, studies of the molecular machinery of conjugation encoded by the pheromone-inducible genes have been initiated. In addition, the system may serve as a useful bacterial model for addressing the evolution of biological complexity.     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

Differential Antibiotic Excretion in Unilateral Structural Pyelonephritis

Differential antibiotic excretion studies in eight patients with unilateral structural pyelonephritis showed that in all instances there was impairment in antibiotic excretion on the affected side compared with the normal kidney. Both peak and mean urinary concentrations were decreased on the structurally abnormal side. The degree of defect in antibiotic concentration was proportional to the amount of unilateral medullary damage, as measured by differential renal function studies. While the antibiotic concentrations achieved had an inconsistent relationship to cure in three patients with proven unilateral infections, the striking decreases in urinary antibiotic levels may argue against oral penicillin-G treatment of unilateral pyelonephritis in which there is renal parenchymal damage.     

Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity

A radiochemical method was developed to estimate cholyltaurine hydrolase potentials and rates of cholyltaurine hydrolysis in chicken intestinal homogenates. This method was used to monitor the effects of antibiotic feed additives on cholyltaurine hydrolase activity. Avoparcin, bacitracin methylenedisalisylic acid, efrotomycin, lincomycin, penicillin G procaine, and virginiamycin improved rate of weight gain and feed conversion of chicks and decreased cholyltaurine hydrolase activity in ileal homogenates relative to those of nonmedicated control birds. The results provided the first evidence that feeding selected antibiotics at subtherapeutic levels can affect bile acid-transforming enzymes in small-intestinal homogenates. The inverse relationship between growth performance and cholyltaurine hydrolase activity raises the possibility that specific inhibitors of this enzyme may promote weight gain and feed conversion in livestock and thereby reduce or eliminate the need for antibiotic feed additives.     

Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity.

A radiochemical method was developed to estimate cholyltaurine hydrolase potentials and rates of cholyltaurine hydrolysis in chicken intestinal homogenates. This method was used to monitor the effects of antibiotic feed additives on cholyltaurine hydrolase activity. Avoparcin, bacitracin methylenedisalisylic acid, efrotomycin, lincomycin, penicillin G procaine, and virginiamycin improved rate of weight gain and feed conversion of chicks and decreased cholyltaurine hydrolase activity in ileal homogenates relative to those of nonmedicated control birds. The results provided the first evidence that feeding selected antibiotics at subtherapeutic levels can affect bile acid-transforming enzymes in small-intestinal homogenates. The inverse relationship between growth performance and cholyltaurine hydrolase activity raises the possibility that specific inhibitors of this enzyme may promote weight gain and feed conversion in livestock and thereby reduce or eliminate the need for antibiotic feed additives.     

Identification of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> genes associated with antibiotic susceptibility

Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.     

翻译后修饰与结核分枝杆菌耐药调控网络

目前对于结核分枝杆菌(Mycobacterium tuberculosis,Mtb)耐药产生机制研究得较多,但对其调控机制的研究较少。翻译后修饰(Post-translational modifications,PTMs)在结核菌多种生理途径(如代谢、应激反应等)中发挥重要调控作用,而它们和结核菌耐药之间的关系逐渐引起了研究者的关注。文中介绍了结核菌抗生素耐受机制以及存在的一些PTMs,重点讨论了PTMs在调控结核杆菌耐药机制中的潜在作用,以期为新型抗结核药物研发提供新的切入点。     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Identification of Nor-��-Lapachone Derivatives as Potential Antibacterial Compounds against Enterococcus faecalis Clinical Strain

A broad-spectrum antibiotic therapy has led to medical complications and emergence of multiresistant bacteria including Enterococcus faecalis. In this study, we designed, synthesized, and evaluated the antibacterial activity of 13 nor-β-lapachone derivatives against a drug resistant E. faecalis strain. Two triazole substituted compounds (1e = 8 μg/ml and 1c = 16 μg/ml) and the non-substituted derivative (1a = 8 μg/ml) were promising compared to chloramphenicol (12 μg/ml), an antibiotic currently available in the market. We also performed a structure-activity relationship analysis using a molecular modeling approach that pointed the low HOMO energy values; HOMO density concentrated on the nor-β-lapachone ring, lipophilicity, solubility and number HBA as important stereoelectronic features for the antibacterial profile. In addition the triazole compounds presented low theoretical toxicity profile, and drug-score higher than commercial antibiotics also fulfilling the Lipinski "Rule of Five", which pointed them as promising candidates for further studies in infections caused by multiresistant E. faecalis hospital strains.     

Impact of Fertilizing with Raw or Anaerobically Digested Sewage Sludge on the Abundance of Antibiotic-Resistant Coliforms,Antibiotic Resistance Genes,and Pathogenic Bacteria in Soil and on Vegetables at Harvest

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.     

First-step mutations for adaptation at elevated temperature increase capsid stability in a virus

The relationship between mutation, protein stability and protein function plays a central role in molecular evolution. Mutations tend to be destabilizing, including those that would confer novel functions such as host-switching or antibiotic resistance. Elevated temperature may play an important role in preadapting a protein for such novel functions by selecting for stabilizing mutations. In this study, we test the stability change conferred by single mutations that arise in a G4-like bacteriophage adapting to elevated temperature. The vast majority of these mutations map to interfaces between viral coat proteins, suggesting they affect protein-protein interactions. We assess their effects by estimating thermodynamic stability using molecular dynamic simulations and measuring kinetic stability using experimental decay assays. The results indicate that most, though not all, of the observed mutations are stabilizing.