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1.
Microbes Infect ; 26(4): 105306, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38316375

RESUMO

Staphylococcus aureus is one of the major pathogens isolated from the airways of people with cystic fibrosis (pwCF). Recently, we described a mucoid S. aureus phenotype from respiratory specimens of pwCF, which constitutively overproduced biofilm that consisted of polysaccharide intercellular adhesin (PIA) due to a 5bp-deletion (5bp-del) in the intergenic region of the intercellular adhesin (ica) locus. Since we were not able to identify the 5bp-del in mucoid isolates of two pwCF with long-term S. aureus persistence and in a number of mucoid isolates of pwCF from a prospective multicenter study, these strains were (i) characterized phenotypically, (ii) investigated for biofilm formation, and (iii) molecular typed by spa-sequence typing. To screen for mutations responsible for mucoidy, the ica operon of all mucoid isolates was analyzed by Sanger sequencing. Whole genome sequencing was performed for selected isolates. For all mucoid isolates without the 5 bp-del, various mutations in icaR, which is the transcriptional repressor of the icaADBC operon. Mucoid and non-mucoid strains belonged to the same spa-type. Transformation of PIA-overproducing S. aureus with a vector expressing the intact icaR gene restored the non-mucoid phenotype. Altogether, we demonstrated a new mechanism for the emergence of mucoid S. aureus isolates of pwCF.


Assuntos
Biofilmes , Fibrose Cística , Mutação , Infecções Estafilocócicas , Staphylococcus aureus , Fibrose Cística/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Humanos , Biofilmes/crescimento & desenvolvimento , Infecções Estafilocócicas/microbiologia , Óperon/genética , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Proteínas Repressoras/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estudos Prospectivos , Sequenciamento Completo do Genoma , Sistema Respiratório/microbiologia
2.
J Bacteriol ; 190(3): 834-42, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17905979

RESUMO

Trimethoprim-sulfamethoxazole (SXT)-resistant Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a cofactor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to previously published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, and nupC) was performed by quantitative reverse transcription-PCR. The previously published sequences of thyA and two normal clinical strains were highly conserved, while thyA of four normal strains and four SCVs had nonsynonymous point mutations. In 8/10 SCVs, deletions occurred, resulting in stop codons which were located in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics, and transcription patterns. In conclusion, we demonstrated that mutations of the thyA gene were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA protein is necessary and sufficient to change the SCV phenotype and behavior back to normal.


Assuntos
Fibrose Cística/microbiologia , Mutação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Timidina/metabolismo , Timidilato Sintase/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , Farmacorresistência Bacteriana , Humanos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Timidilato Sintase/metabolismo , Combinação Trimetoprima e Sulfametoxazol/farmacologia
3.
J Clin Microbiol ; 43(1): 502-5, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15635028

RESUMO

Variation of the polymorphic region of the protein A gene (spa) was observed during long-term persistence of Staphylococcus aureus in the airways of 10 cystic fibrosis patients and occurred at a rate of one genetic change every 70 months. Independent mutational events were observed eight times in 142 isolates: four deletions, two duplications of repeats, and two point mutations.


Assuntos
Fibrose Cística/microbiologia , Variação Genética , Mutação , Polimorfismo Genético , Proteína Estafilocócica A/genética , Staphylococcus aureus/genética , Doença Crônica , Eletroforese em Gel de Campo Pulsado , Humanos , Infecções Estafilocócicas/microbiologia , Proteína Estafilocócica A/química , Staphylococcus aureus/classificação
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