Recombinant lysostaphin

Total RNA was extracted using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions, using 50 U recombinant lysostaphin (Sigma) followed by incubation for 5 minutes with 1 ml of hot Qiazol (Qiagen) to lyse bacteria. Bacteria were further disrupted by vibration with 50 mg of acid-washed glass beads (Sigma) using a Mickle Vibratory Tissue Disintegrator (Mickle Laboratory Engineering) at maximum speed. Contaminating DNA was removed using DNA-free™ Kit (Applied Biosystems) and RNA quality tested on an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA concentration and purity were determined by Nanodrop® ND-1000 spectrophotometer (Thermo Scientific). For each strain, at least 4 RNA samples were prepared from independent cultures.

All isolates were subjected to PFGE, in order to test clonality of the isolates from infection (INF isolates) and to type corresponding nasal isolates of the same patient in relation to the infectious isolate. Nasal isolates which showed patterns identical to the infectious isolate were assigned as CloNo isolate (clonal nose isolate). Isolates showing patterns that differed in PFGE by at least one band from the infectious isolate were assigned as nCloNo isolate (non-clonal nose isolate). Of the nCloNo isolates, only one isolate per PFGE-pattern was selected for further analysis.
PFGE was performed by a modification of the method described previously [99 (link)], using a contour-clamped homogenous electric field (CHEF DR III, BioRad, Hercules, CA, USA). Bacterial cells were grown overnight in TSB, pelleted and poured into low melting point agarose blocks. The cell wall was digested with recombinant lysostaphin (Sigma Aldrich, St. Louis, USA). Restrictions was performed with the endonuclease SmaI (Thermo Fisher Scientific, Waltham, MD, USA). Pulse time was 5.0–50.0 s for 20h. DNA restriction patterns obtained by PFGE were interpreted manually according to the criteria published previously[100 (link)]. One band difference was accepted for clonally related isolates.

Peptidoglycan was prepared from exponentially growing cells as previously described [56 (link)]. Muropeptides were prepared by digestion with mutanolysin and glycan strands were isolated from purified peptidoglycan by sequential digestion with recombinant lysostaphin (1 μg/ml, Sigma) and purified pneumococcal amidase (LytA, 50–100 μg/ml) essentially as previously described [32 (link)] and detailed in the supplementary materials and methods (S1 Text).