Mapping of pbp4 gene mutations to the crystal structure of the PBP4 protein of <i>Staphylococcus aureus</i> complexed with a cephalosporin antibiotic (cefaxotaxime).

<p>The left panel shows the entire complex, whereas the right panel shows a zoomed image of the cephalosporin binding pocket. The <i>Staphylococcus aureus</i> strain depicted in the crystal structure (PDB 3HUM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149541#pone.0149541.ref030" target="_blank">30</a>], Col, is the parental strain of the Colnex strain used in the current study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149541#pone.0149541.t001" target="_blank">Table 1</a>). Mutant residues in PBP4 identified by selection with ceftaroline or ceftobiprole are highlighted in blue. The ligand marked in yellow is cefotaxime.</p

Additional coding mutations present in at least three selections in this study.

<p>Mutated genes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149541#pone.0149541.t001" target="_blank">Table 1</a> are not included.</p

Strains used in this study and mutations detected in penicillin binding proteins, gdpP and acrB genes.

<p>Strains used in this study and mutations detected in penicillin binding proteins, gdpP and acrB genes.</p

Transcription of <i>vraS</i> in strains SG-S, SG-R and SG-rev and their various genetic constructs.

<p>The expression of <i>vraS</i> mRNA was determined by qRT-PCR. As internal control, the expression was also determined in each sample for the <i>pta</i> mRNA.</p

Transcription of virulence related genes in strains SG-S(pGC2), SG-R(pGC2) and SG-R(pGC2::<i>yvqF</i> wt).

<p>The expression of each mRNA was determined by qRT-PCR. As internal control, the expression was also determined in each sample for the <i>pta</i> mRNA. <i>orf</i> SA1007 encodes for alpha-hemolysin, and <i>orf</i> SA1813 encodes for an hypothetical protein, similar to leukocidin chain LukM.</p

Number of genes with significant transcriptional changes and respective functions in SG-R and SG-rev derived by Microarrays analysis.

<p>Number of genes with significant transcriptional changes and respective functions in SG-R and SG-rev derived by Microarrays analysis.</p

Phenotypes of strains SG-S, SG-R and SG-rev.

<p>Phenotypes of strains SG-S, SG-R and SG-rev were characterized by phase-contrast microscopy (<b>A</b>), and electron microscopy of thin sections (<b>B & C</b>).</p

Vancomycin and oxacillin susceptibility profiles of strains SG-S, SG-R and SG-rev.

<p>Strains SG-S, SG-R and SG-rev were grown overnight in TSB and were used to determine vancomycin MIC values using the E-test with bacteria plated on TSA (<b>A</b>). Vancomycin and oxacillin susceptibility were also determined by population analysis (<b>B</b>) and (<b>C</b>).</p

Relevant phenotypic properties of the strains used in this study.

<p>Van – vancomycin; Oxa – oxacillin; DP – daptomycin, FM – fosfomycin, MO – moenomycin, LZ – linezolid, and TGC - tigecycline. (a) MIC values in µg/ml. (b) strain USA300 is a heterogeneously resistant MRSA strain in which – similarly to other heteroresistant MRSA – the majority of cells expressed only marginal level of resistance to oxacillin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002505#ppat.1002505-Chung1" target="_blank">[69]</a>. Complete population analysis profiles of vancomycin and oxacillin resistance for the SG strains are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002505#ppat-1002505-g001" target="_blank">Figs 1 B and C</a>, respectively.</p

Recovery of vancomycin resistance in strain SG-rev complemented with the wild type <i>vraSR</i> genes.

<p>Vancomycin susceptibilities were evaluated by the E-test and by population analysis – as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002505#s4" target="_blank">Methods</a>.</p