A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus

Charbonnier, Yvan; Gettler, Brian; François, Patrice; Bento, Manuela; Renzoni, Adriana; Vaudaux, Pierre; Schlegel, Werner; Schrenzel, Jacques

doi:10.1186/1471-2164-6-95

Cited by 97 publications

(68 citation statements)

References 43 publications

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“…The totality of purified RNA was subjected to in vitro transcription using the MessageAmp II-Bacteria kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions [20]. Amplified RNA was labeled during in vitro transcription in the presence of Cy-3 or Cy-5 cyanine dyes (Perkin-Elmer, Boston, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Huyghe

François

Mombelli³

et al. 2013

PLoS Negl Trop Dis

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Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma.

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Section: Methodsmentioning

confidence: 99%

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Huyghe

François

Mombelli³

et al. 2013

PLoS Negl Trop Dis

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“…The microarray was manufactured by in situ synthesis of 60-base-long oligonucleotide probes (Agilent), selected as previously described (25). The microarray consists of a glass slide with 15,000 printed oligonucleotides, covering Ͼ95% of all open reading frames (ORFs) annotated in strains NCTC 8325 (26), UAMS-1 (27), and SA564 (28) as well as Newman (29), including their respective plasmids.…”

Section: Methodsmentioning

confidence: 99%

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon

Mechler

Bonetti

Reichert

et al. 2016

Antimicrob Agents Chemother

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dUnderstanding the mechanisms of how bacteria become tolerant toward antibiotics during clinical therapy is a very important object. In a previous study, we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (P i ) and polyphosphate (polyP). DAP tolerance in the pitA6 mutant differs from classical resistance mechanisms since there is no increase in the MIC. In this follow-up study, we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that 234 genes were at least 2.0-fold differentially expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism, and replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt operon, which is induced by the accumulation of intracellular P i . The GraXRS system, known as an activator of the dlt operon (D-alanylation of teichoic acids) and of the mprF gene (multiple peptide resistance factor), is not involved in DAP tolerance of the pitA6 mutant. In conclusion, DAP tolerance of the pitA6 mutant is due to an upregulation of the dlt operon, triggered directly or indirectly by the accumulation of P i .

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“…The microarray was manufactured by in situ synthesis of 15,600 60-base-long oligonucleotide probes (Agilent), selected as previously described (34). The probe set covered multiple genomes and plasmids, as previously published (35).…”

Section: Methodsmentioning

confidence: 99%

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

et al. 2014

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In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16-to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H 2 O 2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human wholeblood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

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A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus

Cited by 97 publications

References 43 publications

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

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