Molecular epidemiology and antimicrobial susceptibility of Clostridium difficile isolates from two Korean hospitals

Clostridium difficile is one of the main etiological agents causing antibiotic-associated diarrhea. This study investigated the genetic diversity of 70 toxigenic C. difficile isolates from two Korean hospitals by employing toxinotyping, ribotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Toxin gene amplification resulted in 68 A⁺B⁺ and two A-B+ isolates. Most isolates (95.7–100%) were susceptible to daptomycin, metronidazole, and vancomycin. Seventy C. difficile isolates were classified into five toxinotypes, 19 ribotypes, 16 sequence types (STs), and 33 arbitrary pulsotypes. All C. difficile isolates of ribotype 018 (n = 38) were classified into ST17, which was the most prevalent ST in both hospitals. However, C. difficile isolates of ST17 (ribotype 018) exhibited pulsotypes that differed by hospital. ST2 (ribotype 014/020), 8 (ribotypes 002), 17 (ribotype 018), and 35 (ribotypes 015) were detected in both hospitals, whereas other STs were unique to each hospital. Statistical comparison of the different typing methods revealed that ribotyping and PFGE were highly predictive of STs. In conclusion, our epidemiological study indicates that C. difficile infections in both hospitals are associated with the persistence of endemic clones coupled with the emergence of many unique clones. A combination of MLST with PFGE or ribotyping could be useful for monitoring epidemic C. difficile strains and the emergence of new clones in hospitals.


Genomic DNA extraction
Genomic DNA was isolated from two or three colonies of C. difficile grown on BHI medium overnight in anaerobic conditions. DNA was extracted using a DNA Extraction Kit (Biofact Co., Korea) according to the manufacturer's instructions. The extracted DNA was used for MLST, toxinotyping, and PCR analysis of toxin genes.

PFGE
PFGE was performed according to previously described protocol with some modifications [19]. Briefly, bacteria were cultured in BHI broth until the OD 600 reached 1.8-2.0. The bacterial cells were lysed with 100 μl of 2× lysis buffer (2 M NaCl 2 , 200 mM EDTA (pH 8.0), 1% Brij-58, 0.4% sodium deoxycholate, 1% sodium lauryl sarcosine, lysozyme 4 mg/ml, RNase 100 mg/ml, and mutanolysin 40 U/ml). Plugs containing the lysed bacterial cells were prepared using 2% PFGE-grade low melting point agarose. The plugs were then incubated in 1× lysis buffer over night at 37˚C, followed by incubation with TE buffer (10 mM Tris-HCl, 1 mM EDTA) containing 10 mg/ml proteinase K, for 24 h at 50˚C, with buffer changes. The plugs were then washed with TE buffer (10 mM Tris-HCl, 1 mM EDTA) and digested with 50 U of SmaI (Thermo Fisher Scientific, Waltham, MA, USA) for 6 h at 25˚C. The digested products were electrophoresed on a 1% PFGE-grade agarose gel using a CHEF Mapper system (Bio-Rad). The band patterns were analyzed with GelCompar II software (Applied Maths, Kortijk, Belgium) to produce a dendrogram. A dendrogram was constructed using the unweighted pair-group method with arithmetic mean clustering. Pulsotypes were arbitrarily classified based on a similarity value of 0.85.

PCR ribotyping
PCR ribotyping of C. difficile isolates was performed as previously described [20]. PCR amplification was performed using the primers 5 0 -CTG GGG TGA AGT CGT AAC AAG G-3 0 and 5 0 -GCG CCC TTT GTA GCT TGA CC-3 0 . The PCR conditions comprised an initial denaturation step at 95˚C for 3 min, 30 cycles of denaturation at 95˚C for 30 S, annealing at 55˚C for 40 S, and extension at 72˚C for 70 S, and a final elongation step at 72˚C for 5 min. Amplification products were electrophoresed on 2% agarose gels. The electrophoretic patterns were compared with those available in a published library [21]. Nomenclature of new ribotypes that did not match with any UK ribotypes was based on the Kim's ribotyping scheme [22].

Toxinotyping
The 19-kb PaLoc region comprises toxin genes (tcdA and tcdB) and additional open reading frames (tcdC, tcdD, and tcdE) [23]. Toxinotyping is a restriction fragment length polymorphism typing method used to differentiate C. difficile isolates based on sequence distinctions in the PaLoc region [24]. Toxinotyping was conducted on both toxigenic and non-toxigenic C. difficile isolates as described by Rupnik (http://www.mf.uni-mb.si/tox/). PCR products and their restriction fragments were analyzed on 1% agarose gels. Toxinotypes of C. difficile isolates were determined by comparing their band patterns with that of the standard strain ATCC 43255. Patterns that visually differed from the pattern of this strain were compared with other known toxinotypes.

Comparison of the typing methods
The Simpson's index of diversity (SID), which measures the probability that two subjects randomly selected from a sample population belong to dissimilar types, was used as a discriminatory index. The adjusted Wallace coefficient (WC) was used to evaluate the bidirectional agreement between typing methods [29]. The SID and adjusted Wallace coefficient were calculated using a web-based tool (http://www.comparingpartitions.info/).

Toxin gene profiles
Sixty-eight isolates were A + B + and two were A -B + (Fig 1A). The cdt gene encoding the binary toxin was identified in one isolate (CDN7725), and toxin profile of this isolate was A + B + CDT + . Two A -B + -type C. difficile isolates were detected in KNUH.

Toxinotypes
Sixty-six isolates exhibited band patterns identical to that of the standard strain ATCC 43255; they were assigned to toxinotype 0 ( Fig 1B). Four isolates were found to carry a whole or part of PaLoc and were classified into toxinotypes I, III, VIII, and XII. C. difficile isolates of toxinotypes I, III, and VIII were from the internal medicine ward in KNUH, whereas the single toxinotype XII isolate was from the emergency room in BPH.

Antimicrobial resistance
The distribution of MICs of antimicrobial agents against C. difficile isolates is shown in Statistical comparison of the performance of the epidemiological typing tools SID was used to assess the ability of each epidemiological tool to differentiate between two test samples picked randomly from the sample population. Of the typing methods used in this study, PFGE, which yielded 33 arbitrary pulsotypes, exhibited the highest SID of 0.907, whereas toxinotyping, which defined five different types, showed the lowest SID of 0.112 ( Table 2). The WC was used to analyze the degree of concordance between typing schemes (Table 3). Ribotyping's congruence with MLST was greatest, with a WC of 0.976 (95% CI, 0.957-0.995), and PFGE showed agreement with MLST, with WC of 0.961 (95% CI, 0.938-0.985). However, low congruence was observed between toxinotyping and PFGE (WC = 0.010; 95% CI, 0.000-0.079) and toxinotyping and ribotyping (WC = 0.031; 95% CI, 0.000-0.211).

Discussion
This study reports, for the first time, the distribution of STs among C. difficile isolates from Korean hospitals. In addition, toxin gene profiling, toxinotyping, PFGE, and ribotyping were performed to explore the epidemiological characteristics of C. difficile isolates from two Korean hospitals. Not only were international epidemic C. difficile clones, including ribotypes Genomic DNA was digested with SmaI. The unweighted pair group method with arithmetic mean clustering method was used with the Dice coefficient with position tolerance and optimization of 1.2%. Pulsotypes were arbitrarily 018 (ST17), 014/020 (ST2/ST110), and 015 (ST26/ST35) found in both study hospitals, but diverse sporadic clones unique to each hospital were also detected.
Toxigenic C. difficile strains secrete one or both TcdA and TcdB toxins. A -B + C. difficile variants cannot secrete TcdA owing to deletions in the repetitive sequence of the tcdA gene. This toxin variant strain was first described in the early 1990s [30,31]. A -B + variants were found to constitute less than 10% of the toxigenic isolates from European countries and Japan, but were detected in up to 40% of isolates in Korea and Thailand [3,[10][11][12]. In this study, two A -B + C. difficile isolates were identified only in KNUH. Earlier studies demonstrated a high correlation between toxin types and STs; A -B + variants from China and Thailand were found to belong to ST37 and ST45 (ribotype 017), respectively. Moreover, A -B + variants from Korea belonged to ribotype 017 [22]. However, two A -B + variants tested in this study represented ribotypes 014/ 020 (ST110) and 017 (ST37), respectively.
PCR ribotyping of C. difficile isolates revealed that ribotype 018 (54.3%) was the most prevalent in both hospitals. Our results were consistent with those of a previous study conducted in a single hospital in Korea, which also reported a high prevalence of ribotype 018 [14]. Another report showed that a ribotype from Japan, smz, which corresponds to ribotype 018, was a major clone occurring in both outbreak and non-outbreak CDIs [32]. Ribotype 018 is reported to be one of the most prevalent strains causing complicated infections in France and Italy [33][34][35]. The prevalence and clinical relevance of ribotype 018 can be attributed to the fact that strains belonging to this genotype are relatively resistant to fluoroquinolones and have a unique variant of the splA gene, which is speculated to confer a high degree of adhesiveness that contributes to the success of this genotype [36]. In this study, the resistance rate of ribotype 018 C. difficile isolates to MXF was 84.2% (32 of 38 isolates). Moreover, all C. difficile isolates of ribotype 018, except for one isolate, exhibited the highest CIP MICs (> 32 μg/ml). Ribotypes 017, 014/020, 015, 002, 001, and 012 are increasingly becoming potential epidemic assigned based on a similarity value of 0.85. Undefined, isolates that were not assigned to any known ribotype; AB and C, ribotype nomenclature adopted by Kim's ribotyping scheme [3,22].   strains. These ribotypes were prevalent among C. difficile isolates obtained over the past decade in Asian as well as European countries [5,14,32,37], suggesting that they are globally circulating strains. Ribotypes 027, 014/020, UM11, 053/163, 002, 001, 078-126, 017, and 014 were the chief agents in outbreaks and epidemics in North American countries [38][39][40][41]. In an Australian survey of the hospital-acquired and community-acquired CDIs, ribotypes 014/020, 002, 054, 056, and 070 were the most prevalent in 2010, and C. difficile isolates of ribotypes 014/020, 056, 002, and 018 were the prevalent between 2012 and 2014 [42,43]. Regarding Asian countries, the most common ribotypes in China have been 017, 012, and 046 [44], whereas ribotypes 018, 001, 017, and 014/020 are prevalent in Japan and Korea [11,14]. We also found that most of the ribotypes were unique to a given hospital, with four ribotypes (AB7, AB15, AB32, and AB35) found in KNUH and seven ribotypes (AB9, AB18, AB24, AB25, AB42, AB46, and AB68) found in BPH. Fourteen C. difficile isolates from community-acquired CDIs, two from outpatients and 12 from emergency room, were analyzed. Two isolates from outpatients were A + B + and ribotype 018. Twelve A + B + isolates from emergency room showed five different ribotypes, 018 (n = 6), 014/020 (n = 3), AB15 (n = 1), AB35 (n = 1), and AB46 (n = 1). These results suggest that there is no difference in major clones of C. difficile between communityacquired and hospital-acquired CDIs. Our ribotyping results suggest that not only are epidemic C. difficile clones such as 018, 014/020, and 015 circulating in the study hospitals, but also that many sporadic clones are being introduced. MLST of the 70 C. difficile isolates generated 16 different STs. We determined that ST17 (ribotype 018) was predominant, accounting for 38 isolates. One isolate of ribotype AB68 and pulsotype CD33 was found to belong to ST17. The other frequent types were ST2 (n = 6), ST8 (n = 6), and ST35 (n = 6). Eleven STs were represented by single isolates. MLST yielded a diverse pool of allelic polymorphisms, ranging from 4-10 per housekeeping gene locus. The adk gene, with four distinctive alleles, was the most conserved among the seven loci, whereas the recA gene had the highest diversity, exhibiting a total of 10 alleles. There was a high correlation between STs and ribotypes. For example, ST2, ST17, ST8, and ST35 correlated with the ribotypes 014/020 (100%), 018 (97.4%), 002 (66.7%), and 015 (66.7%), respectively. On the other hand, ST2 and ST110, which differed in the recA gene, belonged to the same ribotype, 014/020. PFGE analysis of C. difficile isolates showed the highest discriminatory power of the epidemiological tools tested, with an SID of 0.907. Of the 33 arbitrary pulsotypes, CD01 (n = 20) and CD05 (n = 6) were the most prevalent; these pulsotypes correlated with ribotype 018 and ST17. Interestingly, 18 of the 20 CD01 isolates and all six CD05 isolates were from BPH and KNUH, respectively. ST2 was highly correlated with respective pulsotypes. However, the remaining 13 ST17 isolates were divided into 10 arbitrary pulsotypes. Moreover, ST8 and ST35 isolates showed diverse PFGE patterns and ribotypes. These results suggest that PFGE analysis is more valuable for intra-hospital epidemiological studies than other typing methods.
A statistical comparison of the different typing methods using the WC revealed that PCR ribotyping and PFGE results were highly predictive of MLST results; in other words, most isolates belonging to one ribotype or pulsotype grouped together in a single ST. However, ribotyping and MLST did not always yield similar outcomes. Occasionally, multiple STs were observed in a given ribotype [25,45]. Conversely, multiple ribotypes clustered in a single ST. This observation suggests that no single epidemiological typing scheme can perfectly predict the outcome of the other typing methods. PFGE analysis yielded more groupings than any of the other three methods tested. However, a low correlation was found between the results of PFGE and these other methods. These results suggest that a combination of band-based typing (PFGE or PCR ribotyping) and sequence-based typing (MLST) could be a powerful tool for epidemiological study of C. difficile isolates.
This study has several limitations. First, we tested limited number of isolates from two hospitals. C. difficile isolates collected in our culture collection system for pathogens were from representative cases of CDIs, but it was possible that clonal diversity of C. difficile isolates is underestimated. However, in this study, 70 C. difficile isolates from two hospitals were classified into 19 ribotypes, 16 STs, and 33 arbitrary pulsotypes. Second, we did not analyze incidence of CDIs and presence of outbreaks in the study hospitals and clinical characteristics of the patients with CDIs regarding risk factors, severity of CDIs, and association with antibiotic exposure. These clinical parameters may influence the clonal distribution of C. difficile in the hospitals. However, despite these limitations, our study provides important epidemiological information of C. difficile isolates from two Korean hospitals concerning the persistence of specific international clones and the emergence of sporadic clones.
This study found that C. difficile strains of ST17/ ribotype 018 were the most prevalent in two Korean hospitals. No hypervirulent 027 strain was detected in this study. Interestingly, C. difficile strains of the epidemic ST17/ribotype 018 exhibited different pulsotypes in each hospital, with arbitrary pulsotype CD01 in BPH and CD05 in KNUH. With the persistence of epidemic clones and the emergence of sporadic clones, CDIs are becoming one of the important nosocomial infections in Korean hospitals. A combination of MLST with PFGE or PCR ribotyping could be useful for monitoring epidemic C. difficile strains and the emergence of new clones in hospitals.