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1.
Int J Antimicrob Agents ; 54(5): 647-651, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476434

RESUMO

A lincosamide-resistant and macrolide-susceptible phenotype has not been described to date in Streptococcus pyogenes [group A streptococcus (GAS)]. The aim of this study was to characterize a GAS isolate susceptible to macrolides but resistant to lincosamide, streptogramin A and pleuromutilin antibiotics. Antimicrobial susceptibility was tested using the microdilution broth method and the resistance phenotype was tested by D-test. The GAS2887HUB isolate was subjected to whole-genome sequencing. The isolate showed a positive Gots' test (clindamycin inactivation). Whole-genome sequencing revealed that the strain was ST10 and emm93, and had five resistance genes [lnu(B), ant(6)-Ia, aph(3')-III, tet(M) and dfrG]. The tet(M) gene was located in a Tn916-like transposon. The lsa(E)-lnu(B)-containing sequence (inserted downstream of the rumA gene) was formed by a 39.6-kb prophage, followed by a gene cluster encoding aminoglycoside-streptothricin resistance [ant(6)Ia-sat4-aph(3')III] and lsa(E)-lnu(B) genes. This structure was not transferred by conjugation. This study identified a new genetic element carrying a determinant of lincosamide resistance in a GAS. Further molecular epidemiological surveys are needed to determine the prevalence of this mechanism of resistance in GAS.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Ilhas Genômicas/genética , Prófagos/genética , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Vírus Defeituosos/genética , Diterpenos/farmacologia , Genoma Bacteriano/genética , Humanos , Lincosamidas/farmacologia , Testes de Sensibilidade Microbiana , Compostos Policíclicos/farmacologia , Streptococcus pyogenes/isolamento & purificação , Estreptogramina A/farmacologia , Sequenciamento Completo do Genoma
2.
Food Chem ; 250: 127-133, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29412901

RESUMO

A reliable UPLC-MS/MS method with high sensitivity was developed and validated for the determination of virginiamycin M1 in muscle, fat, liver, and kidney samples of chicken and swine. Analytes were extracted using acetonitrile and extracts were defatted by N-hexane. Chromatographic separation was performed on a BEH C18 liquid chromatography column. The analytes were then detected using triplequadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration plots were constructed using standard working solutions and showed good linearity. Limits of quantification ranged from 2 to 60 ng mL-1.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Resíduos de Drogas/análise , Estreptogramina A/análise , Espectrometria de Massas em Tandem/métodos , Animais , Galinhas , Especificidade de Órgãos , Suínos
3.
Artigo em Inglês | MEDLINE | ID: mdl-28415016

RESUMO

Antibiotics are used in ethanol production to discourage the growth of bacteria that would result in lower ethanol content and a lower quality product. A survey conducted by the FDA (FY 2010 Nationwide Survey of Distillers Grains for Antibiotic Residues, 2009 [1]) revealed that the residues of these antibiotics can remain in the distillers grains (DG) by-product, which is used as an animal feed ingredient. The low levels of antibiotic residues in DG could be a public health concern, as they could lead to antimicrobial resistance. To enable the quantitative determination of these antibiotics (erythromycin, penicillin G, virginiamycin M1 and virginiamycin S1), we developed a sensitive LC-MS/MS method. The residues were extracted from distillers grains with a mixture of acetonitrile and buffer followed by acetonitrile. The combined extract was diluted with water and washed with hexane. An aliquot was cleaned up on an Oasis HLB solid phase extraction cartridge. Extracts were analyzed by LC-tandem mass spectrometry. The method was successfully validated using a variety of different matrices such as corn DG, corn & milo DG, and deoiled corn DG. Absolute recoveries of the analytes ranged from 53 to 106%. Accuracy ranged from 90 to 101% based on calibration by matrix standards. The limits of quantitation and relative standard deviation were all satisfactory to support future surveillance studies.


Assuntos
Ração Animal/análise , Antibacterianos/análise , Eritromicina/análise , Penicilina G/análise , Estreptogramina A/análise , Estreptogramina Grupo B/análise , Espectrometria de Massas em Tandem/métodos , Virginiamicina/análise , Acetonitrilos/química , Animais , Cromatografia Líquida/métodos , Grão Comestível/química , Hexanos/química , Limite de Detecção , Extração em Fase Sólida/métodos
4.
J Antimicrob Chemother ; 70(12): 3205-13, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26410170

RESUMO

OBJECTIVES: In group B Streptococcus (GBS), cross-resistance to lincosamides, streptogramin A and pleuromutilins (LSAP) is mediated by the acquisition of lsa genes. Here, we characterized the diversity, mobility and ecology of lsa genes in this species. METHODS: lsa variants were systematically identified by BLAST searches in the genomes of 531 GBS strains from different hosts and geographical origins. The associated phenotypes were determined by a microdilution MIC method. Acquisition of resistance genes was deduced from comparative genomics and phylogeny. Their mobility was tested by conjugation experiments. RESULTS: lsa(E) and three variants of lsa(C) were identified in GBS strains. Two lsa(C) variants had not been previously reported. All four variants conferred LSAP phenotypes. lsa(E) was located in a multiresistance gene cluster of a single human strain. This gene was transferred by a high-frequency recombination-type mechanism between GBS strains. Two lsa(C) variants are carried in six unrelated human strains by two similar elements specifically integrated in the oriT site of four different classes of integrative and conjugative elements (ICEs). Strikingly, the acquisition of the resistance gene always occurred by the integration of the element into a resident ICE. The third lsa(C) variant was located at the same site in the core genome of 11 genetically distant bovine strains and was likely propagated by horizontal transfer of the corresponding chromosomal region. CONCLUSIONS: lsa genes in GBS show distinct host specificities and modes of transfer. In general, their dissemination is mediated by recombination rather than by the transfer of conjugative elements.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Transferência Genética Horizontal , Genes Bacterianos , Especificidade de Hospedeiro , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Diterpenos/farmacologia , Variação Genética , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Lincosamidas/farmacologia , Testes de Sensibilidade Microbiana , Compostos Policíclicos , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Streptococcus agalactiae/isolamento & purificação , Estreptogramina A/farmacologia
5.
Vet Microbiol ; 177(3-4): 353-8, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25891423

RESUMO

The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Animais , Bovinos , Coagulase/genética , Diterpenos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Lincosamidas/farmacologia , Mastite Bovina/tratamento farmacológico , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Compostos Policíclicos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/enzimologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Staphylococcus hominis/efeitos dos fármacos , Staphylococcus hominis/genética , Estreptogramina A/farmacologia , Estreptograminas/farmacologia
6.
Vet Microbiol ; 177(1-2): 162-7, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25759293

RESUMO

Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes erysipelas in swine. In recent years, erysipelas infection among swine in China has been increasing. A combined resistance phenotype to pleuromutilins, lincosamides, and streptogramin A (PLSA phenotype) was found in some E. rhusiopathiae isolates. The aim of this study was to identify the resistance genes responsible for the PLSA phenotype in E. rhusiopathiae strains and to map the genetic environment of the identified resistance gene. A total of 46 E. rhusiopathiae isolates from 31 pig farms in China were studied. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by broth microdilution method. Seven were highly resistant to tiamulin (MICs 32 µg/ml) and clindamycin (MICs 64 µg/ml). Resistance genes responsible for the PLSA phenotype were screened by PCR. The lsa(E), spw, lnu(B), aadE and aphA3 genes were detected in strains had the PLSA phenotype, whereas none was detected in susceptible strains. The genetic environment of lsa(E) gene was determined by whole-genome sequencing and overlapping PCR assays. A novel multiresistance gene cluster, orf1-aadE-apt-spw-lsa(E)-lnu(B)-rec-orf2-orf1-aadE-sat4-aphA3, was found. Horizontal gene transfer experiments and whole-genome sequencing suggested that the lsa(E)-carrying multiresistance gene cluster was located in the chromosome. This is the first molecular characterization of PLSA resistance in E. rhusiopathiae. The lsa(E), spw and lnu(B) genes were found in E. rhusiopathiae for the first time. A novel lsa(E)-carrying multiresistance gene cluster was found. The location of lsa(E) in different gene cluster facilitates its persistence and dissemination.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Erysipelothrix/efeitos dos fármacos , Erysipelothrix/genética , Genes MDR/genética , Suínos/microbiologia , Animais , Sequência de Bases , China , Clindamicina/farmacologia , Diterpenos/farmacologia , Lincosamidas/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Família Multigênica/genética , Compostos Policíclicos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Estreptogramina A/farmacologia
7.
Metab Eng ; 29: 12-25, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25708513

RESUMO

Pristinamycin, which is a streptogramin antibiotic produced by Streptomyces pristinaespiralis, contains two chemically unrelated compounds, pristinamycin I (PI) and pristinamycin II (PII). Semi-synthetic derivatives of PI and PII have been approved for use in human medicine to treat a broad range of drug-resistant pathogens. In this study, we design and implement a combinatorial metabolic engineering strategy for improving PII production. First, an extra copy of the PII biosynthetic gene cluster, which was assembled using a modified Gibson assembly method for cloning large DNA fragments with high GC contents, was introduced into a high-producing strain S. pristinaespiralis HCCB10218. This duplication of the PII biosynthetic gene cluster resulted in a maximum increase in PII titer by 45%. Second, all seven cluster-situated regulatory genes (from papR1 to papR6 and spbR) were systematically manipulated. Higher PII titers were achieved by deleting either one of the two repressor genes papR3 or papR5 in combination with overexpression of both activator genes papR4 and papR6, and the resulting strains ∆papR3+R4R6 and ∆papR5+R4R6 showed maximum increases in PII production by 99% and 75%, respectively. A combination of the above two different approaches was employed. Integration of the assembled PII gene cluster (BAC-F1F15) into ∆papR5+R4R6 led to the highest PII titer improvement, which was approximately 1.5-fold higher than the parental strain. By adding the macroreticular resin, which can separate pristinamycin in situ and thereby lessen end-product feedback inhibition and toxic effects, PII titers of the final engineered strain ∆papR5+R4R6/BAC-F1F15 reached 1.13 and 1.16g/L in the Erlenmeyer flask and 5-L bioreactor, respectively, with 5.13- and 5.26-fold improvements over the parental strain. Taken together, this combinatorial strategy is an efficient method to optimize PII biosynthesis of S. pristinaespiralis and may be extended to other industrially used streptomycetes for strain improvement.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Estreptogramina A/biossíntese , Streptomyces , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Humanos , Engenharia Metabólica , Streptomyces/genética , Streptomyces/metabolismo
8.
J Bacteriol ; 197(3): 441-50, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25404695

RESUMO

There are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster of Streptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of the papR6 gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, either papR6 deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region of snaF, the first gene of the snaFE1E2GHIJK operon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C11 αß-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5'-GAGG-4 nt-CCTC-3') was identified. Transcriptional analysis showed that inactivation of the papR6 gene led to markedly decreased expression of snaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ΔpapR6 strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C11 αß-unsaturated thioester.


Assuntos
Regulação Bacteriana da Expressão Gênica , Estreptogramina A/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Família Multigênica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética
9.
Antimicrob Agents Chemother ; 58(12): 7083-92, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25223995

RESUMO

Combinations of group A and B streptogramins (i.e., dalfopristin and quinupristin) are "last-resort" antibiotics for the treatment of infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Resistance to streptogramins has arisen via multiple mechanisms, including the deactivation of the group A component by the large family of virginiamycin O-acetyltransferase (Vat) enzymes. Despite the structural elucidation performed for the VatD acetyltransferase, which provided a general molecular framework for activity, a detailed characterization of the essential catalytic and antibiotic substrate-binding determinants in Vat enzymes is still lacking. We have determined the crystal structure of S. aureus VatA in apo, virginiamycin M1- and acetyl-coenzyme A (CoA)-bound forms and provide an extensive mutagenesis and functional analysis of the structural determinants required for catalysis and streptogramin A recognition. Based on an updated genomic survey across the Vat enzyme family, we identified key conserved residues critical for VatA activity that are not part of the O-acetylation catalytic apparatus. Exploiting such constraints of the Vat active site may lead to the development of streptogramin A compounds that evade inactivation by Vat enzymes while retaining binding to their ribosomal target.


Assuntos
Acetiltransferases/química , Antibacterianos/química , Proteínas de Bactérias/química , Estreptogramina A/química , Acetilcoenzima A/química , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Farmacorresistência Bacteriana/genética , Expressão Gênica , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência
10.
Antimicrob Agents Chemother ; 58(9): 5269-79, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24957822

RESUMO

Streptogramin antibiotics are divided into types A and B, which in combination can act synergistically. We compared the molecular interactions of the streptogramin combinations Synercid (type A, dalfopristin; type B, quinupristin) and NXL 103 (type A, flopristin; type B, linopristin) with the Escherichia coli 70S ribosome by X-ray crystallography. We further analyzed the activity of the streptogramin components individually and in combination. The streptogramin A and B components in Synercid and NXL 103 exhibit synergistic antimicrobial activity against certain pathogenic bacteria. However, in transcription-coupled translation assays, only combinations that include dalfopristin, the streptogramin A component of Synercid, show synergy. Notably, the diethylaminoethylsulfonyl group in dalfopristin reduces its activity but is the basis for synergy in transcription-coupled translation assays before its rapid hydrolysis from the depsipeptide core. Replacement of the diethylaminoethylsulfonyl group in dalfopristin by a nonhydrolyzable group may therefore be beneficial for synergy. The absence of general streptogramin synergy in transcription-coupled translation assays suggests that the synergistic antimicrobial activity of streptogramins can occur independently of the effects of streptogramin on translation.


Assuntos
Antibacterianos/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Estreptograminas/uso terapêutico , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Cristalografia por Raios X , Combinação de Medicamentos , Sinergismo Farmacológico , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Haemophilus influenzae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Estreptogramina A/administração & dosagem , Estreptogramina A/farmacologia , Estreptogramina A/uso terapêutico , Estreptogramina B/administração & dosagem , Estreptogramina B/farmacologia , Estreptogramina B/uso terapêutico , Estreptograminas/administração & dosagem , Estreptograminas/química , Estreptograminas/farmacologia , Virginiamicina/administração & dosagem , Virginiamicina/farmacologia , Virginiamicina/uso terapêutico
11.
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
13.
J Antimicrob Chemother ; 69(4): 919-23, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24324222

RESUMO

OBJECTIVES: To investigate the genetic basis of pleuromutilin resistance in coagulase-negative staphylococci of porcine origin that do not carry known pleuromutilin resistance genes and to determine the localization and genetic environment of the identified resistance gene. METHODS: Plasmid DNA of two pleuromutilin-resistant Staphylococcus cohnii and Staphylococcus simulans isolates was transformed into Staphylococcus aureus RN4220. The identified resistance plasmids were sequenced completely. The candidate gene for pleuromutilin resistance was cloned into shuttle vector pAM401. S. aureus RN4220 transformants carrying this recombinant shuttle vector were tested for their MICs. RESULTS: S. cohnii isolate SA-7 and S. simulans isolate SSI1 carried the same plasmid of 5584 bp, designated pSA-7. A variant of the vga(E) gene was detected, which encodes a 524 amino acid ATP-binding cassette protein. The variant gene shared 85.7% nucleotide sequence identity and the variant protein 85.3% amino acid sequence identity with the original vga(E) gene and Vga(E) protein, respectively. The Vga(E) variant conferred cross-resistance to pleuromutilins, lincosamides and streptogramin A antibiotics. Plasmid pSA-7 showed an organization similar to that of the apmA-carrying plasmid pKKS49 from methicillin-resistant S. aureus and the dfrK-carrying plasmid pKKS966 from Staphylococcus hyicus. Sequence comparisons suggested that recombination events may have played a role in the acquisition of this vga(E) variant. CONCLUSIONS: A novel vga(E) gene variant was identified, which was located on a small plasmid and was not associated with the transposon Tn6133 [in contrast to the original vga(E) gene]. The plasmid location may enable its further dissemination to other staphylococci and possibly also to other bacteria.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Lincosamidas/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Estreptogramina A/farmacologia , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , Diterpenos/farmacologia , Genes Bacterianos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Plasmídeos , Compostos Policíclicos , Análise de Sequência de DNA , Staphylococcus/isolamento & purificação , Suínos , Transformação Bacteriana
14.
Lett Appl Microbiol ; 57(5): 393-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23815812

RESUMO

A newly reduced macrocyclic lactone antibiotic streptogramin A, 5,6-dihydrovirginiamycin M1 was created by feeding virginiamycin M1 into a culture of recombinant Streptomyces venezuelae. Its chemical structure was spectroscopically elucidated, and this streptogramin A analogue showed twofold higher antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) compared with its parent molecule virginiamycin M1. Docking studies using the model of streptogramin A acetyltransferase (VatA) suggested that the newly generated analogue binds tighter with overall lower free energy compared with the parent molecule virginiamycin M1. This hypothesis was validated experimentally through the improvement of efficacy of the new analogue against MRSA strains. The biotransformation approach presented herein could have a broad application in the production of reduced macrocyclic molecules.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Estreptogramina A/análogos & derivados , Estreptogramina A/biossíntese , Estreptogramina A/química , Estreptogramina A/farmacologia , Virginiamicina/metabolismo
15.
Antimicrob Agents Chemother ; 57(9): 4463-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23836170

RESUMO

As opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faecium is naturally susceptible. Since this phenotype may be selected for in vivo by quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitro mutants of E. faecium HM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitro mutant (E. faecium UCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitro and in vivo resistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for Enterococcus ABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)v from UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Enterococcus faecium/genética , Lincosamidas/farmacologia , Estreptogramina A/farmacologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Diterpenos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/metabolismo , Teste de Complementação Genética , Genótipo , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Compostos Policíclicos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
16.
J Antimicrob Chemother ; 68(6): 1251-5, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23386262

RESUMO

OBJECTIVES: To investigate the genetic basis of pleuromutilin resistance in porcine methicillin-resistant Staphylococcus aureus (MRSA) and to map the genetic environment of the identified plasmid-borne resistance gene. METHODS: Seventy porcine MRSA isolates, which exhibited high MICs of tiamulin, valnemulin and retapamulin, were investigated for pleuromutilin resistance genes and mutations. They were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). Plasmid DNA was extracted from the lsa(E)-positive strains and transferred to S. aureus RN4220 for selection of resistance plasmids. The plasmid-borne lsa(E) gene region was sequenced and 10 overlapping PCR assays for the analysis of the genetic environment of lsa(E) were developed. RESULTS: All 70 MRSA isolates were ST9 (MLST)-t899 (spa)-IVa (SCCmec). Sixteen isolates carried the lsa(E) gene; all others were negative for known pleuromutilin resistance mechanisms. An lsa(E)-carrying plasmid of ∼41 kb was detected in a single isolate. Sequence analysis revealed that the lsa(E) gene was located in a multiresistance gene cluster, which showed partial homology to clusters identified in MRSA, methicillin-susceptible S. aureus (MSSA) and Enterococcus faecalis. PCR analysis of the remaining isolates revealed a partly deleted multiresistance gene cluster in 6/15 isolates and solely the lsa(E) gene without the known flanking regions in 9/15 isolates. CONCLUSIONS: We identified the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in porcine MRSA isolates. The multiresistance gene cluster in which lsa(E) was located differed from the previously described ones found in human MRSA/MSSA or in E. faecalis. The location of lsa(E) on a multiresistance plasmid facilitates its persistence and dissemination.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Lincosamidas/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Estreptogramina A/farmacologia , Doenças dos Suínos/microbiologia , Animais , China , Clonagem Molecular , DNA Bacteriano/genética , Diterpenos/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/genética , Compostos Policíclicos , Infecções Estafilocócicas/tratamento farmacológico , Suínos , Doenças dos Suínos/tratamento farmacológico , Transformação Bacteriana
18.
Se Pu ; 30(5): 463-7, 2012 May.
Artigo em Chinês | MEDLINE | ID: mdl-22934408

RESUMO

A liquid chromatography-tandem mass spectrometry method was established for the determination of virginiamycin M1 and S1 residues in livestock and poultry products. The sample was extracted by methanol-acetonitrile solution (1:1, v/v). The supernatant was diluted with 0.01 mol/L ammonium dihydrogen phosphate solution, then purified and concentrated on an Oasis HLB cartridge. The separation of virginiamycin M1 and S1 was performed on a Luna C18 column with the mobile phases acetonitrile and 5 mmol/L ammonium acetate aqueous solution (containing 0.1% (v/v) formic acid) in a gradient elution mode. The identification and quantification of the drugs were carried out by positive electrospray ionization (ESI + ) in the multiple reaction monitoring (MRM) mode using external standard method. The calibration curves showed good linearity in the range of 0.15-10.0 microg/L with correlation coefficients (r2) above 0. 999. The limits of quantities (LOQs) were both 0.25 microg/kg. The average recoveries of the two drugs spiked at 0.25, 0.5 and 2.5 microg/kg levels in different matrices were between 71.2% and 98.4%, and the relative standard deviations (RSDs) were between 3.6% and 15.4%. The method is simple, rapid, sensitive and accurate. It is suitable for the confirmation and quantification of virginiamycin M1 and S1 residues in livestock and poultry products.


Assuntos
Cromatografia Líquida/métodos , Produtos da Carne/análise , Estreptogramina A/análise , Estreptogramina Grupo B/análise , Espectrometria de Massas em Tandem/métodos , Virginiamicina/análise , Animais , Resíduos de Drogas/análise , Contaminação de Alimentos/análise , Gado , Aves Domésticas
19.
Antimicrob Agents Chemother ; 56(7): 3563-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22547628

RESUMO

The cfr gene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisome cfr gene and functionally similar genes. Three cfr-like genes from the order Bacillales were identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced in Escherichia coli, and MICs for selected antibiotics indicate that the cfr-like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as the cfr gene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows that cfr-like genes exist in the environment and that Bacillales are natural residences of cfr-like genes.


Assuntos
Antibacterianos/farmacologia , Bacillales/efeitos dos fármacos , Bacillales/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Diterpenos/farmacologia , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Lincosamidas/farmacologia , Testes de Sensibilidade Microbiana , Oxazolidinonas/farmacologia , Compostos Policíclicos , Estreptogramina A/farmacologia
20.
Antimicrob Agents Chemother ; 55(10): 4900-4, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21768510

RESUMO

A novel streptogramin A, pleuromutilin, and lincosamide resistance determinant, Vga(E), was identified in porcine methicillin-resistant Staphylococcus aureus (MRSA) ST398. The vga(E) gene encoded a 524-amino-acid protein belonging to the ABC transporter family. It was found on a multidrug resistance-conferring transposon, Tn6133, which was comprised of Tn554 with a stably integrated 4,787-bp DNA sequence harboring vga(E). Detection of Tn6133 in several porcine MRSA ST398 isolates and its ability to circularize suggest a potential for dissemination.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus aureus Resistente à Meticilina/genética , Animais , Sequência de Bases , Diterpenos/farmacologia , Genes Bacterianos , Lincosamidas/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Plasmídeos/efeitos dos fármacos , Compostos Policíclicos , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Estreptogramina A/farmacologia , Suínos
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