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Stephen Douthwaite - One of the best experts on this subject based on the ideXlab platform.
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macrolide resistance conferred by rrna mutations in field isolates of mannheimia haemolytica and pasteurella multocida
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Anders Steno Olsen, Ralf Warrass, Stephen DouthwaiteAbstract:OBJECTIVES: To determine how resistance to Macrolides is conferred in field isolates of Pasteurella multocida and Mannheimia haemolytica that lack previously identified resistance determinants for rRNA methylation, efflux and macrolide-modifying enzymes. METHODS: Isolates of P. multocida and M. haemolytica identified as being highly resistant (MICs >64 mg/L) to the Macrolides erythromycin, gamithromycin, tilmicosin, tildipirosin and tulathromycin were screened by multiplex PCR for the previously identified resistance genes erm(42), msr(E) and mph(E). Strains lacking these determinants were analysed by genome sequencing and primer extension on the rRNAs. RESULTS: Macrolide resistance in one M. haemolytica isolate was conferred by the 23S rRNA mutation A2058G; resistance in three P. multocida isolates were caused by mutations at the neighbouring nucleotide A2059G. In each strain, all six copies of the rrn operons encoded the respective mutations. There were no mutations in the ribosomal protein genes rplD or rplV, and no other macrolide resistance mechanism was evident. CONCLUSIONS: High-level macrolide resistance can arise from 23S rRNA mutations in P. multocida and M. haemolytica despite their multiple copies of rrn. Selective pressures from exposure to different macrolide or lincosamide drugs presumably resulted in consolidation of either the A2058G or the A2059G mutation.
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macrolide resistance conferred by base substitutions in 23s rrna
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Birte Vester, Stephen DouthwaiteAbstract:Resistance to all major groups of antibiotics has arisen hand in hand with their extensive use in medicine and animal husbandry, and macrolide antibiotics are no exception. The therapeutic utility of Macrolides has been severely compromised by the emergence of drug resistance in many pathogenic bacteria. The molecular mechanisms by which bacteria become resistant are manifold, but in general these can be collectively characterized as involving either drug efflux, drug inactivation, or alterations in the drug target site. The target site for Macrolides is the large (50S) subunit of the bacterial ribosome. Many cases of macrolide resistance in clinical strains can be linked to alteration of specific nucleotides in 23S rRNA within the large ribosomal subunit. Macrolides are natural polyketide products of secondary metabolism in many actinomycete species (51, 140). Clinically useful Macrolides consist of a 14-, 15-, or 16-member lactone ring (Table (Table1)1) that is generally substituted with two or more neutral and/or amino sugars (16). The structures of the 14- and 16-member-ring Macrolides erythromycin and tylosin and of some semisynthetic erythromycin derivatives are shown in Fig. Fig.1.1. The inhibitory action of erythromycin, and probably that of the other 14-member-ring Macrolides, is effected at the early stages of protein synthesis when the drug blocks the growth of the nascent peptide chain (7, 140), presumably causing premature dissociation of the peptidyl-tRNA from the ribosome (85). The antimicrobial action of these drugs is compounded by their inhibition of the assembly of new large ribosomal subunits, which leads to gradual depletion of functional ribosomes in the cell (23). The mode of action of the 16-member-ring Macrolides is less well characterized, although it is clear that they bind to the same region of the large subunit as the 14-member-ring Macrolides and inhibit peptide bond formation in a more direct manner (reviewed in reference 140). TABLE 1 Macrolide antibiotics and their derivatives discussed in this review FIG. 1 Selected clinically important macrolide antibiotics and their derivatives. Two naturally occurring Macrolides are shown: erythromycin A, which was the first therapeutic macrolide and possesses a 14-member ring, and tylosin, a 16-member-ring macrolide ... Shortly after the introduction of erythromycin in therapy in the 1950s, resistance to the drug was observed in bacterial pathogens (reviewed in reference 76). More disquieting was the observation that erythromycin-resistant strains were cross-resistant not only to all other Macrolides but also to the chemically unrelated lincosamide and streptogramin B drugs. This phenomenon was first observed in Staphylococcus aureus and came to be termed the macrolide-lincosamide-streptogramin B (MLSB) antibiotic resistance phenotype. In these S. aureus strains, MLSB resistance can be induced by exposure to low concentrations of erythromycin (151), which leads to expression of a methyltransferase enzyme (ErmC). ErmC specifically methylates 23S rRNA (74) at the N-6 position of adenosine 2058 (A2058) (Escherichia coli numbering) (121), which is a pivotal nucleotide for the binding of MLSB antibiotics (see below). Subsequently, several dozen erm methyltransferase genes have been identified. Many of these are constitutively expressed, and their products all presumably methylate A2058. A new nomenclature system has recently been proposed for the different erm genes, which clarifies their phylogenetic relatedness (105). For a comprehensive account of the action of Erm methyltransferases, see the review by Weisblum (149). Since the discovery of erm genes, another means of resistance involving alteration of rRNA structure has been identified. Under laboratory conditions, single base substitutions introduced into rRNA were shown to confer macrolide resistance. This form of resistance was first observed in the single rRNA (rrn) operon of yeast mitochondria, which was mutated at position A2058 in the large-subunit rRNA (123). Shortly afterwards, similar phenotypes were obtained in E. coli by expression of mutant rrn alleles from multiple-copy plasmids (see, e.g., references 120 and 143). About 6 years ago, reports of rRNA mutations conferring macrolide resistance in clinical pathogens began to appear in the literature. While it is conceptually gratifying to establish that the mutations appearing in pathogens are identical to those previously isolated in laboratory strains, the clinical implications of this are quite disturbing. The 23S rRNA mutations reported so far to cause macrolide resistance are shown in Table Table2.2. Generally, pathogenic species that develop macrolide resistance through mutations at A2058 (or neighboring nucleotides) possess only one or two rrn operons, such as in the case of Helicobacter pylori and Mycobacterium species. Resistance in bacteria with multiple rrn operons, such as Enterococcus, Streptococcus, and Staphylococcus species, is generally conferred by Erm methylation of A2058 (Table (Table3)3) or by efflux (see e.g., references 70 and 110). However, there are cases of macrolide resistance by drug inactivation (reviewed in reference 150), and there are recent reports of macrolide resistance in Streptococcus pneumoniae strains conferred by mutations in ribosomal proteins L4 and L22 and in rRNA (129; P. Appelbaum, personal communication). Macrolide and ketolide resistance is additionally conferred in E. coli by the expression of small, specific peptides (134), although the level of resistance is probably too low to be a problem in the treatment of clinical strains. TABLE 2 23S rRNA mutations reported to confer macrolide resistance TABLE 3 Macrolide resistance mechanisms found in some pathogens and their numbers of rRNA operons In the following sections of this review, we first look at the current state of knowledge of the bacterial ribosome target site for macrolide antibiotics. A detailed model of a drug target site is a prerequisite for understanding the molecular mechanisms of drug binding and drug resistance and for rational design of new drugs. Our present state of knowledge, although far from being complete, supports the view that the macrolide target site is highly conserved within the ribosomes of all bacteria. We then direct attention to the pathogens, and in particular to H. pylori, that have been shown to attain resistance by rRNA mutation, and we consider the possibility of this form of resistance emerging in other pathogens. Finally, some suggestions are made regarding how future macrolide derivatives might be best equipped to combat bacteria with resistant rRNAs.
C Bebear - One of the best experts on this subject based on the ideXlab platform.
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emergence of a 23s rrna mutation in mycoplasma hominis associated with a loss of the intrinsic resistance to erythromycin and azithromycin
Journal of Antimicrobial Chemotherapy, 2006Co-Authors: Sabine Pereyre, H Renaudin, A Charron, C BebearAbstract:Objectives: Mycoplasma hominis is intrinsically resistant to 14- and 15-membered Macrolides and to the ketolide telithromycin but is susceptible to josamycin, a 16-membered macrolide, and lincosamides. The aim of our study was to investigate the in vitro development of macrolide resistance in M. hominis and to study the impact of ribosomal mutations on MICs of various Macrolides and related antibiotics. Methods: Selection of macrolide-resistant mutants was performed by serial passages of M. hominis PG21 in broth medium containing subinhibitory concentrations of clindamycin, pristinamycin, quinupristin/ dalfopristin and telithromycin. Stepwise selection of josamycin-resistant mutants was performed onto agar medium containing increasing inhibitory concentrations of josamycin. Resistant mutants were characterized by PCR amplification and DNA sequencing of 23S rRNA, L4 and L22 ribosomal protein genes. Results: Various mutations in domain II or V of 23S rRNA were selected in the presence of each selector antibiotic and were associated with several resistance phenotypes. Josamycin was the sole antibiotic that selected for single amino acid changes in ribosomal proteins L4 and L22. Unexpectedly, the C2611U transition selected in the presence of clindamycin and the quinupristin/dalfopristin combination was associated with decreased MICs of erythromycin, azithromycin and telithromycin, leading to a loss of the intrinsic resistance of M. hominis to erythromycin and azithromycin. Conclusions: Ribosomal mutations were associated with resistance to Macrolides and related antibiotics in M. hominis. Some mutants showed a loss of the intrinsic resistance to erythromycin and azithromycin.
Yeo Joon Yoon - One of the best experts on this subject based on the ideXlab platform.
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Recent advances in the discovery and combinatorial biosynthesis of microbial 14-membered Macrolides and macrolactones
Journal of Industrial Microbiology & Biotechnology, 2019Co-Authors: Je Won Park, Yeo Joon YoonAbstract:Macrolides, especially 14-membered Macrolides, are a valuable group of antibiotics that originate from various microorganisms. In addition to their antibacterial activity, newly discovered 14-membered Macrolides exhibit other therapeutic potentials, such as anti-proliferative and anti-protistal activities. Combinatorial biosynthetic approaches will allow us to create structurally diversified macrolide analogs, which are especially important during the emerging post-antibiotic era. This review focuses on recent advances in the discovery of new 14-membered Macrolides (also including macrolactones) from microorganisms and the current status of combinatorial biosynthetic approaches, including polyketide synthase (PKS) and post-PKS tailoring pathways, and metabolic engineering for improved production together with heterologous production of 14-membered Macrolides.
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exploiting the natural metabolic diversity of streptomyces venezuelae to generate unusual reduced Macrolides
Chemical Communications, 2008Co-Authors: Je Won Park, Sung Ryeol Park, Won Seok Jung, Yeon-hee Ban, Eunji Kim, Ah Reum Han, Hanyoung Kang, Yeo Joon YoonAbstract:An unusual set of reduced macrolide antibiotics was discovered by combination of organic synthesis and a biosynthetic approach using the unique metabolic diversity of Streptomyces venezuelae; two unnatural 16-membered ring Macrolides are also created by employing this bio-catalyst.
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enhanced production of hydroxylated Macrolides from the pikromycin pathway of streptomyces venezuelae
Enzyme and Microbial Technology, 2006Co-Authors: Sang Kil Lee, Cha Yong Choi, Jong Seog Ahn, Jay Sung Joong Hong, Yeo Joon YoonAbstract:The post-PKS (polyketide synthase) modification reactions, including the hydroxylation step catalyzed by cytochrome P450 monooxygenases, are often crucial to the structural diversity and biological potency of the macrolide polyketides. In this study, we describe a strategy for enhancing the productivity of a set of desired hydroxylated Macrolides. In a liquid culture of Streptomyces venezuelae, the intermediate Macrolides, YC-17 and narbomycin, accumulate, and a small amount of hydroxylated compounds, namely neomethymycin and pikromycin, is produced. The improved generation of hydroxylated polyketides mediated by the PikC cytochrome hydroxylase from S. venezuelae was accomplished via the overexpression of the pikC gene, the supplementation of ferrous sulfate into the liquid medium, and feeding with the aglycones, 10-deoxymethynolide and narbonolide. In particular, this enhancement of production was achieved with a considerable reduction in culture time. In a liquid culture of a mutant strain (YJ029) that overexpresses the pikC gene, the bioconversion of the 12-membered ring macrolide YC-17 to methymycin and neomethymycin increased by approximately three-fold as compared to that of wild-type S. venezuelae. In the case of the 14-membered ring macrolide, narbomycin, bioconversion to pikromycin increased by approximately five-fold. In addition, the addition of ferrous sulfate and the feeding of aglycones into the medium resulted in a significantly higher generation of the desired hydroxylated Macrolides.
Thomas A Steitz - One of the best experts on this subject based on the ideXlab platform.
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the structures of four macrolide antibiotics bound to the large ribosomal subunit
Molecular Cell, 2002Co-Authors: J L Hansen, Peter B. Moore, Poul Nissen, Thomas A Steitz, Joseph A IppolitoAbstract:Abstract Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered Macrolides and the N6 of A2103 (A2062, E. coli ). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to Macrolides.
Denis Fourches - One of the best experts on this subject based on the ideXlab platform.
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Cheminformatics-based enumeration and analysis of large libraries of macrolide scaffolds
Journal of Cheminformatics, 2018Co-Authors: Phyo Phyo Kyaw Zin, Gavin Williams, Denis FourchesAbstract:We report on the development of a cheminformatics enumeration technology and the analysis of a resulting large dataset of virtual macrolide scaffolds. Although Macrolides have been shown to have valuable biological properties, there is no ready - to - screen virtual library of diverse Macrolides in the public domain. Conducting molecular modeling (especially virtual screening) of these complex molecules is highly relevant as the organic synthesis of these compounds, when feasible, typically requires many synthetic steps, and thus dramatically slows the discovery of new bioactive Macrolides. Herein, we introduce a cheminformatics approach and associated software that allows for designing and generating libraries of virtual macrocycle/macrolide scaffolds with user-defined constitutional and structural constraints (e.g., types and numbers of structural motifs to be included in the macrocycle, ring size, maximum number of compounds generated). To study the chemical diversity of such generated molecules, we enumerated V1M (Virtual 1 million Macrolide scaffolds) library, each containing twelve common structural motifs. For each macrolide scaffold, we calculated several key properties, such as molecular weight, hydrogen bond donors/acceptors, topological polar surface area. In this study, we discuss (1) the initial concept and current features of our PKS (polyketides) Enumerator software, (2) the chemical diversity and distribution of structural motifs in V1M library, and (3) the unique opportunities for future virtual screening of such enumerated ensembles of Macrolides. Importantly, V1M is provided in the Supplementary Material of this paper allowing other researchers to conduct any type of molecular modeling and virtual screening studies. Therefore, this technology for enumerating extremely large libraries of macrolide scaffolds could hold a unique potential in the field of computational chemistry and drug discovery for rational designing of new antibiotics and anti-cancer agents.
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MOESM2 of Cheminformatics-based enumeration and analysis of large libraries of macrolide scaffolds
2018Co-Authors: Phyo Zin, Gavin Williams, Denis FourchesAbstract:Additional file 2. Table S1: Common Structural Motif (CSM) Type Distribution and Occurrence per Macrolide Scaffold among Eighteen Bioactive Macrolide (BM) Scaffolds. Figure S1: Sixteen structural motifs (nine CSMs and seven RSMs) currently employed in PKS Enumerator software, along with the bioactive Macrolides from which they were derived. Figure S2: Modified structures of eighteen well-known bioactive Macrolides (BMs). Figure S3: Structural simplification of Erythromycin for a comparative study with enumerated virtual macrolide scaffolds from V1M. Figure S4: Percentage of (A) macrolide scaffolds in which associated CSM types were found, and (B) CSM type composition, in 18 BMs and V1M. Figure S5: Distribution of (A) rotatable bonds, and (B) heavy atoms in V1M. Figure S6: Color-coded map to demonstrate the molecular properties of eighteen bioactive macrolide scaffolds in correlation to Lipinski’s and Veber’s rules. Figure S7: Pearson’s pair-wise correlation heatmap of all eight molecular descriptors of V1M library: MW – molecular weight, SlogP – hydrophobicity, TPSA - topological polar surface area, HBA – hydrogen bond acceptors, HBD – hydrogen bond donors, NRB – rotatable bonds, heteroatoms, heavy atoms. Figure S8: Modified structures of Rokitamycin and Spiramycin. The computed Tanimoto score between these two structures is 1, based on MACCS fingerprint method
