The microbiome of diabetic foot osteomyelitis

We consecutively obtained 34 bone samples from patients admitted to our hospital with moderate–severe DFI according to the diabetic foot infection classification of the Infectious Diseases Society of America (IDSA) [12]. We included patients who were 21 years or older and had high suspicion of DFO based on their IDSA classification. Exclusion criteria included other infectious diseases, active, previously diagnosed DFO in the study foot, immunosuppressive therapy, organ and/or haematological malignancies, and end-stage renal disease requiring dialysis. We performed a percutaneous biopsy using a 16 gauge Jamshidi needle introduced at least 2 cm from the ulcer site [6] (n = 7) or we obtained intraoperative bone samples from the patients that required surgical debridement or amputation (n = 27). We sent the obtained bone samples to the laboratory for conventional culturing and histopathological tests. We used our hospital microbiology laboratory’s established protocol for anaerobic sampling and transport. The bone specimens were placed in sterile cups without any transport medium and processed within 1 h of collection. Laboratory technicians were kept unaware of the clinical data. We stored a part of the obtained samples promptly at −80 °C until the end of the study. Informed consent was obtained from all individual participants included in the study.

After thawing, we recovered portions of the bone samples using sterile forceps. We extracted genomic DNA using the Roche High Pure PCR Template Preparation Kit (Roche Life Sciences, Indianapolis, IN, USA) with a modified lysis step. We lysed our samples by combining 25 mg of sample, 200 μL each of lysis buffer and binding buffer (kit buffers 1 and 2), 500 μL of zirconium oxide beads and a single 5-mm steel bead in a 200-μL screw-cap tube. We shook the tubes using a TissueLyser II (Qiagen, Inc., Valencia, CA, USA) for 5 min at 30 Hz. We continued the extraction using the manufacturer’s instructions. We used the Illumina MiSeq Personal Sequencer (Illumina, Inc., San Diego, CA, USA) in collaboration with PathoGenius Laboratories (PathoGenius, Lubbock, TX, USA) to assess the distribution of 16S rRNA gene sequences. We amplified the samples for sequencing using a forward and a reverse fusion primer. The forward primer was constructed with the (5′-3′) Illumina i5 adapter, an 8–10-bp barcode, a primer pad and the 28F primer. The reverse fusion primer was constructed with the (5′-3′) Illumina i7 adapter, an 8–10-bp barcode, a primer pad and the 519R primer. We used the HotStarTaq Plus Master Mix Kit (Qiagen, Inc., Valencia, CA, USA) for polymerase chain reaction (PCR) under the following conditions: 94 °C for 3 min; followed by 30 cycles of 94 °C for 30 s, 60 °C for 40 s and 72 °C for 1 min; and a final elongation step at 72 °C for 5 min.

In preparation for 16S sequencing, we denoised the DNA to remove short sequences, singleton sequences and noisy reads [13]. With the bad reads removed, we performed chimera detection using the de novo method built into UCHIME [14] to aid in the removal of chimeric sequences [15]. We corrected the remaining sequences base by base to help remove noise from within each sequence. We quality checked and demultiplexed the denoised and chimera-checked reads generated during sequencing. We then clustered these sequences into operational taxonomic units (OTUs) using the UPARSE algorithm [16]. We ran the centroid sequence from each cluster against a database of high-quality sequences derived from the National Center for Biotechnology Information (NCBI) database, March 2015. We used an internally developed Python program that assigns taxonomic information to each sequence to analyse the output and write the final analysis files.