A survey of the microbiome, culturome and AMR resistance profile of a cohort of chronic Diabetic Foot lesions

16S metagenomic sequencing of DNA extracted from swabs from 21 Diabetic Foot Heel ulcers from a set of chronic ulcer patients (median 8 weeks) was performed using Oxford Nanopore Technology. Patients were selected as follows: over 18yr age, Grade 3-4 DFU according to IDSA/IWG criteria, willing to consent. All attended clinics at the Sheffield Northern General Diabetic care unit, Sheffield, UK. Of the 30 patients swabbed, 19/30 were taking antibiotics for unresolved infections with median ulcer durations of 8 weeks. Patients were swabbed twice (in the same place) with standard cotton swabs in charcoal transport medium (for clinical standard culture, see below) or using S–MM™ swabs (for DNA extraction). S–MM™ swabs were placed in their proprietary DNA preservation solution and stored at -80C before processing for DNA extraction. To remove the possibility that the order of swabbing may influence results, we alternated the order of collection of the culture and DNAswabs. Alongside the swab, patient information regarding antibiotic treatment and the details of this treatment, will be collected. The demographic characteristics of the cohort are summarised in table 1. The average age of the patient was 65.61 years.. All patients were of white ethnicity, with no representations from other ethnic groups. In terms of sex distribution, 77.78% of the patients were male, while 22.22% were female. DNA extraction: from S–MM™ swabs for 16S Barcoding Nanopore library preparation was achieved using a QIAmp DNA mini kit (Qiagen, UK) and the buffer from a plain S–MM™ swab as control. 500 µl of the preservation solution was transferred into a clean tube and 150 µl lysozyme (10mg/ml), 6 µl mutanolysin (25 000 U/ml), and 3 µl lysostaphin added and incubated at 37oC for 1 hour. 40 µl proteinase K, 8 µl RNAse A (100mg/ml), and 500 µl buffer AL were added and incubated at 56oC for 10 mins. 800 µl of ethanol (100%) was added and the mixture vortexed for 15 secs. Sample was transferred to a spin column in a 2 ml collection tube and centrifuged at 6,000 x g for 1 minute discarding the filtrate. 500 µl buffer AW2 (from Qiamp kit) was added and centrifuged at 20,000 x g for 3 minutes. Filtrate was discarded and the sample centrifuged again for 1 minute, discarding the filtrate. The column was then transferred to a new 1.5 ml microcentrifuge tube and eluted with 50 µl buffer AE. Samples were stored at 4oC and used for Nanopore 16S Barcoding Kit library preparation using the Barcoding Kit 1-24 (SQK-16S024) which includes universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3'). Sequencing: the prepared library was sequenced using the Oxford Nanopore Technologies Flow Cell Priming Kit (EXP-FLP002). MinION reads were basecalled in real-time using a MinION MK1C (Oxford Nanopore Technologies, UK) device after which the FASTq pass data was extracted. Barcodes and adapters were trimmed and demultiplexed using Porechop (https://github.com/rrwick/Porechop/) on the Galaxy Europe server (usegalaxy.org), and reads sorted accordingly. The processed reads were then analysed using Mothur (Version 1.48.0; Schloss PD et al., 2009). The data uploaded represent reads from anonymised patient samples under UK-NHS ethics REC reference: 21/SC/0338 and IRAS project ID: 299735).