1,725,336 research outputs found
Distribution of SNPs in the <i>S. aureus</i> 8325 and 8325-4 strain lineages.
No full text<p>Thirteen of the SNPs including the 63 bp deletion detected in 8325-4seq and described in Table 2 were analyzed by PCR from gDNA and direct sequencing for their distribution using strains collected from various laboratories worldwide. Shown in the matrix are 6 strains of 8325 pedigree group prior to prophage removal (white box), 10 strains from the 8325-4 pedigree after prophage Φ11, 12, and 13 removal (dark gray box), and one intermediate strain, RN25/NRS133, which retains prophage Φ13 (light gray box). Each SNP is indicated (top) together with coordinates from the 8325 reference genome (bottom) deposited as Genbank NC_007795.1. Strain abbreviations used are as described in Table 3. WT: wild type, +T: insertion of T, ‘ -‘ denotes SNP not tested.</p
Impact of homogeneous oxacillin resistance on the biofilm phenotype of <i>S. aureus</i> 8325-4.
No full text<p>(A) Western blot analysis of PBP2a levels in 8325-4, 8325-4 p<i>mecA</i> HeR and 8325-4 p<i>mecA</i> HoR. (B) Biofilm phenotypes of 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured) grown for 24 h at 37°C in BHI, BHI NaCl and BHI glucose in hydrophilic 96-well polystyrene plates. (C–F) Dispersal of 8325-4 (C), 8325-4 p<i>mecA</i> HeR (D), 8325-4 p<i>mecA</i> HoR (E), 8325-4 p<i>mecA</i> HoR (cured) (F) biofilms grown for 24 h in BHI, BHI NaCl and BHI glucose by sodium metaperiodate and proteinase K. (G) Biofilm formation by 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured) grown for 24 h in BHI glucose in the absence or presence of 500 µg/ml PAS, 0.25 mg/ml DNase I and heat-inactivated (HI) 0.25 mg/ml DNase I. Biofilm assays were repeated at least three times and standard deviations are indicated. * indicates a statistically significant difference p<0.01.</p
Genetic variation in the Staphylococcus aureus 8325 strain lineage revealed by whole-genome sequencing.
No full textStaphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation
Catheter colonisation and dissemination of 8325-4 and 8325-4 p<i>mecA</i> HoR in a mouse device-related infection model.
No full text<p>Implanted catheter segments were injected with 1×10<sup>7</sup> 8325-4 and 8325-4 p<i>mecA</i> HoR and animals (<i>n</i> = 8 mice per group) were sacrificed after 18 hours. Colony forming units (CFU) per catheter (A), per g peri-catheter tissue (B), per g liver (C), per ml blood (D), per g spleen (E) and per g kidney (F) recovered from animals infected with 8325-4 and 8325-4 p<i>mecA</i> HoR. Statistical significance (p values) is indicated.</p
Impact of homogeneous oxacillin resistance on PNAG production in 8325-4.
No full text<p>(A) Immunoblot analysis of PNAG production in whole cell extracts of 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured) grown overnight at 37°C in BHI, BHI NaCl and BHI glucose. (B and C) Comparison of relative <i>icaA</i> (B) and <i>icaR</i> (C) transcription by real time RT-PCR in 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured). Total RNA was extracted from cultures grown at 37°C to <i>A</i><sub>600</sub> = 1 (early exponential phase) in BHI glucose. Experiments were repeated at least three times and standard deviations are indicated. Statistical significance (p value) is indicated.</p
Appendix IV: Terra Petraea Chronological Inconsistencies R-script
No full textThis fourth appendix to "Terra Petraea. The Archaeological Landscape of the Petraean Hinterland from the Hellenistic to the Byzantine Period" (ISBN 978-3-8325-5171-1) is a PDF version of an exemplary R-script written by the author for evaluating the chronological inconsistencies inherent to the original survey data (cf. chapter 2)
Confirmed SNPs in 8325-4 compared to NCTC8325.
No full textConfirmed SNPs in 8325-4 compared to NCTC8325.</p
Impact of homogeneous oxacillin resistance on protease activity and expression of the accessory gene regulator locus in 8325-4.
No full text<p>(A) Protease activity in culture supernatants of 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured) grown for 8 h at 37°C in BHI glucose. (B) Comparison of relative <i>RNAIII</i> transcription by real time RT-PCR in 8325-4, 8325-4 p<i>mecA</i> HeR, 8325-4 p<i>mecA</i> HoR and 8325-4 p<i>mecA</i> HoR (cured). Total RNA was extracted from cultures grown at 37°C to <i>A</i><sub>600</sub> = 1 (early exponential phase) and for 8 hours (late exponential/early stationary phase; <i>A</i><sub>600</sub>≈8) in BHI glucose. Experiments were repeated at least three times and standard deviations are indicated. Statistical significance (p value) is indicated.</p
Polysaccharide constitutes the extracellular matrix of 8325-4 aggregates.
No full text<p>Aggregates of 8325-4 cells were treated with DNase (tube 2), proteinase K (tube 3) or sodium metaperiodate (tube 5) at 37°C for 18 hours. Tubes 1 and 4 are untreated controls.</p
High resolution 8325 pedigree based on the SNP analysis depicted in Figure 2.
No full text<p>Strains are shown in white boxes, mutagenesis treatments are shown in red boxes, and SNPs are shown in colored boxes, color coded as in Figure 2, on the line indicating strain evolution from 8325 to 8325-4seq. The placement of the SNPs along the line indicates their entry point into the pedigree. Five digit numbers indicate SAOUHSC locus tags. IG: intergenic.</p
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