Diabetes Volume 70, June 2021 1357

Nanopore 16S Amplicon Sequencing Enhances the Understanding of in Medically Intractable Diabetic Foot

Jangsup Moon,1,2 Narae Kim,1 Han Sang Lee,1 Soon-Tae Lee,1 Keun-Hwa Jung,1 Kyung-Il Park,3 Sang Kun Lee,1 Dong-Oh Lee,4 Dong Yeon Lee,4 and Kon Chu1

Diabetes 2021;70:1357–1371 | https://doi.org/10.2337/db20-0907

Diabetic foot infections (DFIs) cause substantial mor- infected (3,4). Diabetic foot infections (DFIs) are the bidity and mortality. The mainstay of the treatment is most common cause of diabetes-related hospital admis- empiric antibiotics and surgical debridement in severe sions (5). Diabetic foot ulcers usually heal very slowly be- cases. In this study, we performed nanopore 16S rDNA cause of several factors, including diabetes-associated sequencing from the debridement specimens of DFIs. microvascular disease, peripheral neuropathy, progressive Fifty-four surgical debridement specimens obtained

changes of bony structure in the foot, impaired host im- COMPLICATIONS from 45 patients with medically intractable DFI were in- mune response, and involvement of that often cluded. The 16S rDNA PCR was performed on each exist within the wound and are resistant to antimicrobial specimen, and Nanopore sequencing was performed for up to 3 h. The reads were aligned to the BLAST database, treatments (1,6). When foot ulcers and infections fail to and the results were compared with conventional culture heal, critical complications, such as osteomyelitis, occur – – studies. The 16S sequencing results revealed that the (7 9). Diabetic foot osteomyelitis occurs in 44 68% of pa- majority of the DFIs (44 of 54, 81.5%) were polymicrobial tients who are admitted to the hospital because of DFI infections. All bacteria isolated by conventional culture and is the leading cause of foot amputation in these studies were detected by 16S sequencing. Several anae- patients (3). robes (Prevotella, Finegoldia, , Bacter- Molecular-based methods have been increasingly ap- oides) were commonly identified by 16S sequencing but plied for identification because of recent advan- were frequently missed by culture studies. In many ces in sequencing technologies (10,11). The 16S rDNA cases, certain bacteria only revealed by the 16S se- amplicon sequencing is particularly useful for the detec- quencing were more abundant than the bacteria isolated tion of bacteria (12,13) and has many advantages over by the culture studies. In conclusion, nanopore 16S se- conventional culture studies. As well as a rapid turn- quencing was capable of pathogen identification in DFIs around time, 16S sequencing is capable of the detection and has many advantages over conventional culture of unculturable bacteria and polymicrobial at studies. Nanopore 16S sequencing enables a compre- hensive understanding of the bacteria involved in DFIs. once (14,15). Nanopore sequencing is one of the new-gen- eration sequencing technologies produced by Oxford Nanopore Technologies (ONT; Oxford, U.K.). It is carried One of the most serious complications of diabetes is foot out by predicting nucleotide sequences from the electrical ulcers (1,2). Patients with diabetes have a 12–25% life- current patterns that are affected by the bases passing time risk of developing a foot ulcer, which often becomes through the nanopore. Nanopore sequencing has many

1Department of Neurology, Laboratory for Neurotherapeutics, Biomedical Corresponding authors: Dong Yeon Lee, [email protected], and Kon Chu, Research Institute, Seoul National University Hospital, College of Medicine, [email protected]. Seoul National University, Seoul, South Korea Received 7 September 2020 and accepted 15 March 2021 2Department of Genomic Medicine, Seoul National University Hospital, Seoul, © 2021 by the American Diabetes Association. Readers may use this article South Korea as long as the work is properly cited, the use is educational and not for 3Department of Neurology, Seoul National University Hospital Healthcare System profit, and the work is not altered. More information is available at https:// Gangnam Center, College of Medicine, Seoul National University, Seoul, South www.diabetesjournals.org/content/license. Korea 4Department of Orthopedic Surgery, Seoul National University Hospital, College of Medicine, Seoul National University, Seoul, South Korea 1358 16S rDNA Sequencing in Diabetic Foot Infection Diabetes Volume 70, June 2021 advantageous characteristics, including a simple library min. All PCRs were performed in a C1000 Touch Thermal preparation procedure that could be extremely useful for Cycler (Bio-Rad, Hercules, CA). A negative control (dis- clinical metagenomics (16,17). Nanopore sequencing ena- tilled water) and a positive control (bacterial genomic bles real-time analysis of reads and long-read sequencing, DNA) were included in every PCR. The PCR products which can be very useful for rapid pathogen detection were electrophoresed on a 1.5% agarose gel containing (10,18), and is applicable for bacterial detection by 16S 0.05 mL/mL RedSafe (Intron Biotechnology, Seoul, South amplicon sequencing (17,19,20). Thus, nanopore 16S am- Korea) and were visualized using a Bio-Rad Gel Doc EZ plicon sequencing is being applied to the detection of vari- Imager. When the negative control demonstrated a PCR- ous bacterial infections, including bacterial meningitis, positive band, contamination was suspected, and the PCR brain abscess, pneumonia, and others (21–24). was repeated from the initial step. In the current study, we performed nanopore 16S am- plicon sequencing on the tissues obtained during surgery Nanopore Library Preparation and Sequencing from patients with medically intractable DFI. We investi- When the result of the 16S rDNA PCR was positive, se- gated whether nanopore 16S amplicon sequencing is capa- quencing libraries were prepared from the PCR products ble of pathogen identification in patients with DFI and using the Rapid Barcoding Sequencing Kit (SQK-RBK004; compared its efficacy with conventional culture studies. ONT). The input DNA was end repaired and A-tailed us- ing the Ultra II End Prep Enzyme (New England Biolabs  RESEARCH DESIGN AND METHODS [NEB], Hertfordshire, U.K.) incubated at 20 C for 5 min  Patients and Sample Collection and at 65 C for 5 min. The end-prepared DNA was puri- fi Among the patients who visited the orthopedics depart- ed with AMPure XP (Beckman Coulter, High Wycombe, ment of Seoul National University Hospital (SNUH) U.K.), and the DNA was eluted in nuclease-free water fol- between June 2018 and December 2019, those with med- lowed by ligation with a 1D adapter using Blunt/TA Li- ically intractable DFI requiring surgical debridement or gase Master Mix (NEB) at room temperature for 10 min. fi amputation were included. Medically intractable DFI was The 1D adapter DNA puri cation was achieved with defined as failure of conservative treatment, including Adapter Binding Buffer (ONT) using the magnetic stand, dressing, off-loading techniques such as total contact cast, and the DNA library was eluted with elution buffer and antibiotics therapy for at least 1 month. Cases com- (ONT). The presequencing mix was loaded onto an R9.5 bined with osteomyelitis, which necessitated copious elim- flow cell (FLO-MIN107) in a mix of running buffer with ination of infected bone and soft tissues, were included. fuel mix and library loading buffer (ONT). Finally, se- Patients having foot ulcers with uncertain infections or quencing was performed for 2 or 3 h, and base calling was patients who refused consent were excluded. Debrided tis- performed using MinKNOW software. sues were obtained during surgery from the deep central portion of lesions, including bone and adjacent soft tissue, 16S rDNA Analysis by a skillful orthopedic surgeon (D.Y.L.). Then, samples During or after sequencing, the sequenced reads were an- for culture studies were directly placed in blood culture alyzed by the cloud-based Metrichor/EPI2ME platform bottles for aerobic and anaerobic culture and were (Metrichor Ltd., Oxford, U.K.). The 16S analysis workflow promptly transported to the microbiology laboratory. of EPI2ME was used, which is designed to Basic Local Samples for sequencing were stored in sterile tubes from Alignment Search Tool (BLAST) base-called reads against the operating room and kept at 4C before the experi- the National Center for Biotechnology Information 16S mental procedures. The study was approved by the insti- bacterial database. Generated reads were classified to cer- tutional review board of SNUH (IRB No. 1806-032-949), tain bacteria at the species level on the basis of the per- and informed written consent was obtained from all cent coverage and identity. The list of the bacteria was patients. arranged in descending order according to the number of aligned reads, and the pathogens from the top of this list DNA Extraction and 16S rDNA PCR were determined by clinicians. The species identification The DNA was extracted from the surgical specimens using within a certain genus was determined on the basis of the the PureLink Genomic DNA Mini Kit (Invitrogen, Carls- number of aligned reads, with the one with the largest bad, CA) following the manufacturer’s instructions. For number being selected as the answer. each sample, the 16S rDNA PCR was performed as de- scribed previously (23). In brief, the full length of 16S Relative Abundance Calculation rDNA was amplified by PCR using a bacterial 16S rDNA The nanopore sequencing throughput is affected by many PCR kit (Takara, Tokyo, Japan). The 16S rDNA primer factors, which include the number of active pores remain- mix (Takara) was added to the genomic DNA. PCR was ing in the flow cell, DNA purity and integrity, library then performed with an initial denaturation at 94Cfor1 concentration, and sequencing time (25). Therefore, the min followed by 35 cycles at 94Cfor30s,55Cfor30s, absolute number of the aligned reads does not reflect the and 72C for 1 min, with a final extension at 72Cfor3 absolute abundance of certain bacteria. However, in cases diabetes.diabetesjournals.org Moon and Associates 1359 of polymicrobial infection, the relative abundance of each Nanopore 16S Sequencing Was Useful for bacterium can be estimated by comparing the number of Differentiating Polymicrobial Infections From reads aligned to each bacterium within a single sequenc- Monomicrobial Infections ing run. The relative abundance score of certain bacteria After 16S sequencing, multiple bacteria were revealed in the majority of cases (44 of 54, 81.5%). In addition, the (A) was calculated by the aligned read counts divided by sequenced reads were aligned to a single bacterium in 10 the read counts of the most abundant bacteria (number of 54 cases, leading to the diagnosis of monomicrobial in- of reads aligned to certain bacteria [A]/number of reads fections (illustrative case in Fig. 1B). Thus, the 16S se- aligned to the most abundant bacteria). quencing was capable of differentiating polymicrobial infections from monomicrobial infections. According to the results of the culture studies, polymi- Data and Resource Availability crobial infections were only suspected in 32 cases, and a The data sets generated and/or analyzed during the cur- single bacterium was isolated in 18 cases. Among these rent study are available from the corresponding authors 18 cases, 10 turned out to be polymicrobial infections by upon reasonable request. 16S sequencing. In four cases, no bacteria were cultivated despite the confirmation of bacterial infections by 16S se- quencing. Two of these four cases actually had polymicro- RESULTS bial infections that were only revealed by 16S sequencing The Majority of the Medically Intractable Infections (Table 1 and Fig. 2). In summary, the 16S sequencing was Were Caused by Polymicrobial Infections superior to the culture studies in differentiating polymi- A bacterial culture study and 16S rDNA sequencing were crobial infections from monomicrobial infections. both performed on 54 samples obtained from 45 patients with medically intractable DFI (Table 1). In every case, Nanopore 16S Sequencing Provided Clues to the antibiotics were administered before the collection of the Dominant Pathogen Among the Polymicrobial samples. More than 80% of the cases (44 of 54, 81.5%) Infections turned out to be caused by polymicrobial infections (Fig. In cases of polymicrobial infections, the sequenced reads 1A). A total of 92 bacteria belonging to 18 genera were were aligned to multiple bacteria (illustrative case in Fig. isolated in 41 patients by conventional culture studies. 1C). Assuming that the 16S PCR would amplify the se- The bacteria most frequently identified by culture studies quence of each bacterium at a similar rate, the bacterium were Staphylococcus (n = 18) followed by (n = with the largest number of aligned reads could be consid- 16), Escherichia (n =10),andEnterococcus (n =9).Atotal ered as the most abundant pathogen in the sample. In ad- of 290 bacteria belonging to 43 genera were revealed by dition, the relative abundance of certain bacteria was assessed by comparing the number of reads aligned to 16S sequencing. The bacteria most frequently identified each bacterium. by 16S sequencing were also Staphylococcus (n = 25) fol- The 16S sequencing was useful for monitoring the re- lowed by Finegoldia (n =23),Prevotella (n = 21), Strepto- maining pathogen. In some cases, 16S sequencing was (n =21),Anaerococcus (n =19),andBacteroides (n = repeated after prolonged antibiotic treatment. The com- 15) (Table 2 and Fig. 2). position of the pathogens was changed in the follow-up 16S sequencing (illustrative case in Fig. 1D).

Nanopore 16S Sequencing Was More Sensitive Than Uncultured Anaerobes Were the Dominant Pathogens Conventional Culture Studies in the Majority of the Cases All bacteria isolated by culture studies were identified by Among the 44 polymicrobial infections revealed by 16S 16S sequencing except for one case. In this patient (pa- sequencing, the dominant pathogens were frequently tient 7, Table 1 and Fig. 2), ,afre- missed by the culture studies. In 23 of 44 (55%) cases, quent source of contamination, was only isolated in the the most abundant pathogen revealed by 16S sequencing culture studies. Overall, the 16S sequencing revealed the was not isolated in the culture studies. The most fre- presence of many anaerobes that were not isolated by cul- quently missed dominant pathogens were Prevotella (n = ture studies from the samples. 6) and Bacteroides (n =6)followedbyAnaerococcus (n =3) The bacteria that were frequently missed by the culture and Parvimonas (n =2)(Fig.3andTable2). studies were Prevotella (n = 20), Finegoldia (n = 19), Anae- Moreover, the majority of the pathogens isolated by rococcus (n =18),andBacteroides (n = 15). Bacteroides, Pep- the culture studies were located in the lower ranks of the toniphilus (n = 12), Fusobacterium (n = 10), Campylobacter list of pathogens (illustrative case in Fig. 1C). Within the (n =7),Haemophilus (n =6),Parvimonas (n =6),Peptos- cases with polymicrobial infections, the culture studies treptococcus (n = 6), and Porphyromonas (n = 5) were only isolated 88 bacteria. Among these, 33 (37.5%) bacteria identified by 16S sequencing and not by culture studies isolated from the culture studies were actually below third (Table 2 and Fig. 2). place in the 16S sequencing (Table 1). Table 1—Basic demographics and a list of pathogens identified by culture studies and 16S sequencing 1360 Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before † ‡ no. no. (years) Diagnosis Foot Operation name (days) Culture result 16S result (relative abundance, rank) culture Infection Foot Diabetic in Sequencing rDNA 16S Polymicrobial infections 1 1 M/49 Foot ulcer, OM R Debridement of 5th 20 Veillonella (V. parvula)* (0.64, 3rd) 1 metatarsal bone Corynebacterium (E. avium)* dominant Corynebacterium species polymicrobial (C. tuberculostearicum)* (<0.01, below 5th) 2 2 M/57 Foot ulcer and R Amputation of 66 Escherichia coli Escherichia Streptococcus (S. anginosus)* 1 gangrene 1st–5th Streptococcus viridans (E. fergusonii > (0.13, 3rd) transmetatarsal group E. coli)* dominant joint polymicrobial 3 3 M/70 Foot ulcer, OM L Amputation of 5th 18 S. aureus Staphylococcus Streptococcus (S. agalactiae)* – transmetatarsal Staphylococcus (S. capitis > (0.93, 2nd) joint epidermidis S. aureus > Streptococcus S. caprae > agalactiae S. epidermidis)* dominant polymicrobial 4 4 M/78 Foot abscess R BK amputation 15 Citrobacter freundii Parvimonas (P. micra) Citrobacter (C. freundii)* 1 dominant polymicrobial (0.76, 2nd) Enterococcus (E. faecalis)* (0.02, below 6th) 5 4' M/79 Foot abscess L BK amputation 29 C. freundii Bacteroides Enterococcus (E. faecalis)* 1 E. faecalis (B. xylanisolvens) (0.75, 2nd) dominant polymicrobial Citrobacter (C. freundii)* (0.15, 5th) 6 5 M/65 Foot ulcer and L Amputation of 1st 26 E. coli Pseudomonas Escherichia (E. fergusonii > – gangrene transmetatarsal Pseudomonas (P. aeruginosa)* E. coli) * (0.09, 2nd) joint aeruginosa dominant polymicrobial Corynebacterium (C. striatum) Corynebacterium (<0.01, below 3rd) striatum 7 6 M/49 Foot ulcer, OM L Amputation of 4th toe 32 S. viridans group Prevotella Streptococcus (S. constellatus)* 1 Finegoldia magna (P. melaninogenica) (0.34, 3rd) Diabetes dominant polymicrobial Finegoldia (F. magna)* (0.31, 4th) 8 7 M/66 Foot ulcer, OM R Debridement of ankle, 64 Enterococcus avium Parvimonas (P. micra) Enterococcus (E. avium) 1 foot S. aureus dominant polymicrobial (0.02, 11th) 9 7' M/67 Foot ulcer, OM R Bone trimming, 14 Morganella morganii Proteus (P. mirabilis) Morganella (M. morganii)* 1 2021 June 70, Volume amputation stump P. aeruginosa dominant polymicrobial (0.66, 2nd) E. faecalis Pseudomonas (P. aeruginosa)* (0.01, 9th) Enterococcus (E. faecalis)* (0.01, 11th) Continued on p. 1361 diabetes.diabetesjournals.org Table 1—Continued Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before no. no. (years) Diagnosis Foot Operation name (days)† Culture result 16S result (relative abundance, rank) culture‡ 10 08 M/64 Foot ulcer, OM R Lisfranc amputation 38 Enterobacter cloacae Enterobacter (E. cloacae)* Serratia (S. nematodiphila > 1 Serratia marcescens dominant polymicrobial S. marcescens)* (0.86, 2nd) 11 09 M/79 Foot ulcer, OM R Debridement of ankle 37 E. faecalis Prevotella (P. oris) Enterococcus (E. faecalis)* – and foot C. freundii dominant polymicrobial (0.11, 2nd) Citrobacter (C. freundii)* (0.08, 4th) 12 10 M/79 Foot ulcer and R BK amputation 32 Aeromonas hydrophila Aeromonas (A. hydrophila)* Escherichia (E. fergusonii > 1 gangrene E. coli dominant polymicrobial E. marmotae > E. coli)* (0.01, 3rd) 13 11 F/94 Foot ulcer and R Amputation of 1st 9 S. agalactiae Bacteroides (B. fragilis) Streptococcus (S. agalactiae)* 1 gangrene transmetatarsal S. aureus dominant polymicrobial (0.16, 4th) joint Staphylococcus Staphylococcus (S. aureus > lugdunensis S. lugdunensis)* (0.05, 6th) E. faecalis Enterococcus (E. faecalis)* (0.01, 8th) 14 12 M/79 Foot ulcer and L Amputation of 2nd 12 S. aureus Staphylococcus Escherichia (E. fergusonii > 1 gangrene toe E. coli (S. aureus)* dominant E. coli)* (0.19, 2nd) polymicrobial 15 12'' M/80 Foot ulcer, OM L Amputation of 1st toe 12 S. aureus Achromobacter Staphylococcus (S. aureus)* 1 F. magna (A. xylosoxidans)* (0.48, 2nd) dominant polymicrobial Finegoldia (F. magna)* (<0.01, 4th) 16 13 M/80 Foot ulcer, OM L Amputation of 1st toe 31 E. faecalis Anaerococcus Enterococcus (E. faecalis)* (0.03, 1 S. aureus (A. lactolyticus) 8th) dominant polymicrobial Staphylococcus (S. aureus)* (0.01, 9th) 17 13' M/81 Foot ulcer, OM L Amputation of 1st 18 E. cloacae complex Prevotella (P. timonensis) Enterobacter (E. cloacae)* 1 transmetatarsal Anaerococcus species dominant polymicrobial (0.49, 3rd) joint Klebsiella oxytoca Anaerococcus (A. vaginalis)* S. aureus (0.33, 4th) Klebsiella (K. oxytoca)* (0.26, 5th) onadAssociates and Moon Staphylococcus (S. aureus)* (0.03, 17th) 18 14 M/47 Foot ulcer, OM R Amputation of 5th 42 Streptococcus Prevotella (P. intermedia) Streptococcus (S. anginosus > 1 transmetatarsal anginosus dominant polymicrobial S. pyogenes)* (0.55, 3rd) joint Continued on p. 1362 1361 Table 1—Continued 1362 Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before no. no. (years) Diagnosis Foot Operation name (days)† Culture result 16S result (relative abundance, rank) culture‡ Infection Foot Diabetic in Sequencing rDNA 16S 19 15 M/68 Foot abscess, L Amputation of 2nd 22 S. viridans group Finegoldia (F. magna) Streptococcus (S. constellatus)* 1 OM toe S. aureus dominant polymicrobial (0.26, 3rd) Staphylococcus (S. caprae > S. aureus)* (0.03, 6th) 20 16 F/64 Foot abscess, L Amputation of 33 S. agalactiae Lactobacillus (L. iners) Streptococcus (S. agalactiae)* 1 OM transmetatarsal Klebsiella pneumoniae dominant polymicrobial (0.37, 4th) joint Klebsiella (K. pneumoniae)* (<0.01, 8th) 21 17 M/77 Foot ulcer and R BK amputation 15 P. aeruginosa Pseudomonas Staphylococcus (S. aureus)* 1 gangrene S. aureus (P. aeruginosa)* (0.73, 2nd) dominant polymicrobial 22 18 F/66 Foot ulcer, OM R Debridement of ankle 53 K. pneumoniae Klebsiella (K. pneumoniae)* Enterobacter (E. asburiae > 1 and foot E. cloacae complex dominant polymicrobial E. cloacae)* (0.29, 2nd) 23 19 M/56 Foot ulcer, OM R Amputation of 46 E. coli Bacteroides (B. fragilis) Escherichia (E. fergusonii > 1 3rd–5th E. faecium dominant polymicrobial E. coli)* (0.01, 3rd) transmetatarsal Enterococcus (E. faecalis > joint E. faecium)* (<0.01, 6th) 24 20 M/78 Foot ulcer, OM L Amputation of 39 M. morganii Anaerococcus Morganella (M. morganii)* 1 2nd–3rd Streptococcus (A. murdochii) dominant (0.56, 2nd) transmetatarsal dysgalactiae polymicrobial Streptococcus (S. dysgalactiae)* joint (0.01, 9th) 25 21 M/71 Foot ulcer, OM R Amputation of 2nd 16 S. dysgalactiae Streptococcus Staphylococcus (S. epidermidis)* 1 toe S. epidermidis (S. dysgalactiae)* (0.01, 2nd) dominant polymicrobial 26 22 M/60 Foot ulcer, OM R Amputation of 4th 109 S. marcescens Fusobacterium Serratia (S. marcescens)* 1 transmetatarsal F. magna (F. nucleatum) dominant (0.06, 2nd) joint E. coli polymicrobial Finegoldia (F. magna)* (0.02, 4th) Escherichia (E. fergusonii > E. coli)* (0.01, 5th) * 1 27 23 F/72 Foot ulcer, OM R Drainage of abscess 17 S. agalactiae Streptococcus Serratia (S. marcescens) Diabetes S. marcescens (S. agalactiae)* (0.06, 3rd) dominant polymicrobial 28 24 F/35 Foot ulcer, OM R Amputation of 4th toe 25 Prevotella bivia Prevotella (P. bivia)* Escherichia (E. fergusonii > 1 E. coli dominant polymicrobial E. marmotae > E. coli > 2021 June 70, Volume E. albertii)* (0.28, 2nd) Continued on p. 1363 diabetes.diabetesjournals.org Table 1—Continued Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before no. no. (years) Diagnosis Foot Operation name (days)† Culture result 16S result (relative abundance, rank) culture‡ 29 25 M/61 Foot ulcer, OM L Debridement of 5th 11 E. cloacae complex Enterobacter Finegoldia (F. magna)* 1 metatarsal bone F. magna (E. xiangfangensis)* (0.28, 3rd) dominant polymicrobial 30 26 M/64 Foot ulcer, OM R Amputation of 2nd 12 Proteus vulgaris Prevotella (P. intermedia) Enterococcus (E. avium)* 1 toe E. avium dominant polymicrobial (0.05, 14th) M. morganii Morganella (M. morganii)* (0.02, 17th) 31 27 F/69 Foot ulcer, OM R Amputation of 1st 26 S. viridans group Prevotella (P. oralis) Streptococcus (S. oralis > 1 transmetatarsal S. agalactiae dominant polymicrobial S. mitis > S. pneumoniae > joint S. aureus S. agalactiae)* (0.04, 9th) Staphylococcus (S. capitis > S. caprae >> S. aureus)* (0.01, 11th) 32 28 M/56 Foot ulcer, OM L Drainage of abscess 27 S. agalactiae Bacteroides (B. fragilis) Streptococcus (S. agalactiae)* 1 E. coli dominant polymicrobial (0.09, 7th) Escherichia (E. fergusonii > E. coli > E. albertii)* (0.02, 8th) Polymicrobial infections only identified by 16S sequencing 33 08' M/64 Foot ulcer, OM R Debridement of ankle 38 S. marcescens Serratia (S. marcescens > 1 and foot S. nematodiphila)* dominant polymicrobial 34 29 M/58 Foot ulcer, OM L Debridement of ankle 18 No growth Bacteroides (B. fragilis) 1 and foot dominant polymicrobial 35 29' M/59 Foot ulcer, OM R Amputation of 1st 31 Enterobacter asburiae Enterobacter (E. cloacae > transmetatarsal E. xiangfangensis > joint E. asburiae)* dominant polymicrobial 36 30 M/61 Foot ulcer and R Amputation of 1st toe 38 S. dysgalactiae Streptococcus 1 gangrene (S. dysgalactiae)*

dominant polymicrobial Associates and Moon 37 31 M/72 Foot ulcer, OM L Amputation of 1st toe 31 Staphylococcus Bacteroides (B. fragilis) Staphylococcus 1 hominis dominant polymicrobial (S. pettenkoferi >> S. hominis)* (0.01, 3rd) 38 32 M/63 Foot ulcer, OM R Amputation of 27 M. morganii Morganella (M. morganii)* 1 1st–5th dominant polymicrobial transmetatarsal joint Continued on p. 1364 1363 1364 Table 1—Continued Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before no. no. (years) Diagnosis Foot Operation name (days)† Culture result 16S result (relative abundance, rank) culture‡ Infection Foot Diabetic in Sequencing rDNA 16S 39 42' M/47 Foot ulcer, OM R Amputation of 27 P. aeruginosa Pseudomonas 1 2nd–5th (P. aeruginosa)* transmetatarsal dominant polymicrobial joint 40 33 M/73 Foot ulcer, OM L Amputation of 2nd 16 E. coli Anaerococcus (A. vaginalis) Escherichia (E. fergusonii > 1 toe dominant polymicrobial E. coli)* (0.01, 9th) 41 34 M/64 Foot ulcer, OM R Debridement of 5th 48 S. epidermidis Staphylococcus 1 metatarsal bone (S. epidermidis)* dominant polymicrobial 42 35 M/56 Foot ulcer, OM L Amputation of 4–5th 11 No growth (Candida Streptococcus (S. mitis) 1 transmetatarsal tropicalis) dominant polymicrobial joint 43 36 M/78 Foot ulcer, OM L Amputation of 1st toe 8 S. aureus Peptoniphilus Staphylococcus (S. aureus)* 1 (P. grossensis) dominant (0.08, 7th) polymicrobial 44 37 M/70 Foot ulcer, OM L Amputation of 5th toe 9 Corynebacterium Corynebacterium 1 species (C. striatum)* dominant polymicrobial Monomicrobial infections 45 02' M/58 Foot ulcer and L Amputation of 42 E. coli Escherichia (E. fergusonii > 1 gangrene 1st–5th E. albertii > E. coli)* transmetatarsal monomicrobial joint 46 38 M/69 Foot ulcer, OM L Amputation of 39 Klebsiella aerogenes Klebsiella (K. aerogenes) – 1st–5th monomicrobial transmetatarsal joint 47 12' M/79 Foot ulcer and L Partial excision of 3rd 12 No growth Anaerococcus (A. vaginalis) 1 gangrene toe monomicrobial Diabetes 48 39 F/76 Foot ulcer, OM R Resection of shaft of 9 Acinetobacter Acinetobacter 1 5th metatarsal baumannii (A. baumannii)* bone monomicrobial

49 40 M/49 Septic ankle R Debridement of ankle 15 S. viridans group Streptococcus 1 2021 June 70, Volume (S. vestibularis)* monomicrobial 50 41 M/86 Foot ulcer and L BK amputation 18 S. aureus Staphylococcus 1 gangrene (S. aureus)* monomicrobial Continued on p. 1365 diabetes.diabetesjournals.org Table 1—Continued Admin. Cultured pathogen 16S result Case Patient Sex/age duration in 16S sequencing before no. no. (years) Diagnosis Foot Operation name (days)† Culture result 16S result (relative abundance, rank) culture‡ 51 42 M/47 Foot ulcer, OM L Drainage of abscess 20 S. aureus Staphylococcus 1 (S. aureus)* monomicrobial 52 43 M/77 Foot ulcer, OM L Amputation of 1st toe 16 No growth Staphylococcus 1 (S. epidermidis) monomicrobial 53 44 M/76 Foot ulcer, OM R Amputation of 4th toe 21 P. vulgaris Proteus (P. vulgaris)* 1 monomicrobial 54 45 M/81 Foot abscess R Drainage of abscess 25 K. pneumoniae Klebsiella (K. pneumoniae)* 1 monomicrobial Admin., administrative; BK, below knee; F, female; L, left; M, male; OM, osteomyelitis; R, right. ' and '' in the patient no. represent experiments performed on different samples in the same patient. †Number of days each patient was hospitalized when the surgery was performed to obtain samples. ‡Experiments for 16S sequencing was only performed during the working hours of weekdays. Despite these shortcomings, this column demonstrates whether the result of 16S sequencing came out before the culture studies. *These bacteria identi- fied by 16S sequencing were also isolated by culture studies. onadAssociates and Moon 1365 1366 16S rDNA Sequencing in Diabetic Foot Infection Diabetes Volume 70, June 2021

Figure 1—The proportion of monomicrobial and polymicrobial infections, and illustrative cases of 16S sequencing. A: According to 16S sequencing, 81% (44 of 54) of the cases were polymicrobial infections. Meanwhile, conventional culture studies identified polymicrobial infections in only 59% (32 of 54) of the cases. Monomicrobial infection was suggested in 33% (18 of 54) of cases, and no bacteria were isolated in 4 cases. Twelve cases of polymicrobial infections were misdiagnosed as monomicrobial infections (n = 10) or no growth (n = 2). B: An illustrative case of monomicrobial infection (patient 40). Genus-level alignment of the reads revealed a Streptococcus monomicro- bial infection. Other genera listed are the result of misalignment. On a species-level alignment, S. vestibularis was at the top of the list and considered to be the pathogen. C: An illustrative case of polymicrobial infection (patient 13). After read alignment to the 16S database, Anaerococcus had the largest number of reads and thus was considered to be the dominant pathogen. In the species-level analysis, A. lactolyticus was suggested since it was the top ranked among the Anaerococcus species. In addition to Anaerococcus, Peptoniphilus (P. harei), Finegoldia (F. magna), Prevotella (P. timonensis), and others were present in the sample. Enterococcus (E. faecalis) and Staphylo- coccus (S. aureus) were isolated by the culture studies in this sample; however, they were ranked eighth and ninth in the 16S sequencing analysis. D: An illustrative case of follow-up study in polymicrobial infection (patient 8). On the initial 16S sequencing (case 10), Entero- bacter (E. cloacae) was found to be the most abundant in the sample, followed by Serratia, Klebsiella, and Citrobacter. In the follow-up 16S sequencing (case 33), performed after a 4-week antibiotic treatment, Serratia was the most abundant pathogen in the samples. This reflects that Enterobacter was successfully treated by the antibiotics but that Serratia was resistant to the treatment.

Nanopore 16S Sequencing Could Be Faster Than the surgery was performed during nighttime or during Culture Studies the weekend, samples were stored at 4C until the experi- The 16S sequencing was much faster than the culture mental procedures. Nevertheless, 16S sequencing revealed studies. Since the reduction in turnaround time was not the results earlier than the culture studies in most of the the primary objective of this study, the 16S sequencing cases. In 50 of 54 (92.6%) cases, the 16S sequencing was was conducted only during weekday working hours. When faster than the culture studies (Table 1). In most of the diabetes.diabetesjournals.org Moon and Associates 1367

Table 2—Pathogens that were frequently observed by culture studies and 16S rDNA sequencing Frequently isolated Frequently missed Dominant pathogen by Frequently isolated bacteria by 16S rDNA bacteria by 16S rDNA sequencing (n = 44), bacteria by culture, n sequencing, n culture (≥5), n n (missed by culture) Staphylococcus 18 Staphylococcus 25 Prevotella 20 Prevotella 7 (6) Streptococcus 16 Finegoldia 23 Finegoldia 19 Bacteroides 6 (6) Escherichia 10 Prevotella 21 Anaerococcus 18 Streptococcus 4 (1) Enterococcus 9 Streptococcus 21 Bacteroides* 15 Anaerococcus 3 (3) Enterobacter 5 Anaerococcus 19 Peptoniphilus* 12 Enterobacter 3 (0) Klebsiella 5 Bacteroides 15 Fusobacterium* 10 Pseudomonas 3 (0) Finegoldia 4 Enterococcus 13 Staphylococcus 8 Staphylococcus 3 (0) Morganella 4 Escherichia 12 Campylobacter* 7 Parvimonas 2 (2) Pseudomonas 4 Peptoniphilus 12 Streptococcus 7 Proteus 1 (1) Serratia 4 Fusobacterium 10 Haemophilus* 6 Achromobacter 1 (1) Citrobacter 3 Corynebacterium 8 Parvimonas* 6 Aeromonas 1 (0) Corynebacterium 3 Klebsiella 8 * 6 Corynebacterium 1 (0) Proteus 2 Campylobacter 7 Corynebacterium 5 Enterococcus 1 (1) Acinetobacter 1 Enterobacter 6 Porphyromonas* 5 Escherichia 1 (0) Aeromonas 1 Haemophilus 6 Veillonella 5 Finegoldia 1 (1) Anaerococcus 1 Parvimonas 6 Fusobacterium 1 (1) Prevotella 1 Peptostreptococcus 6 Klebsiella 1 (0) Veillonella 1 Pseudomonas 6 Lactobacillus 1 (1) Serratia 6 Morganella 1 (0) Veillonella 6 Peptoniphilus 1 (1) Citrobacter 5 Serratia 1 (0) Porphyromonas 5 Morganella 4 Proteus 4 Solobacterium 4 Atopobium 3 Dialister 3 Helcococcus 3 Pectobacterium 3 Providencia 3 Acinetobacter 2 Gemella 2 Mogibacterium 2 Stenotrophomonas 2 Achromobacter 1 Aeromonas 1 Arcanobacterium 1 Cutibacterium 1 Lachnoclostridium 1 Lactobacillus 1 Raoultella 1 Vibrio 1 Xenorhabdus 1 *These bacteria were never cultivated in culture tests. 1368 16S rDNA Sequencing in Diabetic Foot Infection Diabetes Volume 70, June 2021

Figure 2—The overall distribution of bacteria identified in the patients. The color of the square indicates which bacterium was identified in each patient. Any color except yellow means that the bacterium was both isolated by culture study and 16S sequencing. Yellow squares display the bacteria that were only identified by 16S sequencing. Exceptionally, the light blue square designated with the letter c in case 8 was the only result obtained by culture studies but not by 16S sequencing, which could be the result of contamination. The square des- ignated with the letter D indicates that the bacterium was the dominant pathogen on the basis of the abundancy of aligned reads in 16S sequencing. For example, in case 1, Corynebacterium and Veillonella were both revealed by culture studies and 16S sequencing. The 16S sequencing additionally identified Finegoldia, Bacteroides, and Enterococcus, and Enterococcus was assumed to be the dominant pathogen.

cases, the sequencing was run for 2 h; however, when se- bacteria isolated by the conventional culture studies, was quencing and analysis were performed simultaneously, more sensitive than the culture studies, and was capable the results were obtained within 30 min of sequencing. of detecting multiple anaerobes that were not cultivated This finding implies that even shorter sequencing time by the culture studies. Moreover, 16S sequencing was par- (<1 h) would be sufficient for pathogen identification, ticularly useful in cases of polymicrobial infections. In thus shortening the turnaround time. At best, the turn- some cases of polymicrobial infections, the dominant around time could be reduced to 6 h from DNA extraction pathogens were only identified by 16S sequencing and to the analysis of the reads (23). not by culture studies. In addition, in most of the cases, 16S sequencing was faster than conventional culture studies. DISCUSSION Nanopore 16S sequencing was more sensitive than We performed nanopore 16S amplicon sequencing in sur- conventional culture studies in identifying pathogens. A gical specimens from patients with medically intractable substantial number of pathogens were only identified by DFI. The 16S sequencing successfully identified all the 16S sequencing and not by culture studies. The 16S diabetes.diabetesjournals.org Moon and Associates 1369

Figure 3—The list of dominant pathogens among the polymicrobial infections. Among the polymicrobial infections identified by 16S se- quencing (n = 44), the dominant pathogen could be identified according to the number of aligned reads. Prevotella (n = 7, 16%) and Bac- teroides (n = 6, 14%) were most frequently presented as the dominant pathogens by 16S sequencing. However, more than one-half (n = 23, 55%) of these dominant pathogens were not cultivated in the conventional culture studies. Prevotella (n = 6) and Bacteroides (n =6) were frequently missed by the culture studies, although they were in fact the most abundant in the samples.

sequencing was particularly useful for detecting pathogens specimen, it is hard to obtain quantitative data from that are difficult or impossible to cultivate, especially culture studies. In addition, 16S sequencing could give when antibiotics were given before sample acquisition. information on the relative abundance of certain bacte- This was in line with previous studies that showed that ria among the listed pathogens. Although the abundance 16S sequencing is more sensitive than culture studies for and virulence of certain bacteria are not related to each pathogen detection in DFIs (1,3,26,27). Moreover, in other, these data would be considerably useful during most DFIs, antibiotics are prescribed before the debride- the medical treatment of patients with DFI. When fol- ment surgery (28–30), which would be another reason for low-up samples can be obtained regularly, antibiotics the lower diagnostic yield of the culture studies (31–33). could be adjusted to target the predominant pathogen Thus, 16S sequencing would be very useful for pathogen according to 16S sequencing results. identification in DFIs. Because of nanopore 16S sequencing, we demonstrated Nanopore 16S sequencing was particularly useful for that the majority of the medically intractable DFIs are the detection of polymicrobial infections. In most of the caused by polymicrobial infections. Staphylococcus and cases of polymicrobial infections, the culture studies Streptococcus were the most frequently isolated pathogens only isolated one or two bacteria from the various lists. in medically intractable DFIs. Meanwhile, 16S sequencing Certain bacteria that are difficult to cultivate were fre- revealed that Finegoldia, Prevotella, Anaerococcus,andBac- quently missed by the culture studies. Moreover, some teroides are highly prevalent in medically intractable DFIs bacteria grow dominantly during the culture procedure, and seldom cultivated in culture studies. Previous studies so the results may not reflect the true bacterial compo- using molecular techniques have also demonstrated that sition within the sample (34–36). Even when culture chronic DFIs are largely caused by anaerobes and polymi- studies can isolate multiple bacteria from a single crobial infections (1,37). Johani et al. (38) conducted 16S 1370 16S rDNA Sequencing in Diabetic Foot Infection Diabetes Volume 70, June 2021 amplicon sequencing in the intraoperative bone speci- Moreover, 16S sequencing cannot provide information mens of 20 patients with diabetic foot osteomyelitis. about antibiotics susceptibility, and the abundance of cer- Their results revealed that 70% had polymicrobial infec- tain pathogens is not directly related to their virulence. tions, with Corynebacterium being the leading cause. Nevertheless, obtaining accurate information about patho- Additionally, nanopore 16S sequencing was even faster gens in a timely manner with 16S sequencing will help in than culture studies for the detection of pathogens in managing patients with DFI. The prospective application of DFIs. Although we did not put maximum effort into re- 16S sequencing for making refined adjustment of antibiot- ducing the turnaround time in this study, the 16S se- ics in DFIs and their impact on prognosis should be investi- quencing method revealed the pathogen much earlier gated in the near future. than the culture studies in most cases. At best, we believe In conclusion, nanopore 16S sequencing was particu- that the turnaround time of 16S sequencing from DNA larly useful for pathogen identification in medically intrac- extraction to pathogen identification could be reduced to table DFI. Because of superior sensitivity over culture 6–9 h. This is comparable to our previous reports per- studies, a greater variety of bacteria, including uncultura- formed with other types of clinical samples (23). Nano- ble anaerobes, was revealed by the nanopore 16S sequenc- pore sequencing is suitable for small, rapid sequencing ing with a shorter turnaround time. Therefore, nanopore tests; thus, rapid turnaround time will be appropriate for 16S sequencing should be applied more widely in the case-by-case applications. management of DFIs, and its effect on the outcome of pa- Accurate diagnosis of pathogens could help to im- tients should be evaluated in subsequent studies. prove the poor prognosis of DFIs. Several factors are re- lated to the poor prognosis of DFI, including the presence of microvascular disease, an impaired host im- Funding. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT, Republic of mune response, and the occurrence of multilayered mi- fi Korea (NRF-2019R1A2C4070284). crobial communities within the wound known as bio lm Duality of Interest. No potential conflicts of interest relevant to this (6,34,39). Empiric antibiotics are widely prescribed in article were reported. the early stages of DFI on the basis of available clinical Author Contributions. J.M., D.Y.L., and K.C. contributed to the con- and epidemiological data (40–43); however, the standard ception and design of the study. J.M. and K.C. wrote the manuscript. N.K., treatment of DFIs involves debridement of the necrotic S.-T.L., K.-H.J., and D.O.L. contributed to the acquisition and analysis of tissue and antimicrobial treatment targeting the patho- data. H.S.L. contributed to the literature search and figure generation. K.-I.P. gens isolated by culture-dependent methods (28,29,44). and S.K.L. contributed to the data interpretation. All authors reviewed the fi Although the need for surgery is caused by various fac- manuscript for scholarly content and accuracy and gave approval for the nal draft. 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