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1.
Artigo em Inglês | MEDLINE | ID: mdl-33253083

RESUMO

Genome sequence analysis of two strains collected in Canada at the end of the 1970s and deposited in 1998 at the Collection de l'Institut Pasteur has led to the taxonomic description of a novel fish-associated species in the genus Flavobacterium. Both strains, CIP 105534T and CIP 105535, were yellow-pigmented, Gram-stain-negative, non-spore-forming rod-shaped bacteria that exhibited gliding motility. They grew aerobically in a temperature range from 5 to 30 °C with optimal growth at 25 °C on trypticase soy or Reasoner's 2A agar but they did not grow on marine agar. Their major fatty acid profiles were similar, consisting of iso-C15 : 0, C16 : 1 ω7c and/or iso-C15 : 0 2-OH (shown as summed feature 3), C16 : 0 3-OH, iso-C17 : 0 3-OH and C16 : 0. The major polyamine was sym-homospermidine. Phosphatidylethanolamine and, most notably, ornithine-containing lipid OL2 and unidentified aminophospholipid APL1 were major polar lipids. A yellow pigment spot was visible after chromatographic analysis. The predominant respiratory quinone was MK-6. The G+C content of the two genomes was 34 mol% and their size was around 5.8 Mb. Comparison of the 16S rRNA gene sequences with those of the closely related type strains showed high levels of relatedness with Flavobacterium collinsii and Flavobacterium pectinovorum. All average nucleotide identity (ANI) and digital DNA-DNA hybridization values estimated against publicly available Flavobacterium genome assemblies were lower than 90 and 30 %, respectively. Phylogenetic, phenotypic and chemotaxonomic data indicated that the two strains represent a novel species of the genus Flavobacterium, for which the name Flavobacterium bizetiae sp. nov. is proposed. The type strain is CIP 105534T (=LMG 1342T). The unique ability of F. bizetiae to use melibiose as a sole source of carbon could provide a simple phenotypic test to discriminate F. bizetiae from its closest relatives.

2.
Nature ; 586(7827): 145-150, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968273

RESUMO

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

3.
Int J Syst Evol Microbiol ; 70(3): 1961-1962, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31971498

RESUMO

A previous 16S rRNA gene sequence comparison had demonstrated that the type strains of Serratia vespertilionis and Serratia ficaria shared 99.5 % sequence similarity. Despite the 56.2 % homology by DNA-DNA hybridization previously found between these strains, the results of an in silico whole-genome sequence comparison and a new DNA-DNA hybridization study have clearly demonstrated that the genomes of the type strain of S. vespertilionis deposited in different Culture Collections (52T=CECT 8595T=DSM 28727T) and the type strain of S. ficaria (culture DSM 4569T), cannot support such a species differentiation. Tests for substrate utilization redone on the deposited cultures of these strains has also shown very few differences between the type strains of both species. Based on these results, and since the name S. ficaria was validly published earlier, S. vespertilionis should be considered as a later heterotypic synonym of S. ficaria, in application of the priority rule. The type strain of the species S. ficaria is strain 4024T=DSM 4569T=NCTC 12148T=ATCC 33105T=CIP 79.23T=ICPB 4050T.


Assuntos
Filogenia , Serratia/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
4.
Res Microbiol ; 170(8): 435-447, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31563533

RESUMO

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Escherichia coli/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Transporte Biológico/fisiologia , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Biossíntese de Proteínas/genética , Conformação Proteica , Domínios Proteicos , Ribossomos/metabolismo
5.
Genome Announc ; 6(14)2018 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-29622616

RESUMO

Flavobacterium columnare strain PH-97028 (=CIP 109753) is a genomovar III reference strain that was isolated from a diseased Ayu fish in Japan. We report here the analysis of the first available genomovar III sequence of this species to aid in identification, epidemiological tracking, and virulence studies.

6.
Syst Appl Microbiol ; 40(7): 401-410, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28890241

RESUMO

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.


Assuntos
Genoma Bacteriano/genética , Oxirredutases/metabolismo , Staphylococcus , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Bases , Farmacorresistência Bacteriana/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ácido Mevalônico/metabolismo , Novobiocina/farmacologia , Fenótipo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Análise de Sequência de DNA , Fator sigma/genética , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Staphylococcus/metabolismo , Terpenos/metabolismo
7.
Antimicrob Agents Chemother ; 58(6): 3335-41, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24687494

RESUMO

Natural resistance to lincosamides and streptogramins A (LSA), which is a species characteristic of Bacillus subtilis and Enterococcus faecalis, has never been documented in the Staphylococcus genus. We investigate here the molecular basis of the LSA phenotype exhibited by seven reference strains of Staphylococcus sciuri, including the type strains of the three described subspecies. By whole-genome sequencing of strain ATCC 29059, we identified a candidate gene that encodes an ATP-binding cassette protein similar to the Lsa and VmlR resistance determinants. Isolation and reverse transcription-quantitative PCR (qRT-PCR) expression studies confirmed that Sal(A) can confer a moderate resistance to lincosamides (8 times the MIC of lincomycin) and a high-level resistance to streptogramins A (64 times the MIC of pristinamycin II). The chromosomal location of sal(A) between two housekeeping genes of the staphylococcal core genome supports the gene's ancient origins and thus innate resistance to these antimicrobials within S. sciuri subspecies.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Lincosamidas/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética , Estreptogramina A/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Mapeamento Cromossômico , DNA Bacteriano/química , DNA Bacteriano/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fenótipo , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Staphylococcus/efeitos dos fármacos
8.
Antimicrob Agents Chemother ; 57(9): 4543-4546, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23796922

RESUMO

There exist numerous genes disseminated by mobile elements that can confer cross-resistance to lincosamides and streptogramin A compounds in staphylococci. This study investigated the nature and means of dissemination of genes responsible for LSA resistance among 24 French clinical isolates screened for reduced susceptibility to lincomycin. The vga(A)v gene was found to be the most prevalent determinant of LSA resistance, while Tn5406 appeared to be its exclusive gene support.

9.
Res Microbiol ; 164(3): 226-35, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23261969

RESUMO

Macrolides have wide clinical applications in the treatment of community-acquired respiratory tract infections, among which streptococci are the most frequent causative agents. An active efflux-based mechanism of macrolide resistance, referred to as the M phenotype in streptococcal isolates, has been associated with the presence of mef genes that encode a subset of major facilitator superfamily (MFS) transporters like Mef(E). An msr(D) gene, adjacent to and co-transcribed with mef in the presence of erythromycin, has also been implicated in drug efflux, but its role remains elusive. Msr(D) belongs to the ATP binding cassette (ABC) proteins and harbors two fused nucleotide-binding domains with no membrane-spanning domains. The present work indicates that the major resistance traits of the M phenotype in Escherichia coli may be due to Msr(D) and not to Mef(E). Fluorescence microscopy using Mef(E) tagged with GFP linked low efficacy of the chimera in conferring macrolide resistance with improper subcellular localization. The active role of Msr(D) in directing Mef(E)-GFP to the cell poles was demonstrated, as was synergistic effect in terms of levels of resistance when both proteins were expressed. A trans-dominant negative mutation within ABC Msr(D) affecting MFS Mef(E) strongly suggests that both proteins can interact in vivo, and such a physical interaction was supported in vitro. This is the first reported example of a functional interplay between an ABC component and an MFS transporter. The direct involvement of Msr(D) in the efflux of macrolides remains to be demonstrated.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Macrolídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Eritromicina/metabolismo , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Terciária de Proteína
10.
Biochemistry ; 48(40): 9372-83, 2009 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-19711984

RESUMO

A comparative study was designed to evaluate the staphylococcidal efficiency of two sequence-related plasticins from the dermaseptin superfamily we screened previously. Their bactericidal activities against Staphylococcus aureus as well as their chemotactic potential were investigated. The impact of the GraS/GraR two-component system involved in regulating resistance to cationic antimicrobial peptides (CAMPs) was evaluated. Membrane disturbing activity was quantified by membrane depolarization assays using the diS-C3 probe and by membrane integrity assays measuring beta-galactosidase activity with recombinant strain ST1065 reflecting compromised membranes and cytoplasmic leakage. Interactions of plasticins with membrane models composed of either zwitterionic lipids mimicking the S. aureus membrane of CAMP-resistant strains or anionic lipids mimicking the negative charge-depleted membrane of CAMP-sensitive strains were analyzed by jointed Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and differential scanning calorimetry (DSC) to yield detailed information about the macroscopic interfacial organization, in situ conformation, orientation of the peptides at the lipid-solvent interface, and lipid-phase disturbance. We clearly found evidence of distinct interfacial behaviors of plasticins we linked to the distribution of charges along the peptides and structural interconversion properties at the membrane interface. Our results also suggest that amidation might play a key role in GraS/GraR-mediated CAMP sensing at the bacterial surface.


Assuntos
Antibacterianos/química , Antibacterianos/toxicidade , Proteínas do Olho/química , Proteínas do Olho/toxicidade , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Adulto , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Farmacorresistência Bacteriana , Proteínas do Olho/antagonistas & inibidores , Inibidores do Crescimento/antagonistas & inibidores , Inibidores do Crescimento/química , Inibidores do Crescimento/toxicidade , Humanos , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Conformação Proteica , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/crescimento & desenvolvimento
11.
J Biol Chem ; 283(37): 25332-9, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18562322

RESUMO

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Hidrólise , Staphylococcus aureus/metabolismo , Estreptogramina A/química , Estreptogramina A/farmacologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Antibacterianos , Resistência Microbiana a Medicamentos , Cinética , Modelos Químicos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo
12.
FEMS Microbiol Lett ; 269(2): 317-22, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17302923

RESUMO

This study aims to compare the resistance phenotypes conferred by various genes encoding enzymes that phosphorylate erythromycin. The mph genes were cloned into Escherichia coli AG100A susceptible to macrolides and ketolides following disruption of the AcrAB pump. An 882 bp sequence containing a premature stop codon, homologous to the three other previously described mph genes and present widely among Enterobacteriaceae, was found to confer resistance to erythromycin by phosphorylation. The mph(C) gene, as reported for mph(B), also conferred resistance to spiramycin. The mph(A) gene was unique in conferring resistance to azithromycin. The four investigated genes conferred resistance to telithromycin.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Macrolídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Antibacterianos/metabolismo , Farmacorresistência Bacteriana/genética , Eritromicina/metabolismo , Eritromicina/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Cetolídeos/metabolismo , Cetolídeos/farmacologia , Macrolídeos/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmídeos , Recombinação Genética
14.
Antimicrob Agents Chemother ; 49(3): 973-80, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15728891

RESUMO

The Vga and Msr resistance determinants, encoded by mobile genetic elements in various staphylococcal strains, belong to a family of ATP-binding cassette (ABC) proteins whose functions and structures are ill defined. Their amino acid sequences are similar to those of proteins involved in the immunity of streptomycetes to the macrolide-lincosamide-streptogramin antibiotics that they produce. Sequence analysis of the genomes of the gram-positive bacteria with low G+C contents revealed that Lmo0919 from Listeria monocytogenes is more closely related to Vga variants than to Msr variants. In the present study we compared the antibiotic resistance profiles conferred by the Vga-like proteins in two staphylococcal hosts. It was shown that Vga(A), the Vga(A) variant [Vga(A)v], and Lmo0919 can confer resistance to lincosamides and streptogramin A compounds, while only Vga(B) is able to increase the level of resistance to pristinamycin, a mixture of streptogramin A and streptogramin B compounds. By using polyclonal antibodies, we found that the Vga(A) protein colocalized with the beta subunit of the F(1)-F(0) ATPase in the membrane fractions of staphylococcal cells. In order to identify functional units in these atypical ABC proteins, such as regions that might be involved in substrate specificity and/or membrane targeting, we analyzed the resistance phenotypes conferred by various plasmids carrying parts or modified versions of the vga(A) gene and we determined the subcellular localization of the gene products. Only polypeptides composed of two ABC domains were detected in the cell membranes. No region of drug specificity was identified. Resistance properties were dependent on the integrities of both Walker B motifs.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Bactérias/fisiologia , Staphylococcus/efeitos dos fármacos , Estreptogramina A/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Dados de Sequência Molecular , Fenótipo
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