Biotyper version 3

Routine sputum culture was performed using homogenized sputum according to the Health Protection Agency (HPA) standard operating procedures. All cultured organisms were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry on a Microflex LT using Biotyper, version 3.1 (Bruker Daltonics, Ballerica, MA, USA). In addition, prospective Haemophilus species were also identified using X (hemin), V (NAD) and XV disks (hemin and NAD) (BD BBL Taxo differentiation discs for Haemophilus spp., Wokingham, UK) on nutrient agar (Oxoid Ltd, Basingstoke, UK). H. influenzae strains isolated from 4 clinical samples (PID 278, 133, 29 and 288) were cultured overnight (37°C with 5% CO2, shaking at 180 rpm) in 10 mL culture broth (BHI with 10 μg/mL NAD [NAD] and hemin [Sigma-Aldrich, Dorset, UK]). Once optical density at 600 nm reached 0.5 (exponential phase) bacteria were pelleted and resuspended in BHI containing 10% glycerol and stored at −80°C until use. Clinical laboratory results from the John Radcliffe Hospital Microbiology Laboratory, Oxford, UK have been provided for three samples.

A retrospective study was performed at the Cambridge University Hospitals NHS Foundation Trust between October 2012 and April 2013. Stool samples were processed at the on-site Public Health England Clinical Microbiology and Public Health Laboratory. Samples were tested using a commercial assay (C. DIFF QUIK CHEK COMPLETE; Techlab), which detects glutamate dehydrogenase (GDH) antigen and toxins A and B of C. difficile. Positive samples were cultured anaerobically for 48 h onto Brazier’s cycloserine-cefoxitin-egg yolk (CCEY) agar (BioConnections) using alcohol shock (https://www.gov.uk/government/publications/smi-b-10-processing-of-faeces-for-clostridium-difficile) and colonies with typical appearance were identified by MALDI-TOF MS (Biotyper version 3.1; Bruker Daltonics). Stool samples from which C. difficile was isolated were stored at −80 °C prior to repeated recovery of C. difficile, DNA extraction and sequencing. Clinical data were recorded on patient age, gender, date of hospital admission, discharge and ward movement, and previous admission to hospital in the preceding 6 months. Recurrent C. difficile infection was defined as the onset of diarrhoea 4 weeks or more after the end of the prior episode.