17, 20, 21 and 22 The
study was approved by Oxfordshire Research Ethics Committee B (08/H0605/102). Nasal swabs were taken by all participants on recruitment under research nurse supervision. A dry Crenolanib cotton swab was placed in the tip of both nostrils and rotated three times. All S. aureus positive participants, all students and all participants from the last practice were posted a nasal swabbing kit one and two months after recruitment, and then every two months thereafter. The swabbing technique was demonstrated on recruitment and explained in a leaflet included with each kit. Swabs were returned by post in charcoal medium (typically <1 week), and stored at 4 °C on receipt before processing (processing took <1 week; up to two weeks in total). As the study objective was to investigate S. aureus dynamics,
isolation protocols focussed on identifying all strains, even those present at low frequencies. To increase the sensitivity of culture, swabs were therefore incubated overnight at 37 °C in 5% NaCl enrichment broth (E&O Laboratories, Bonnybridge, UK). A 5 mm loop-full of broth was sub-cultured onto SASelect® chromogenic agar (Bio-Rad, Limerick, Ireland) and incubated at 37 °C overnight. Pink colonies were tested further using DNAse, catalase and Staphaurex tests following standard procedures. 23 Samples positive in all three tests were presumed to be S. aureus. A selection of pink colonies from the SASelect agar were resuspended FER in saline from which one aliquot was stored as glycerol stock at −20 °C
and another added Entinostat chemical structure to 10 μl 0.85% Saline (E&O Laboratories) and 50 μl TE buffer (Sigma, Dorset, UK), heated at 99.9 °C for 10 min, then centrifuged to separate the supernatant. From this, 50 μl was removed and stored at −20 °C as a crude chromosomal DNA extract. spa-typing was performed as described, 24 with DNA amplification and sequencing using the Microlab Star Liquid Handling Workstation (Hamilton Robotics Ltd, Birmingham, UK). Chromatograms for the spa gene were assembled using Ridom StaphType. 24 Samples with mixed chromatograms were re-cultured and six-12 colonies separately typed. spa-types were grouped into spa-Clonal Complexes (CCs) using BURP clustering, and CCs labelled as their MLST equivalent for ease of comparison with other studies. 25 Epidemiological and healthcare information was collected from a structured questionnaire at recruitment, general practice and OUH records (see Supplementary Methods). After two years follow-up, general practice and OUH records were re-reviewed to ascertain antimicrobial use and inpatient admissions throughout follow-up. (1) Loss of carriage (primary outcome) Confirmed loss of carriage was defined as two consecutive negative swabs (or two consecutive swabs without the previous spa-type for analysis of spa-types (spa-level)).