Clostridium difficile is a leading cause of both nosocomial and antibiotic-associated diarrhea. The clinical manifestation of C. difficile infection (CDI) ranges from asymptomatic colonization to mild diarrhea to toxic megacolon and fulminant colitis. CDI is now considered a public health threat, with a dramatic rise in the incidence and severity of CDI observed in Europe and North America in the past two decades (Rupnik et al., 2009).

Most CDI cases have largely been attributed to the presence of the hyper-virulent C. difficile strain BI/NAP1/027 [restriction endonuclease analysis (REA) group BI, North American pulse-field type 1, PCR ribotype 027; McDonald et al., 2005]. Although ribotype 027 strains have caused major epidemics in North America and Europe, only sporadic cases have been reported in Asia (Collins et al., 2013). The major virulence factors for C. difficile are enterotoxin A (TcdA) and cytotoxin B (TcdB) (George et al., 1978). However, some strains that produce a binary toxin called C. difficile binary toxin (CDT), and whose role in causing disease is not yet clear, have been described and are associated with increased virulence and recurrence rates (Popoff et al., 1988).

Knowledge of the antimicrobial susceptibility profiles and molecular types of C. difficile is an important first step for monitoring and understanding the epidemiology of this organism. A variety of molecular methods have been applied for genotyping of C. difficile, including PCR ribotyping, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), and sequencing of functional genes such as slpA and tcdC (Collins et al., 2015). Each of these typing methods has its own merits and disadvantages, and may be applied in different occasions according to the aim and scale of the study (Killgore et al., 2008; Collins et al., 2015).

In China, there are limited studies on the molecular epidemiology and antibiotic susceptibility profiles of C. difficile, possibly due to insufficient laboratory diagnostic capacity, low awareness, and lack of high-quality surveillance systems (Collins et al., 2013; Hawkey et al., 2013; Cheng et al., 2015). However, in two previous retrospective studies, PCR ribotype 027 isolates were detected, suggesting that the threat of CDI in China is generally neglected and possibly underestimated (Cheng et al., 2016). To partly address this problem, we previously proposed a glutamate dehydrogenase-based algorithm for improving the clinical laboratory diagnostic capacity for CDI in China (Cheng et al., 2015).

The objective of this study was to evaluate the utility of different molecular typing assays for C. difficile, in relation to toxigenicity and antimicrobial susceptibility, using isolates obtained from one hospital in China. The usefulness of the different assays in different situations was considered, to inform decision making for improving the capacity of clinical laboratories in the management of CDI in developing countries like China.

Clostridium difficile infection has become a significant public health threat in the developed world, with substantial increase in morbidity and mortality reported since the early 2000s. However, due to inadequate laboratory diagnostic capacity, little is known about the magnitude of the CDI problem in developing countries like China, and hence the problem may be much underestimated (Collins et al., 2013; Hawkey et al., 2013; Cheng et al., 2016). In this regard, our group has previously attempted to improve the diagnosis of CDI in China by proposing a practical workflow for future CDI laboratory diagnosis (Cheng et al., 2015). Furthermore, the lack of high-quality CDI surveillance systems and epidemiology studies in developing countries contributes to the low awareness of CDI. Thus further studies are needed to address this problem.

Molecular typing methods are essential for understanding the epidemiology of C. difficile, and are especially important as part of an active surveillance and hospital infection control strategy for this organism. To date, several typing methods have been used to study the epidemiology of C. difficile, and there are pros and cons for each method. Most studies evaluating the utility of different molecular typing methods for CDI are from the developed world. In the present study, we evaluated the performance and utility of the most commonly used genotypic assays for C. difficile, including toxigenic typing, tcdC genotyping, slpA genotyping, MLST, PCR-ribotyping, and MLVA.

Although the dominant C. difficile toxigenic type is A+B+, there has been an increase in the prevalence and significance of the A-B+ type in some regions of the world (Kim et al., 2008; King et al., 2015). In this study, 23.3% of the isolates were A-B+CDT-, which is in agreement with previous studies in China in which the average prevalence rate of A-B+ strains is 21.4% (201/937), ranging from 0 to 53.6% by geographic distribution (Huang et al., 2009, 2010; Dong et al., 2013; Hawkey et al., 2013; Yan et al., 2013; Chen et al., 2014; Fang et al., 2014; Wang et al., 2014; Zhou et al., 2014; Cheng et al., 2015; Ye et al., 2015; Tian et al., 2016). Interestingly, infection with CDT-positive C. difficile strains has been reported to be associated with higher mortality and recurrence rates (Stewart et al., 2013). In this study, 5.1% (6/116) isolates were A+B+CDT+, which is slightly higher than in previous studies in Shanghai (1.6%, 2/110) and Zhejiang provinces (0%, 0/82; Huang et al., 2010; Fang et al., 2014). However, the overall prevalence of A+B+CDT+ C. difficile strains in China is much lower than in North America and Europe (Bassetti et al., 2012), possibly due to low lab diagnostic capacity and subsequent lack of awareness amongst medical personnel. By tcdC sequencing, five of six A+B+CDT+ isolates detected in this study had an 18-bp deletion at nucleotides 330–347, but only two ribotype 027 isolates, characterized by a single deletion at nucleotide 117, were detected.

The predominant ribotype and ST identified in the present study was ribotype 012/ST54 (16.4%), followed by ribotypes 001/ST3 (14.7%) and 017/ST37 (13.8%), whilst the commonest ribotype is ribotype 012 reported overall in China (100 of 589 isolates, 17.0%), and ST54, ST3, ST35, and ST37 (prevalence of 21.4, 12.6, 11.0, and 9.4 of 682 isolates tested, respectively) rank the top four STs (Huang et al., 2009, 2010; Dong et al., 2013; Hawkey et al., 2013; Yan et al., 2013; Chen et al., 2014; Fang et al., 2014; Wang et al., 2014; Zhou et al., 2014; Cheng et al., 2015; Ye et al., 2015; Tian et al., 2016). The remaining ribotypes/STs were distributed sporadically among different departments and with a lower prevalence. Generally, there was no obvious correlation between clonal clusters of the organism generated from the different departments during the study period, suggesting that a wide variety of strains are implicated in this hospital. However, due to lack of consistent data and limited lab diagnostic capacity for CDI at the hospital concerned, these findings may be biased.

Although the major molecular types identified in different C. difficile studies were similar, the prevalence of different molecular types varied amongst studies in China. In agreement to our findings, ribotype 017 C. difficile strains have been reported in several Asian countries (Collins et al., 2013). However, in North America and Europe, many CDI cases have been attributed to C. difficile ribotype 027 and 078 strains which have been described in many hospitals, and were particularly associated with outbreaks or epidemics (Bassetti et al., 2012). The major clone implicated in epidemics varies according to geographic locale, highlighting the significance of establishing a local surveillance network for CDI.

The emergence of increased antibiotic resistance in different C. difficile strain types worldwide may be a contributing factor to increased occurrence of CDI outbreaks in several hospitals. Although several studies have reported on the increasing MIC for metronidazole and vancomycin in C. difficile (Baines et al., 2008; Adler et al., 2015), both drugs showed high in vitro activity against all the isolates in our study. Furthermore, all strains were also susceptible to meropenem and piperacillin/tazobactam. Although a small number (12.9%) of C. difficile strains with high-level resistance to rifampin and rifaximin was observed in the present study, the rate was lower than the 25–29% reported in Shanghai, China (Huang et al., 2009, 2010).

The high rate of drug resistance for ciprofloxacin (100%), erythromycin (69%), and clindamycin (87.9%) among the studied isolates suggests that use of these antibiotics may be a risk factor for the development of CDI in this geographic locale (Bassetti et al., 2012). In addition, A-B+ strains in the present study exhibited higher drug resistance rates compared to A+B+ strains for the antibiotics clindamycin, erythromycin, levofloxacin, rifampicin, rifaximin, and tetracycline. Thus toxigenic typing may be valuable for understanding antibiotic resistance in the management and control of CDI. Furthermore, the drug resistance rates of strains of different PCR ribotypes differed. These findings emphasize the importance of implementing active surveillance and molecular epidemiologic studies on CDI.

All the typing methods used in this study have the advantage of high reproducibility (Table 4). MLVA exhibited extremely high discriminatory power, and thus may be a potential valuable tool for investigating CDI outbreaks (Eyre et al., 2013b). However, a reasonable cut-off value is needed since C. difficile isolates from the same outbreak may belong to genetically closely related but different MLVA types (Table 4) (Marsh et al., 2006).

Based on our findings, we recommend PCR ribotyping as the method of choice for epidemiological surveillance and infection prevention and control, as it delivers high discrimination, accuracy, and reproducibility. However, more work is needed for its standardization and database construction (Table 4) (Indra et al., 2008; Fawley et al., 2015). Although MLST also offers high discriminatory power index and easy inter-laboratory data comparison which may be useful for epidemiology surveillance and phylogenetic studies, the high cost is prohibitive (Table 4) (Killgore et al., 2008; Collins et al., 2015; Tian et al., 2016).

Sequencing of the slpA gene is valuable for comparison of C. difficile strains implicated in epidemics in diverse areas because the typing results are reproducible and can easily be shared. In addition, as the slpA gene is related to the strain’s serogroup, this typing method could be useful in vaccine development (Kato et al., 2005; Tian et al., 2016). tcdC genotyping had the lowest D-value, but sequencing of this gene may be used as an indicator for the hyper-virulent C. difficile clones, e.g., PCR ribotype 027/078 isolates (Curry et al., 2007).

Several other C. difficile typing methods apart from the ones applied in this study have been used, including PFGE, REA, and PCR-restriction fragment length polymorphism (RFLP). Unfortunately, these restriction enzyme-agarose gel electrophoresis-based methods have poor reproducibility, and are difficult to standardize for result interpretation and inter-laboratory data exchange (Killgore et al., 2008; Collins et al., 2015). Recent advances in whole-genome sequencing of C. difficile has the potential to provide even greater epidemiological information, but is still premature for use in large scale epidemiologic studies (Eyre et al., 2013a; Knight et al., 2015).

Our study is the most systematic study to integrate molecular epidemiology and antibiotic susceptibility testing, and provides comprehensive data for a better understanding of CDI in China. The findings highlight the importance of active surveillance using molecular typing techniques for better management and control of CDI in developing countries. Different molecular typing assays could be used depending on the scale and aim of the surveillance.

J-WC, MX, TK, and FK wrote the manuscript; Z-PX, LZ, and XF collaborated in molecular investigations of the strains; L-YS and X-LX summarized the patient’s medical records; Y-CX designed and supervised the study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.