The First Two Clostridium difficile Ribotype 027/ST1 Isolates Identified in Beijing, China–an Emerging Problem or a Neglected Threat?

Clostridium difficile hyper-virulent ribotype 027 strain has become a significant concern globally, but has rarely been reported in Asian countries including China. Recently, a retrospective single-center study in Beijing, China, detected two ribotype 027 C. difficile isolates from two patients coming for outpatient visits in 2012 and 2013. We performed a systematic investigation of the two isolates (and patients). Both C. difficile isolates had the typical PCR ribotype 027 profile; were positive for tcdA, tcdB and binary toxin genes; belonged to multilocus sequence type 1 (ST1); had typical ribotype 027 deletions in the tcdC gene; and were highly-resistant to fluoroquinolones; but had a different MLVA profile and were not genetically related to any previously reported international ribotype 027 clones. A review of the patients’ medical records showed that neither received appropriate antimicrobial treatment and were lost to follow-up after outpatient visits. We propose that C. difficile infections caused by ribotype 027 are probably a neglected problem in China, and the subsequent impact of unawareness of this problem is worrying. Appropriate testing assays and multi-center or national level surveillance for C. difficile infections and specifically for ribotype 027 should be introduced to provide essential data and guide future clinical practice.

Scientific RepoRts | 6:18834 | DOI: 10.1038/srep18834 However, CDI is not widely recognized in Asia including China, possibly due to insufficient laboratory diagnostic capacity, low sample submission rate, and lack of high-quality surveillance systems 8,9 . Compared to North America and Europe, relatively few studies on C. difficile have been performed in Asia, with C. difficile ribotype 027 occasionally reported in the region to date. Thus the genuine extent of the disease and burden of C. difficile ribotype 027 in China and the majority of Asian countries remains largely unknown. Based on this background, and in order to gain some insights on CDI epidemiology in China, we retrospectively tested stool specimens (from suspected CDI cases), from a major hospital in Beijing (Peking Union Medical College Hospital, PUMCH), using culture and molecular tests.
In this retrospective study, two C. difficile ribotype 027 isolates were detected, which are the first reported cases in Beijing, China. Furthermore, we characterized isolates by molecular approaches and antimicrobial susceptibility testing and reviewed patients' medical records, aiming to have a better understanding of the current situation for future clinical practice implications.

Results
Background of C. difficile isolates. Out of 336 stool specimens from suspected CDI cases, 94 (28.0%) were positive by culture, but only 36 (10.7%) were positive for toxin A and/or B, by enzyme immunoassay (EIA). Further toxin testing on the C. difficile isolates yielded four isolates positive for toxin A and B genes (tcdA, tcdB) and the binary toxin genes (cdtA and cdtB).
Multiple-locus variable-number tandem-repeat analysis (MLVA). MLVA was performed to investigate the genetic relatedness among Chinese, European and North American C. difficile ribotype 027 isolates. In three previous studies, 69 ribotype 027 C. difficile isolates from the USA, Canada, United Kingdom, France and Netherlands, were investigated by MLVA 10,14,15 . Regarding the two ribotype 027 isolates, they differed by 3 markers (A6 Cd , B7 Cd and C6 Cd ), and had a similarity of about 57%. As shown in Fig. 1, the two ribotype 027 isolates were also not genetically related with other previously reported ribotype 027 strains (Fig. 1).

Medical records review.
The two ribotype 027 isolates were from two individual patients who visited an outpatient clinic at Peking Union Medical College Hospital. The first isolate, PUCD235, was from a 58-year-old male patient who came to the outpatient clinic on December 11, 2012 post rectal cancer surgery at another hospital. The second isolate, strain PUCD301, was from a 59-year-old female patient who visited the hospital for cholecystitis on March 17, 2013. Both patients had symptoms of diarrhea upon visiting, and increased white blood cell counts and neutrophils were noticed. However, laboratory routine testing for C. difficile toxins A and B by the commercial EIA methods for both patients were negative, and no additional tests (e.g. C. difficile culture) were ordered by the clinicians. Consequently, CDI was not diagnosed, and thus the patients did not receive appropriate antimicrobial treatment and were subsequently lost to follow-up.

Discussion
CDI is a major medical and infection control threat to health care facilities, including hospitals, long-term care facilities, and nursing homes around the world. The majority of CDI cases reported recently are largely associated with the emergence of epidemic ribotype 027 strain, with most of it reported from North America and Europe [36][37][38] . However, according to our literature review, only 142 ribotype 027 cases were reported in Asia from 19 publications in eight regions, with 80.3% of these cases reported from Israel, where ribotype 027 has become the major clone disseminated in that country 17,20,31 . For most Asian countries, C. difficile ribotype 027 cases have only been reported sporadically.
The few reported cases of C. difficile ribotype 027 in Asia may be a tip of the iceberg as many Asian countries have inadequate laboratory diagnostic capacity, low submission rate of samples, and lack high-quality and multiple-center surveillance systems for CDI. According to our previous survey data, as of Dec 2014, only 70 amongst over 1700 tertiary hospitals in China, routinely carried out EIA for detection of C. difficile toxins A and B (unpublished data), which was the only commercial C. difficile testing method approved by China Food and Drug Administration then. Furthermore, few laboratories perform culture or molecular-based detection of C. difficile. Therefore, the magnitude of the CDI problem may be much under-estimated 8,9 . To date, only one CDI case due to C. difficile ribotype 027 has been reported in mainland China, so the two cases identified in the present study are the second and third reported cases in China. Of note, the two patients involved did not get accurate diagnosis of CDI upon outpatient visit, mainly because the EIA testing for C. difficile toxins A and B were negative, although both patients had symptoms of diarrhea and abnormal routine blood test results. It has been reported that EIA-based methods are comparably less sensitive and have lower positive predictive value than toxigenic culture or molecular tests, in detecting C. difficile toxins. Moreover, the VIDAS EIA assay also appears to have lower sensitivity than other EIA-based methods for the detection of CDI 39 , prompting many centers to implement a two-pronged strategy that combines glutamate dehydrogenase (GDH) assay and toxin detection results to diagnose CDI. The two patients did not receive any appropriate treatment and were lost to follow-up which is a concern as these un-recognized C. difficile cases may become important transmission sources and a potential threat to public health 40 . Our findings suggest that C. difficile ribotype 027 has existed in northern China, therefore, it is important to improve laboratory diagnostic capacity and carry out active surveillance for the emergence of C. difficile hyper-virulent clones to avoid potential epidemic spread.
The development of molecular typing methods is of great significance for better understanding of C. difficile epidemiology. Our study identified the similarities and differences between the present C. difficile ribotype 027 isolates and globally described epidemic strains. Specifically, if only looking at MLST data, both strains belonged to ST1, suggesting possible similar origin source. However, a number of the 027 isolates previously reported in Asia were from a "historic" strain, which was fluoroquinolone susceptible, which is in contrast to the strains described in this study that were clearly fluoroquinolone resistant. In addition, sequencing of the slpA gene revealed that the two strains shared a SNP C467T, which differed from most global ribotype 027 strains, but was similar to one USA strain (2007855) recovered from animal rather than human source 12 . Further MLVA typing results also supported that the two ribotype 027 isolates were genetically different as they did not cluster together with strains from previously reported cases in North America and European countries (Fig. 1). The high discriminatory power of MLVA typing, if used properly (with suggested reasonable cut-off value) can be a potential valuable tool for investigating future ribotype 027 outbreaks 41 . In addition, if an association can be established between specific ribotype 027 MLVA subtypes, and virulence and clinical outcome of CDI patients 42 , this would be valuable for infection control and may help explain why our two strains appeared to have low virulence.
In conclusion, our study characterized the first two C. difficile PCR ribotype 027 isolates identified in Beijing, China. Our findings highlight the importance of raising public awareness, improving the laboratory diagnostic capacity, as well as implementing active surveillance systems for CDI in China, so as to capture this neglected potential public health threat and for providing useful indications for future clinical and infection control practices.

Ethics. The study was approved by the Human Research Ethics Committee of Peking Union Medical College
Hospital (No. S-263), and the study was carried out in accordance with the approved guidelines. Informed consents were obtained from all the patients.
Background for local laboratory diagnosis of C. difficile infections. Stool specimens from suspected CDI cases are routinely sent to the PUMCH laboratory in Beijing for C. difficile detection. The available tests for C. difficile detection at this hospital are toxin A and B detection using a commercial enzyme immunoassay (EIA) Figure 2. Summary of C. difficile PCR ribotype 027 reported in Asia. Size of circles indicated the number of cases identified in each region. NA, data not available. The map was generated by GNU Image Manipulation Program (version 2.8.14, the GIMP Team, USA).
Retrospective analysis of faecal specimens for C. difficile, including culture and molecular assays. A total of 336 consecutive, non-repetitive faecal specimens from 336 patients (stored at − 80 °C before use) were collected between August 2012 and July 2014. All the specimens were tested by culture and molecular assays. Generally, the specimens were cultured on cycloserine-cefoxitin fructose agar (CCFA) in anaerobic condition at 35 °C for 48 h, and suspected colonies were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Presence of toxin-encoding genes was then detected by a multiplex PCR assay to all confirmed C. difficile isolates as described below.
DNA extraction and toxin gene detection. One to five typical colonies were picked up from pure cultures of C. difficile isolates, and bacterial suspensions equivalent to 1 McFarland turbidity standard in 200μ l double-distilled water, were made. Genomic DNA was extracted with QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. A 5-plex PCR was used to detect tcdA, tcdB, cdtA and cdtB genes and the 16S rDNA, as previously described by Persson et al. 43 . We also sequenced the tcdC gene, a negative regulator of tcdA and tcdB 43 .
Sequencing of the slpA gene. As described previously by Kato et al. 46 , the partial slpA gene was amplified by the primers slpAcom19 (5′ -GTTGGGAGGAATTTAAGRAATG-3′ ) and slpAcom22 (5′ -GCWGTYTCTATTCTATCDTYWCC-3′ ). Both strands of the amplified products were sequenced and comparisons were made with the amino acid sequences deduced from the DNA sequences.
Multilocus variable-number tandem repeat analysis (MLVA). The genetic relatedness of the toxigenic isolates was investigated by MLVA, using the set of 7 loci (A6 Cd , B7 Cd , C6 Cd , E7 Cd , F3 Cd , G8 Cd , and H9 Cd ) as previously described by van den Berg et al. 15 . Repeat numbers were analyzed using BioNumerics software v6.5 (Applied Maths, Texas, USA) for cluster analysis. A dendrogram was constructed using the unweighted-pair group method with arithmetic mean clustering (UPGMA) with the multistate categorical similarity coefficient (MCSC).
Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed by the agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (document M11-A8) 47 . The following 11 antimicrobial agents were chosen: ciprofloxacin, clindamycin, erythromycin, levofloxacin, meropenem, metronidazole, piperacillin/tazobactam, rifampicin, rifaximin, tetracycline and vancomycin. Interpretation of testing results was based on CLSI M100-S25 48 , or according to the criteria suggested by Huang et al. 49 for the drugs whose breakpoints were not available in CLSI documents, as summarized in Table 1.

Review of C. difficile ribotype 027 reported in Asian countries.
To have a more comprehensive understanding of current epidemiology of C. difficile ribotype 027 in Asia, we reviewed and summarized all related published literature in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) database as of November 6, 2015.