Diversity of binary toxin positive Clostridioides difficile in Korea

The objective of this study is to determine the trend and diversity of binary toxin-positive Clostridioides difficile over 10 years in Korea. Binary toxin-positive strains were selected from a tertiary hospital in Korea in 2009–2018. The multi-locus sequence typing and antibiotic susceptibility test were performed. Among the 3278 isolates in 2009–2018, 58 possessed binary toxin genes (1.7%). The proportion of CDT- positive isolates was 0.51–4.82% in 2009–2018, which increased over the 10-year period (P = 0.023). Thirteen sequence types (STs) were identified; ST5 (14 [24%]), ST11 (11 [19%]), ST221 (10 [17%]), ST201 (7 [12%]) and ST1 (5 [9%]) were popular. All 58 isolates were susceptible to vancomycin and piperacillin/tazobactam, and clindamycin and moxifloxacin were active in 69.0% and 62% of isolates, respectively. ST1 strains were resistant to several antibiotics, including moxifloxacin (80%), clindamycin (60%) and rifaximin (60%). Moreover, four of five ST1 presented a metronidazole minimum inhibitory concentration of 4 µg/mL. Moxifloxacin resistance was highest (72.3%) for ST11. In conclusion, binary toxin-positive strains are non-prevalent in Korea and involve diverse STs. ST1 strains were resistant to several antibiotics.

A neighbor-joining tree was constructed using the concatenated sequences of the seven loci used in MLST (Fig. 3). STs were clustered into four groups, designated as 2, 3, 5, and undetermined. ST122 which was placed on the root of lineage 2, was designated as novel lineage 6 in a previous report 10 .
Antibiotic susceptibility of binary toxin-positive C. difficile isolates. Table 1 presents the resistance rates of the 58 isolates to the six antibiotics. All isolates were susceptible to VAN and TZP, but CLI and MXF were active in 68.9% and 62.1% of the isolates, respectively. Five isolates (8.6%) were resistant to RFX and MTZ.
When we examined antibiotic susceptibility by ST type, ST1 strains showed a higher antibiotic resistance rate than other ST strains: MXF (80%), CLI (60%) and RFX (60%). Furthermore, 4 of 5 ST1 isolates presented an MTZ MIC of 4 µg/mL; thus the geometric mean MIC of MTZ was the highest among the ST1 isolates. One isolate of ST371, a strain closely related to ST1 according to MLST phylogeny, presented similar antibiotic susceptibility

Discussion
Binary toxins (CDTs) belong to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin; and CDTb which binds to host cells and translocates CDTa into the cytosol. ADP-ribosylation induces depolymerization of the actin cytoskeleton, which produces membrane protrusions and aids bacterial adherence 2 . CDT production is associated with increased CDI severity 2,4,6,7 . Some binary toxin-positive strains have a tcdC-truncating mutation, a negative regulator of tcdA and tcdB, which may increase the infection severity 11 . However, truncating mutations in tcdC alone did not cause a difference in patient' outcomes, although it was related to leukocytosis and C-reactive protein elevation 7 .
In this study, during the years of 2009-2018 at a university hospital in Korea, binary toxin-positive strains are not frequently encountered, and the epidemic binary-positive hypervirulent strains are not prevalent. Instead, ST-type strains in diverse clades existed in the hospital. However, ST11 strains showed a tendency to increase in this study, and similar findings were noticed in Taiwan and mainland China, but not in Japan [12][13][14][15] . High resistance to MXF of ST11 was commonly observed 12 , and high resistance to tigecycline was reported 13 . ST11/R078 strains began to be detected in 2011 and 2012 in the Asian countries 12 . R078 families are initially the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Frequent presence of R078 in meat products suggests a possible transmission from animals to humans 16 . In terms of R027 isolates, they have not spread among Asian countries after the first introduction in Korea, Hong Kong and Australia in 2008-2010 [17][18][19] . Despite hospital crowding, high levels of antibiotic (particularly fluoroquinolone) use, and huge  www.nature.com/scientificreports/ aging population with multiple comorbidities, ST1 strains have not increased in these countries without clear explanation. Binary-positive isolates from different clades showed different pattern of antibiotic susceptibility in this study. Despite a similar low prevalence, the epidemic hypervirulent ST1 and ST11 showed the higher resistance to most antibiotics. ST 371 most closely related to ST1 was resistant to MXF and CLI. Especially, ST1 showed 80% (4/5) of MTZ resistance despite MTZ resistance in C. difficile is rare. Those isolates showed the MIC of 4 µg/mL, and they were not considered from a single clone because all of them were from different years (1 isolate per year). It would be interesting to study the clonality and MTZ resistance mechanisms of those isolates.
MTZ resistance mechanisms in C. difficile are not clearly understood but are likely multifactorial processes involving alterations to metabolism such as with nitroreductase, iron uptake, DNA repair or biofilm formation 20,21 . Recently, MTZ resistance mediated by a high-copy number 7 kb plasmid (pCD-METRO) in C. difficile was discovered, which increased the MIC 25-fold 22 . The pCD-METRO was present in RT027 and nontoxigenic RT010 isolates from several countries. C. difficile RTs 027, 106, 001/072, 206, 010 and 356 strains had increased mean metronidazole MICs compared to other strains 20 .
Briefly reviewing the several binary-positive ST-types, all the ST-types below are positive for both toxin A and toxin B genes and the binary toxin genes. The ST67 strain, first reported in Japan did not contain an 18-base pair (bp) deletion or a one-bp deletion at position 117 in in tcdC, although it harbored eight nucleotide substitutions. The cytotoxicity of this strain was similar to that of ATCC BAA-1870 (RT027/ST1) 11 . ST5 and ST201 belonging to clade 3 have been sequenced in China 23 . The ST5 strain harbored a 54-bp consecutive deletion that resulted in a truncated TcdC protein, and ST201 also encodes a truncated TcdC. The ST122 strain, first isolated from Kuwait, contains an 18-bp deletion in the tcdC gene 10 . The phylogenetic analysis using a reference collection (Leeds-Leiden/ECDC) formed a well-separated sister clade to the clade formed by lineages 1 and 2, and the authors suggested a potential new lineage 10 . In our analysis, ST122 formed a separate lineage on the roots of lineage 2.
In summary, the proportion of binary toxin-positive strains was 1.7% among the 3278 isolates in 2009-2018. An epidemic strain of ST1 is not prevalent in Korea. The MLST analysis revealed diverse ST distributions in clades 2, 3, and 5. ST1 strains were more resistant to several antibiotics than other strains.

Material and methods
Study design and definition. This study was conducted at Hanyang University Hospital, a 900-bed tertiary care facility in Seoul, Korea. The study was approved by the institutional review board of Hanyang University Hospital (HYUH IRB 2016-01-031), which waived the need for informed consent.
All patients who had a CDI (as defined in the following paragraph) in 2009-2018 were identified through medical chart review, and the isolates from these patients were collected and stored. Diarrhea was defined as unformed stools more than three times per day on consecutive days or six times within 36 h 24 . The diagnosis of CDI was made using toxigenic culture, a commercial toxin A&B assay kit (VIDASⓇ C. difficile toxin A & B; BioMerieux SA, Marcy l'Etoile, France), and/or pseudomembrane on endoscopy 2 . Isolates from patients with CDI were tested by multiplex PCR 25 , and the isolates with CDT genes (cdtA and cdtB) were included.
Antibiotic susceptibility. The minimum inhibitory concentrations (MICs) of six antibiotics-metronidazole (MTZ), vancomycin (VAN), piperacillin/tazobactam (TZP), clindamycin (CLI), moxifloxacin (MXF) and rifaximin (RFX)-were determined. Brucella agar containing hemin (5 µg/mL), vitamin K1 (10 µg/mL), and 5% horse blood was used 28 . The MICs of CLI, MXF, and VAN were determined using Etest (AB-BIODISK, Solna, Sweden), while those of MTZ, RFX, and TZP were determined using the agar dilution test (Sigma-Aldrich, St. Louis, MO, USA). C. difficile ATCC 700057 was used as the control strain for the susceptibility tests. Resistance breakpoints were defined by the Clinical Laboratory and Standards Institute and European Committee on Antimicrobial Susceptibility Testing 28,29 . Phylogenetic analysis. A phylogenetic tree of the 13 STs was constructed using the seven housekeeping genes of the MLST. Each gene was separately aligned over 13 STs, and seven genes were concatenated for each ST. For the tree construction, the maximum likelihood method with the Tamura-Nei model was applied using MEGA6 30 . The robustness of the nodes was evaluated using the bootstrap method with 1000 replicates.
Statistical methods. SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. A logistic regression analysis was performed, as appropriate. Statistical significance was set at P < 0.05.