Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system

Analysis of Failed Two-Stage Procedures with Resection Arthroplasty as the First Stage in Periprosthetic Hip Joint Infections

Resection arthroplasty can be performed as the first stage of a two-stage procedure in some patients with severe periprosthetic hip joint infections with poor bone stock. This retrospective study aimed to evaluate factors associated with the subsequent failure or success of these patients. Between 2011 and 2020; in 61 (26.4%) of 231 patients who underwent a two-stage protocol of periprosthetic hip joint infections; no spacer was used in the first stage. The minimum follow-up period was 12 months. Patient’s demographics and various infection risk factors were analyzed. In total, 37/61 (60.7%) patients underwent a successful reimplantation, and four patients died within the follow-up period. Patients within the failure group had a significantly higher Charlson comorbidity index (p = 0.002); number of operations prior to resection arthroplasty (p = 0.022) and were older (p = 0.018). Failure was also associated with the presence of a positive culture in the first- and second-stage procedures (p = 0.012). Additional risk factors were persistent high postoperative CRP values and the requirement of a negative-pressure wound therapy (p ≤ 0.05). In conclusion, multiple factors need to be evaluated when trying to predict the outcome of patients undergoing resection arthroplasty as the first stage of a two-stage procedure in patients with challenging periprosthetic hip joint infections

Properties of a novel PBP2a protein homolog from <em>Staphylococcus aureus</em> strain LGA251 and its contribution to the β-lactam-resistant phenotype

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A(LGA), the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A(LGA) was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A(LGA) did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity

Intermediate-Type Vancomycin Resistance (VISA) in Genetically-Distinct <i>Staphylococcus aureus</i> Isolates Is Linked to Specific, Reversible Metabolic Alterations

<div><p>Intermediate (VISA-type) vancomycin resistance in <i>Staphylococcus aureus</i> has been associated with a range of physiologic and genetic alterations. Previous work described the emergence of VISA-type resistance in two clonally-distinct series of isolates. In both series (the first belonging to MRSA clone ST8-USA300, and the second to ST5-USA100), resistance was conferred by a single mutation in <i>yvqF</i> (a negative regulator of the <i>vraSR</i> two-component system associated with vancomycin resistance). In the USA300 series, resistance was reversed by a secondary mutation in <i>vraSR</i>. In this study, we combined systems-level metabolomic profiling with statistical modeling techniques to discover specific, reversible metabolic alterations associated with the VISA phenotype.</p></div

Heat Map of Altered Metabolites in the VISA, SG-R, Versus its Parent VSSA, SG-R and the revertant VSSA, SG-rev.

<p>Heat map displaying the 12 metabolites whose abundance was significantly altered in the VISA isolate, SG-R compared against the parent VSSA, SG-S. Changes in abundance are indicated by color coding with red indicative of increases in mean intracellular abundance relative to the baseline (defined by the abundance in SG-S) and blue indicative of decreases in intracellular abundance on a log (2) scale. Specific <i>p</i>-values for the comparison of SG-R versus SG-S are denoted to the near right of the heat map. Metabolites are grouped according to pathway, denoted to the far right of each metabolite. Metabolites 1–7 reversed directionality in the revertant isolate (SG-rev) indicating a link to vancomycin resistance. Metabolites 8–12, however, did not reverse in SG-rev and are separated by a row to denote this difference. The superscript (<i>a</i>) denotes unable to determine if methylmalate or hydroxyglutarate in the absence of a chemical standard.</p

Principal component and hierarchical cluster analyses show separation of isolates by resistance phenotype.

<p>Principal component analysis <b>(A)</b> of the metabolic profiles of all isolates from both series again shows a separation of isolates by resistance phenotype. Specifically, the metabolic profiles of parent VSSA isolates SG-S (green squares) and JH1 (black dots) form a single cluster (green ellipse), while those corresponding to VISA isolates SG-R (dark blue diamonds) and JH2 (red circles) form two separate clusters (dark blue ellipse and red ellipse, respectively) along the same vector of change (denoted by the black arrow) to the left of the VSSA cluster. The metabolic profiles corresponding to the revertant SG-rev (light blue diamonds) cluster between the susceptible parent (SG-S) and resistant (SG-R) isolates, consistent with its revertant phenotype (light blue ellipse). <b>(B)</b> Results of hierarchical cluster analysis also show that the metabolic profiles of all five isolates from both series separate by resistance phenotype, forming two distinct branches with the VISA isolates JH2 and SG-R on the left and VSSAs SG-S, JH1 and SG-rev on the right, though one replicate of SG-R (SG-R5) was found to cluster with the parental VSSA isolates (red asterix).</p

Principal component analyses of the metabolic profiles of the SG series (A) and the JH series (B) show clustering of replicates by resistance phenotype.

<p>(<b>A</b>) Principal component analysis of the metabolic profiles of isolates from the SG series (SG-S, SG-R and SG-rev) shows a separation of samples (replicates) by resistance phenotype. Specifically, the metabolic profiles corresponding to replicates of the VISA strain, SG-R (red ellipse) cluster separately from those of its parent VSSA, SG-S (black ellipse), while those of SG-rev (green ellipse) cluster between these two, overlapping both. (<b>B</b>) Principal component analysis of the metabolic profiles of isolates from the JH series again shows clustering of replicates by phenotype, with both the parent VSSA JH1 (black circles) and VISA JH2 (red circles) forming non-overlapping clusters (though one outlier, JH2-10, is noted).</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