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Molecular characteristics of Clostridium difficile in children with acute gastroenteritis from Zhejiang



Clostridium difficile infection (CDI) has an increasing pediatric prevalence worldwide. However, molecular characteristics of C. difficile in Chinese children with acute gastroenteritis have not been reported.


A five-year cross-sectional study was conducted in a tertiary children’s hospital in Zhejiang. Consecutive stool specimens from outpatient children with acute gastroenteritis were cultured for C. difficile, and isolates then were analyzed for toxin genes, multi-locus sequence type and antimicrobial resistance. Diarrhea-related viruses were detected, and demographic data were collected.


A total of 115 CDI cases (14.3%), and 69 co-infected cases with both viruses and toxigenic C. difficile, were found in the 804 stool samples. The 186 C. difficile isolates included 6 of toxin A-positive/toxin B-positive/binary toxin-positive (A+B+CDT+), 139 of A+B+CDT, 3 of AB+CDT+, 36 of AB+CDT and 2 of ABCDT. Sequence types 26 (17.7%), 35 (11.3%), 39 (12.4%), 54 (16.7%), and 152 (11.3%) were major genotypes with significant differences among different antimicrobial resistances (Fisher's exact test, P < 0.001). The AB+ isolates had significantly higher resistance, compared to erythromycin, rifampin, moxifloxacin, and gatifloxacin, than that of the A+B+ (χ2 = 7.78 to 29.26, P < 0.01). The positive CDI rate in infants (16.2%) was significantly higher than that of children over 1 year old (10.8%) (χ2 = 4.39, P = 0.036).


CDI has been revealed as a major cause of acute gastroenteritis in children with various genotypes. The role of toxigenic C. difficile and risk factors of CDI should be emphatically considered in subsequent diarrhea surveillance in children from China.

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Clostridium difficile is a Gram-positive, anaerobic, spore forming bacterium that leads to healthcare-associated diarrhea and can be as life-threatening as pseudomembranous colitis, toxic megacolon, intestinal perforation, and septic shock [1]. It has been reported that in 2011 C. difficile was responsible for almost half a million infections and was associated with approximately 29,000 deaths in the United States [2]. The estimated incidence of community-associated Clostridium difficile infection (CA-CDI) and health care–associated CDI in ages one and seventeen was 17.9 and 6.3 per 100, 000, respectively [2].

The rate of pediatric CDI-related hospitalizations has increased in the past decade in North America and Europe [3,4,5,6]. In the USA, the national rates of CDI-related pediatric hospitalizations have increased from 7.24 to 12.80 per 10,000 hospital admissions, in more than 3700 hospitals, between 1997 and 2006 [3]. One retrospective analysis revealed a 53% increase in the annual incidence density from 2001 to 2006, of 2.6 to 4.0 cases per 1000 admissions involved with 4895 CDI children, from 22 tertiary-care pediatric hospitals [4]. The CDI incidence was 6.6 cases/1000 admissions in a large pediatric hospital in Italy, where most symptomatic children less than 3 years old only had positive C. difficile culture without other gastrointestinal pathogens [5]. CDI has been reported in Asian children with inflammatory bowel disease, cancer and acute gastroenteritis [7,8,9]. However, data on C. difficile in children from China, including CDI rates, CDI related hospitalizations etc., were scarce. CDI was more difficult to identify in children than in adults, due to Clostridium difficile colonization and co-infections with various viruses (especially norovirus and caliciviruses) [9,10,11]. A literature review found it hard to draw any meaningful conclusions given the diversity of studies regarding the detection time, methods, the organisms tested for and the lack of cases definition of CDI in children under 5 years [12]. Testing for CDI should be routinely standardized according to new clinical practice guidelines [13]. The C. difficile tests in infants under 1 year were usually not recommended in the USA and UK [13, 14]. However, one retrospective cohort study found that 26% of children hospitalized with CDIs were infants and 5% were neonates [4]. The identification of CDI in pediatric population was quite complicated.

Acute gastroenteritis is still a serious public health problem in China, with more than 10,000 children dying from diarrhea annually [15]. Rotavirus group A, norovirus, Shigella spp., diarrheagenic Escherichia coli and Salmonella spp. are the most frequent pathogens in acute diarrhea in children [15]. Previous studies on CDI in China mostly focused on adult patients with hospitalizations [16], or specific conditions such as cancer [17], hematological malignancies [18], pregnancy [19] and advanced age [20]. Limited information is available regarding pediatric CDI and the molecular characteristics of C. difficile in children from China.

Although C. difficile colonization has been reported in northern Chinese infants [21, 22], C. difficile in children with acute gastroenteritis has not yet been studied, and CA-CDI in children was not mentioned in China. We conducted a five-year cross-sectional study on outpatient children with acute gastroenteritis, in a tertiary children’s hospital from eastern China, and investigated the molecular characteristics of C. difficile, including toxin genes, genotypes and antimicrobial susceptibility. Our study firstly presented the data on CA-CDI in children with acute gastroenteritis in Zhejiang, China, and also provided the pilot evidence to further study clinical significance of routine testing C. difficile in children.


Study design

This cross-sectional study was conducted in the Outpatient and Emergency departments of the Children’s Hospital of Zhejiang University School of Medicine, from February 2013 to December 2017, excluding October to December in 2013 and 2014. This tertiary hospital is the largest comprehensive center for pediatric health care in Zhejiang Province. Clinical stool samples were collected from selected outpatient children with acute diarrhea during the study period and then transported to the Xiacheng District Center for Disease Control and Prevention (XCCDC), within 24 h. Each sample was divided into two aliquots (1 mL/each) for further testing. This study was approved by the institutional review boards of the XCCDC. The informed consent requirement was waived due to no more than minimal risk involved in this study.

Data collection and viral detection

According to the guidelines of the Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America (SHEA/IDSA) [23], inclusion criteria were present as follows. Outpatients who suffered from acute diarrhea with more than 3 fluid, loose, or unformed stools within 24 h were sampled for this study, and all patients belonged to CA-CDI described as below. CA-CDI was defined by the presence of diarrhea symptoms and a positive test for toxigenic C. difficile, of which the onset of diarrhea occurred in the community or within 48 h after hospital admission, and had not been hospitalized within the previous 12 weeks [24]. Exclusion criteria were outpatients over 18 years old, patients with diarrhea who have been admitted over 48 h, and patients with underlying conditions (malignancy, immunodeficiency, abdominal surgery, hematological disease and hematopoietic stem cell transplantation). Duplicated stool samples from the same patients were removed. Clinical information on age, gender, school attendance and presenting symptoms, including fever and diarrhea, were collected.

Viral nucleic acids (DNA or RNA) were extracted from each stool sample using the appropriate kits (QIAgen, Inc., Valencia, CA, USA). The fluorescent real time PCR assays for rotavirus group A and B, norovirus genogroup I and II, astrovirus, sapovirus, adenovirus, and other viruses were used according to the manufacturer’s instructions (Shanghai ZJ Bio-Tech Co., Ltd., Shanghai, China).

C. difficile culture

For C. difficile culture, cefoxitin-cycloserine fructose agar (CCFA, Oxoid, UK), supplemented with 7% sterile defibrinated sheep blood, was used for selective isolation. Stool samples were primarily treated with purified ethanol and plated onto a CCFA medium (as described previously) [25]. After anaerobic culture at 37 °C for 48 h (GENbag anaer, bioMérieux, France), all colonies were identified according to special odor, characteristic morphology, and gram staining, as previously reported [26]. All isolates were stored at − 80 °C in brain-heart infusion broth, supplemented with 10% glycerol, for subsequent analysis [25].

DNA extraction and PCR of C. difficile toxin genes

C. difficile isolates were recovered on blood agar plates and extracted for genomic DNA with the QIAamp DNA blood Mini Kit (Valencia, CA, USA), according to the manufacturer’s instructions. The housekeeping gene tpi, toxin genes A and B (tcdA, tcdB) and binary toxin genes A and B (cdtA, cdtB) were amplified as previously reported [27,28,29]. The PCR product of the tpi gene was 230 bp in C. difficile isolates. The length of the tcdA gene was 369 bp for toxin A+B+ strains, and 110 bp for toxin AB+ strains. The length of the tcdB, cdtA, and cdtB genes was 688 bp, 375 bp, and 478 bp, respectively. C. difficile standard strains, including BAA-1803 and BAA-1870, were used as positive controls for tcdA and tcdB and the binary toxin genes. With BAA-1801 and ATCC-700057 as negative controls for all the toxin genes (American Type Culture Collection, Manassas, VA, USA). The positive, negative, and blank controls were examined in each experiment as parallel.

Multi-locus sequence typing (MLST)

MLST was performed as previously reported [28]. Seven housekeeping loci (adk, atpA, dxr, glyA, recA, sodA, and tpi) were amplified using PCR. The PCR products were identified with a 3730 XL DNA analyzer (Applied Biosystems). Data for C. difficile alleles and sequence types (STs) were submitted to the public MLST online database (

Antimicrobial susceptibility test and drug-resistant genes

A total of 12 antibiotics, including fusidic acid, ciprofloxacin, piperacillin-tazobactam (PIP-TAZ), metronidazole, rifampin, moxifloxacin, gatifloxacin, vancomycin, clindamycin, levofloxacin, tetracycline, and erythromycin were tested with the agar dilution method, according to the CLSI guideline (M11-A8, [30]). The breakpoints were determined according to the previous study [25]. Intermediate resistance was regarded as non-susceptible in later analysis. Multi-drug resistance (MDR) was defined as resistance to at least three classes of antibiotics [31]. Bacteroides fragilis (ATCC 25285) and C. difficile (ATCC 700057) were included in each run for quality control. The erythromycin- and clindamycin-resistant isolates were tested for the presence of the ermB gene. The tetracycline-resistant isolates were tested for the presence of the tetM gene, both according to previous publications [32, 33].

Data analysis

Data was analyzed with Statistical Package for Social Sciences (SPSS, Chicago, IL, USA), version 25.0 and Microsoft Excel. The χ2 test, or Fisher's exact Test, was used to analyze correlations among STs, toxin gene profiles and antimicrobial susceptibility patterns of C. difficile strains. A P value of < 0.05 was considered statistically significant.


Collection of C. difficile isolates

A total of 804 outpatient children were enrolled in this cross-sectional study, from 2013 to 2017. Stool samples were collected from each outpatient and analyzed as designed (Fig. 1). The demographic information is shown in Table 1. The overall median and interquartile range (IQR) of age was 0.67 (0.38–1.00) year old. The number of outpatients grouped by age of under six months, six months to one year, one to two years and over two years were 259, 266, 162 and 117, respectively. Moreover, 777 (96.6%) of the outpatients were scattered children and 141 (17.5%) had a fever (> 38.5 °C). In total, 115 (14.3%) cases were identified as CA-CDI, 69 (8.6%) cases had co-infections with viruses and 393 (48.9%) cases had neither C. difficile nor virus infections. A total of 294 (36.6%) outpatients identified positively for viruses, shown in Table 1.

Fig. 1
figure 1

Flow diagram of data collected during this study (February 2013 to December 2017)

Table 1 Clinical information of outpatients participated in this study

Toxin genes and MLST for C. difficile isolates

A total of 186 C. difficile isolates, including 6 A+B+CDT+, 139 A+B+CDT, 3 AB+CDT+, 36 AB+CDT and 2 ABCDT, were obtained from 804 stool samples. Totally, 27 STs were identified, with 7 new STs (ST513, 515, 526, 627, 628, 629, and 630), and none of the hypervirulent, epidemic ST1 (ribotype 027) isolates were detected in this study. The most prevalent type was ST26 (n = 33, 17.7%), followed by ST54 (n = 31, 16.7%), ST39 (n = 23, 12.4%), ST35 (n = 21, 11.3%), and ST152 (n = 21, 11.3%). ST83 and ST627 were found to be non-toxigenic (ABCDT). All ST37, 39, 81, and 630 isolates were AB+, with the remaining ST types being A+B+. A total of nine CDT+ strains included 3 of ST39, 3 of ST54, 2 of ST152 and 1 of ST15. The distribution of STs was relatively different in outpatient children younger and older than 1 year. ST26 (n = 29, 24.0%), ST54 (n = 21, 17.4%) and ST35 (n = 13, 10.7%) were the major STs in infants, whereas ST39 (n = 11, 17.5%), ST54 (n = 10, 15.9%) and ST152 (n = 10, 15.9%) were the predominant STs in children over 1 year old.

Antimicrobial susceptibility and drug-resistant genes

The antimicrobial susceptibility patterns of all the 186 C. difficile isolates are summarized in Table 2. All isolates were susceptible to metronidazole, vancomycin and PIP-TAZ, and resistant to ciprofloxacin. Resistance rates varied for other antimicrobials. The resistance rates of rifampin, moxifloxacin, gatifloxacin and tetracycline were 3.8, 7.5, 7.5 and 9.1%, respectively. However, the resistance rates to clindamycin, erythromycin, fusidic acid and levofloxacin were 85.5, 86.0, 69.4 and 79.6%, respectively. The 131 isolates were intermediate to levofloxacin, while only 17 isolates were resistant to it. A high resistance rate to MDR (89.2%, 166/186) was observed in these isolates. As for two non-toxigenic isolates, one was resistant to erythromycin and ciprofloxacin, intermediate to levofloxacin, and susceptible to other nine antimicrobials, and the other was resistant to ciprofloxacin, and susceptible to all eleven antimicrobial agents. The ermB gene was detected in 83.9% (141/168) of the erythromycin- and clindamycin-resistant isolates, while the tetM gene was present in 88.2% (15/17) of the tetracycline-resistant isolates.

Table 2 Correlations among MLST types, toxin genotypes, and antimicrobial susceptibility patterns of the 186 C. difficile isolates

The correlations between genotypes and antimicrobial susceptibility patterns are shown in Table 2. The antimicrobial patterns among major STs differed significantly (Fisher's exact test or χ2 = 36.09, P < 0.001) as below. In comparison with other STs, ST26 isolates had low resistance rates to fusidic acid and levofloxacin, ST35 isolates had a high resistance rate to tetracycline, and ST39 isolates had high resistance rates to rifampin, moxifloxacin, and gatifloxacin. Additionally, ST152 isolates exhibited comparatively lower resistance to clindamycin and erythromycin than the other major STs. The clindamycin resistance rate of the two non-toxigenic isolates was lower than that of toxigenic isolates (Fisher's exact test, P = 0.020). Furthermore, the resistance rates of erythromycin, rifampin, moxifloxacin, and gatifloxacin in AB+ isolates were higher than those in the A+B+ isolates (χ2 = 7.78–29.26, P < 0.005).


A total of 294 (36.6%) stools tested positive for viral infections, of which 123 (41.8%) were positive for rotavirus group A, 2 (0.7%) for norovirus genotype GI and 96 (32.7%) for GII, 7 (2.4%) for astrovirus, 16 (5.4%) for sapovirus, 10 (3.4%) for adenovirus, and 40 (13.6%) for multiple virus infections. None of them tested positive for rotavirus group B. The positive rate for viruses was much higher in children older than 1 year (59.9%, 167/279) than in infants (24.2%, 127/525) (χ2 = 99.91, P < 0.001).

For the 69 (8.6%) pediatric outpatients co-infected with both viruses and toxigenic C. difficile, 17 (24.6%) were co-infected with rotavirus, 30 (43.5%) with norovirus, 1 (1.4%) with astrovirus, 7 (10.1%) with sapovirus, 2 (2.9%) with adenovirus and 12 (17.4%) were co-infected with multiple viruses, including rotavirus and one or more other viruses. The co-infection rate was significantly higher in children older than 1 year (11.8%, 33/279) than in infants (6.9%, 36/525) (χ2 = 5.74, P = 0.017). However, the positive rate of CA-CDI in infants (16.2%, 85/525) was significantly higher than that in children older than 1 year (10.8%, 30/279), in the 115 CA-CDI cases (χ2 = 4.39, P = 0.036). Two non-toxigenic C. difficile isolates in 2014 and 2015, respectively, were not associated with CDI according to the published guideline [23].

In CA-CDI cases, the positive rates of toxigenic C. difficile from 2013 to 2017 were 11.9, 22.5, 17.9, 12.8, and 8.3%, respectively, revealing a significantly declining trend (trend chi-square χ2 = 5.84, P = 0.016), while a notable uptrend was observed in the viral infections (trend chi-square χ2 = 73.53, P < 0.001), with rates of 4.8, 22.5, 39.5, 31.0, and 60.4% (Fig. 2a). Analysis of the age distribution of pathogens revealed that co-infections were more common in 6 months to 2 years of age (χ2 = 21.38, P < 0.001). The positive rates of viral infections were much higher in children aged over 1 year (χ2 = 99.91, P < 0.001), while the CDI rates were relatively stable among different age groups (χ2 = 5.22, P = 0.156) (Fig. 2b).

Fig. 2
figure 2

Line chart of the positive rates of only toxigenic C. difficile (CA-CDI), total viral infections, and co-infections with different horizontal groups; a: in five years. b: in different age groups


Despite a colonization rate of toxigenic or non-toxigenic C. difficile of 30–40% in newborns, 30% in infants between 1 and 6 months of age and a reduction to 14% between 6 and 12 months of age, the incidence of CDI has dramatically increased in pediatric populations [34, 35]. Meanwhile, CA-CDI cases have increased in young children [6, 36, 37], along with a simultaneous global increase in studies on CA-CDI in children, in recent years [38,39,40]. However, studies have been more focused on the colonization of C. difficile in children from China [21, 22]. Only one study, on CA-CDI in southwest China, compared the clinical features and molecular characteristics in both children and adults [41]. The prevalence of CA-CDI in young children from China remains unknown.

The positive rate of CA-CDI (14.3%) from this study was consistent with the findings in southwest China (14.3% for children) [41], but higher than that reported in the USA [36, 37, 42] and Europe [43, 44]. According to a meta-analysis, the mean positive rate of toxigenic C. difficile in diarrheal adult patients from mainland China was 14% (95% confidence interval = 12–16%) [45]. In our study, the positive rate of CA-CDI showed a downward trend with the increase of viral infections. This was consistent with reports stating that diarrhea in children under 5 years old was mainly caused by viruses, both in China and worldwide [15, 46]. Notably, the positive rate of CA-CDI found in infants was higher than in children over 1 year old in this study, and only two non-toxigenic isolates found were not associated with CA-CDI in children with acute gastroenteritis. Children under 1 year were not recommended to conduct CDI tests due to high rates of C. difficile colonization as the new clinical practice guideline described [13]. So, the role of toxigenic C. difficile in infants and children has still been controversial in CDI cases. However, there were no any data supporting the standard of CDI diagnosis for children in China. The diversity of individual gut microbiomes was distinctly different among human beings from different geographical regions [47], and stewardship of antimicrobial use was implemented at different time in different counties [48]. Thus, it was speculated that the principle of CDI test for children under 1 year might be differentiated in China. Due to the high positive rate detected in this study, toxigenic C. difficile seemed to play an important role in acute gastroenteritis in children from eastern China. Thus, further investigation is required to confirm its role in infants, in order to guide or determine whether clinical CDI tests should be performed.

Our results indicated that the distribution of C. difficile genotypes in children from eastern China was distinctly different from those in adult hospitalized patients and in other regions [22, 45, 49]. All the toxigenic C. difficile isolates in this study were clustered in clade 1 and 4 in children, with toxigenic ST26 being one of the major genotypes in children mostly under 1 year old in China, and however a molecular epidemiology study in the UK showed that the most common genotypes in children were the non-toxigenic ST26 and ST15 [50]. Furthermore, a systematic review and meta-analysis in mainland China showed that ST2 and ST37 were the dominant genotypes in mainland China [45], and our previous study also identified ST37 as one of the most dominant genotypes associated with sever CDI in hospitalized adult patients from eastern China [25]. However, ST37 was not one of the major genotypes, and accounted for 30.8% of the AB+ strains in children in this study, which is similar to the report from southwest China [41]. It was speculated that ST37 might be transmitted into gastrointestinal tract along with individual growing up, which need to be further studied through monitoring continuous changes in intestinal flora. We also found that ST152 rarely reported in adult patients was identified as another major genotype in children. Even though most of the ST152 isolates were obtained in 2015, there were no relationships among these children, including geographical address and daily interaction. Thus, whole genome sequencing should be further performed to investigate the genetic relationships among these C. difficile isolates.

High consistencies between antimicrobial related genes and resistance phenotype was found in this study, indicating that the erythromycin- and clindamycin-resistant and the tetracycline-resistant isolates were mainly mediated by ermB and tetM genes, respectively. The antimicrobial resistance pattern on C. difficile isolates presented the low resistance rates to rifampin, moxifloxacin, gatifloxacin and tetracycline, which were similar to those of C. difficile isolates from diarrheal adults with healthcare acquired CDI [25, 45]. The antimicrobial resistance data were also compared with those on C. difficile isolates from adults with CA-CDI published in our team. The results showed that the CA-CDI associated A+B+ isolates from children presented significantly higher resistance rate to erythromycin (82.8%, 120/145) than that in adults with CA-CDI (60.8%, 62/102)(χ2 = 14.91, P < 0.001), and however AB+ isolates in children (87.2%, 34/39) exhibited distinctly lower resistance rate to clindamycin than that from adults with CA-CDI (100.0%, 88/88) (Fisher's exact test, P = 0.002) in Zhejiang [51]. It was speculated that frequent and inappropriate antimicrobial usage and different intestinal flora might be main reasons to lead to the differences on antimicrobial resistance in between adults and children in Zhejiang, China, which need be studied in the near future. Notably, we also found that the resistance rates of erythromycin, rifampin, moxifloxacin and gatifloxacin in AB+ isolates were significantly higher than in A+B+ isolates in children, indicating that exposures to clindamycin, erythromycin, fusidic acid, levofloxacin and ciprofloxacin might potentially exist in pediatrics in eastern China. Therefore, antimicrobial resistance mechanisms should be investigated and more antimicrobial resistance genes such as mefA, cfrB, and cfrC should be detected as the previous report [52] later in order to obtain the complete molecular characterization of the C. difficile isolates from pediatrics. Furthermore, partial C. difficile AB+ isolates led to clinical severe CDI as we previously reported [25]. Thus, CDI cases induced by AB+ isolates should be treated under the guidance of antimicrobial resistance tests in clinical therapy.

Only two non-toxigenic isolates were found in this study, indicating that acute diarrhea might mainly be induced by toxigenic C. difficile in children in Zhejiang, China. The relationship between toxigenic and non-toxigenic isolates was still unclear. Even though no significant differences on the resistance patterns were found between the non-toxigenic and toxigenic isolates except clindamycin, small numbers of non-toxigenic isolates might result in possible in our data analysis results among them. Thus, more non-toxigenic isolates should be collected to explore the correlation of C. difficile between with and without toxin genes, and supplement the resistance characteristics of C. difficile from children in Zhejiang, China.

There are some limitations in this study. Firstly, only outpatients from one tertiary children’s hospital was enrolled, making selection bias inevitable. Inpatient children should also be involved to disclose the intact molecular characteristics of C. difficile in this region. Secondly, a concrete medical record history was unavailable. Clinical information, including history of antibiotic use and clinical diagnosis, should be recorded in order to analyze the risk factors of the increasing prevalence of CDI in diarrheal children. Thirdly, the actual cause of diarrhea in outpatients, co-infected with both viruses and toxigenic C. difficile, was still unknown. Thus, we are going to conduct another study with a large scale of diarrheal children from outpatients and inpatients including clinical information and a questionnaire including more risk factors such as environmental exposure with pets [53] in order to analyze molecular epidemiology, transmission routes and risk factors of CDI in children in China, and meanwhile the role of toxigenic C. difficile in young children should be investigated later.


This was the first study on the molecular characteristics of C. difficile in outpatient children with acute gastroenteritis, from Zhejiang, eastern China. A wide variety of STs, including ST26, ST54, ST35, ST39, and ST152, were found to be major genotypes with differing antimicrobial resistance profiles in children, which differed distinctly from adults in China. AB+ isolates need to be considered due to the high antimicrobial resistance rates. Further studies and surveillance should be performed to investigate the role and risk factors of C. difficile in children with diarrhea.

Availability of data and materials

All data generated and/or analyzed during the current study are available from the corresponding authors on reasonable request.



Clostridium difficile infection


Community-associated Clostridium difficile infection


Xiacheng District Center for Disease Control and Prevention


Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America


Cefoxitin-Cycloserine Fructose Agar


American Type Culture Collection


Multi-locus sequence typing


Sequence type




Clinical and Laboratory Standards Institute


Multi-drug resistance


Interquartile range


  1. Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol. 2009;7(7):526–36.

    Article  CAS  PubMed  Google Scholar 

  2. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, et al. Burden of Clostridium difficile infection in the United States. N Engl J Med. 2015;372(9):825–34.

    Article  CAS  PubMed  Google Scholar 

  3. Zilberberg MD, Tillotson GS, McDonald LC. Clostridium difficile infections among hospitalized children, United States, 1997-2006. Emerg Infect Dis. 2010;16(4):604–9.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Kim J, Smathers SA, Prasad P, Leckerman KH, Coffin S, Zaoutis T. Epidemiological features of Clostridium difficile-associated disease among inpatients at children's hospitals in the United States, 2001–2006. Pediatrics. 2008;122(6):1266–70.

    Article  PubMed  Google Scholar 

  5. Spigaglia P, Barbanti F, Castagnola E, Diana MC, Pescetto L, Bandettini R. Clostridium difficile causing pediatric infections: new findings from a hospital-based study in Italy. Anaerobe. 2017;48:262–8.

    Article  PubMed  Google Scholar 

  6. Malmqvist L, Ullberg M, Hed Myrberg I, Nilsson A. Clostridium difficile infection in children: epidemiology and trend in a Swedish tertiary care hospital. Pediatr Infect Dis J. 2019;38(12):1208–13.

    Article  PubMed  Google Scholar 

  7. Kim DH, Jin MC, Yang HR. Clostridium difficile infection at diagnosis and during the disease course of pediatric inflammatory bowel disease. Pediatr Gastroenterol Hepatol Nutr. 2018;21(1):43–50.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Daida A, Yoshihara H, Inai I, Hasegawa D, Ishida Y, Urayama KY, et al. Risk factors for hospital-acquired Clostridium difficile infection among pediatric patients with Cancer. J Pediatr Hematol Oncol. 2017;39(3):e167–e72.

    Article  PubMed  Google Scholar 

  9. Kim A, Chang JY, Shin S, Yi H, Moon JS, Ko JS, et al. Epidemiology and factors related to clinical severity of acute gastroenteritis in hospitalized children after the introduction of rotavirus vaccination. J Korean Med Sci. 2017;32(3):465–74.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Stokely JN, Niendorf S, Taube S, Hoehne M, Young VB, Rogers MA, et al. Prevalence of human Norovirus and Clostridium Difficile Coinfections in adult hospitalized patients. Clin Epidemiol. 2016;8:253–60.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lees EA, Carrol ED, Ellaby NAF, Roberts P, Corless CE, Lenzi L, et al. Characterization of circulating Clostridium difficile strains, host response and intestinal microbiome in hospitalized children with diarrhea. Pediatr Infect Dis J. 2020;39(3):221–8.

    Article  PubMed  Google Scholar 

  12. Graaf HD, Pai S, Burns DA, Karas JA, Enoch DA, Faust SN. Co-infection as a confounder for the role of Clostridium difficile infection in children with diarrhoea: a summary of the literature. Eur J Clin Microbiol Infect Dis. 2015;34(7):1281–7.

    Article  PubMed  Google Scholar 

  13. McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, et al. Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018;66(7):e1–e48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pai S, Aliyu SH, Enoch DA, Karas JA. Five years experience of Clostridium difficile infection in children at a UK tertiary hospital: proposed criteria for diagnosis and management. PLoS One. 2012;7(12):e51728.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hao R, Li P, Wang Y, Qiu S, Wang L, Li Z, et al. Diversity of pathogens responsible for acute diarrheal disease in China. Clin Infect Dis. 2013;57(12):1788–90.

    Article  PubMed  Google Scholar 

  16. Chen YB, Gu SL, Shen P, Lv T, Fang YH, Tang LL, et al. Molecular epidemiology and antimicrobial susceptibility of Clostridium difficile isolated from hospitals during a 4-year period in China. J Med Microbiol. 2017;67(1):52–9.

    Article  PubMed  CAS  Google Scholar 

  17. Fang WJ, Jing DZ, Luo Y, Fu CY, Zhao P, Qian J, et al. Clostridium difficile carriage in hospitalized cancer patients: a prospective investigation in eastern China. BMC Infect Dis. 2014;14(1):523.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Gu SL, Chen YB, Lv T, Zhang XW, Wei ZQ, Shen P, et al. Risk factors, outcomes and epidemiology associated with Clostridium difficile infection in patients with haematological malignancies in a tertiary care hospital in China. J Med Microbiol. 2015;64(Pt 3):209–16.

    Article  PubMed  Google Scholar 

  19. Ye GY, Li N, Chen YB, Lv T, Shen P, Gu SL, et al. Clostridium difficile carriage in healthy pregnant women in China. Anaerobe. 2016;37:54–7.

    Article  PubMed  Google Scholar 

  20. Huang H, Wu S, Chen R, Xu S, Fang H, Weintraub A, et al. Risk factors of Clostridium difficile infections among patients in a university hospital in Shanghai. China Anaerobe. 2014;30:65–9.

    Article  PubMed  Google Scholar 

  21. Cui QQ, Yang J, Niu YN, Qiang CX, Li ZR, Xu KY, et al. Epidemiological investigation of Clostridioides difficile colonization in Chinese community infants. Anaerobe. 2019;56:116–23.

    Article  PubMed  Google Scholar 

  22. Tian TT, Zhao JH, Yang J, Qiang CX, Li ZR, Chen J, et al. Molecular characterization of Clostridium difficile isolates from human subjects and the environment. PLoS One. 2016;11(3):e0151964.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, Mcdonald LC, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol. 2010;31(5):431–55.

    Article  PubMed  Google Scholar 

  24. McDonald LC, Coignard B, Dubberke E, Song X, Horan T, Kutty PK. Recommendations for surveillance of Clostridium difficile–associated disease. Infect Control Hosp Epidemiol. 2007;28(2):140–5.

    Article  PubMed  Google Scholar 

  25. Jin D, Luo Y, Huang C, Cai J, Ye J, Zheng Y, et al. Molecular epidemiology of Clostridium difficile infection in hospitalized patients in eastern China. J Clin Microbiol. 2017;55(3):801–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McDonald LC, Killgore GE, Thompson A, Owens RCJ, Kazakova SV, Sambol SP, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med. 2005;353(23):2433–41.

    Article  CAS  PubMed  Google Scholar 

  27. Ludovic L, Dhalluin A, Testelin S, Mattrat MA, Maillard K, Lemeland JF, et al. Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (toxin a), and tcdB (toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol. 2004;42(12):5710–4.

    Article  CAS  Google Scholar 

  28. Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW, Fung R, et al. Multilocus sequence typing of Clostridium difficile. J Clin Microbiol. 2010;48(3):770–8.

    Article  CAS  PubMed  Google Scholar 

  29. Gonçalves C, Decré D, Barbut F, Burghoffer B, Petit J-C. Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile. J Clin Microbiol. 2004;42(5):1933–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing of anaerobic bacteria. M11-A8: National Committee for Clinical and Laboratory Standards. Wayne, PA; 2017.

  31. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81.

    Article  CAS  PubMed  Google Scholar 

  32. Spigaglia P, Barbanti F, Mastrantonio P. Detection of a genetic linkage between genes coding for resistance to tetracycline and erythromycin in Clostridium difficile. Microb Drug Resist. 2007;13(2):90–5.

    Article  CAS  PubMed  Google Scholar 

  33. Schmidt C, Löffler B, Ackermann G. Antimicrobial phenotypes and molecular basis in clinical strains of Clostridium difficile. Diagn Microbiol Infect Dis. 2007;59(1):1–5.

    Article  CAS  PubMed  Google Scholar 

  34. Enoch DA, Butler MJ, Pai S, Aliyu SH, Karas JA. Clostridium difficile in children: colonisation and disease. J Inf Secur. 2011;63(2):105–13.

    Google Scholar 

  35. Jangi S, Lamont JT. Asymptomatic colonization by Clostridium difficile in infants: implications for disease in later life. J Pediatr Gastr Nutr. 2010;51(1):2–7.

    Article  Google Scholar 

  36. Centers for Disease Control and Prevention. Surveillance for community-associated Clostridium difficile--Connecticut, 2006. MMWR Morb Mortal Wkly Rep. 2008;57(13):340–3.

    Google Scholar 

  37. Benson L, Song X, Campos J, Singh N. Changing epidemiology of Clostridium difficile-associated disease in children. Infect Control Hosp Epidemiol. 2007;28(11):1233–5.

    Article  PubMed  Google Scholar 

  38. Adams DJ, Eberly MD, Rajnik M, Nylund CM. Risk factors for community-associated Clostridium difficile infection in children. J Pediatr. 2017;1(Suppl 1):105–9.

    Article  Google Scholar 

  39. Predrag S, Branislava K, Nikola S, Niko R, Zorica SR, Stanković-Đorđević D. Community-acquired Clostridium difficile infection in Serbian pediatric population. Eur J Clin Microbiol Infect Dis. 2018;37(6):1061–9.

    Article  PubMed  Google Scholar 

  40. Pechal A, Lin K, Allen S, Reveles K. National age group trends in Clostridium difficile infection incidence and health outcomes in United States community hospitals. BMC Infect Dis. 2016;16(1):682–7.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Liao F, Li W, Gu W, Zhang W, Liu X, Fu X, et al. A retrospective study of community-acquired Clostridium difficile infection in Southwest China. Sci Rep. 2018;8(1):3992–4002.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Denno DM, Stapp JR, Boster DR, Qin X, Clausen CR, Del Beccaro KH, et al. Etiology of diarrhea in pediatric outpatient settings. Pediatr Infect Dis J. 2005;24(2):142–8.

    Article  PubMed  Google Scholar 

  43. Taori SK, Wroe A, Hardie A, Gibb AP, Poxton IR. A prospective study of community-associated Clostridium difficile infections: the role of antibiotics and co-infections. J Inf Secur. 2014;69(2):134–44.

    Google Scholar 

  44. Borali E, Ortisi G, Moretti C, Stacul EF, Lipreri R, Gesu GP, et al. Community-acquired Clostridium difficile infection in children: a retrospective study. Dig Liver Dis. 2015;47(10):842–6.

    Article  PubMed  Google Scholar 

  45. Tang C, Cui L, Xu Y, Xie L, Sun P, Liu C, et al. The incidence and drug resistance of Clostridium difficile infection in mainland China: a systematic review and meta-analysis. Sci Rep. 2016;6:37865.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Boschi-Pinto C, Lanata CF, Black RE. The global burden of childhood diarrhea. Matern Child Health: Springer; 2009. p. 225–43.

  47. Mobeen F, Sharma V, Tulika P. Enterotype variations of the healthy human gut microbiome in different geographical regions. Bioinformation. 2018;14(9):560–73.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Luo Y, Cheong E, Bian Q, Collins DA, Ye J, Shin JH, et al. Different molecular characteristics and antimicrobial resistance profiles of Clostridium difficile in the Asia-Pacific region. Emerg Microbes Infec. 2019;8(1):1553–62.

    Article  CAS  Google Scholar 

  49. Luo Y, Zhang W, Cheng JW, Xiao M, Sun GR, Guo CJ, et al. Molecular epidemiology of Clostridium difficile in two tertiary care hospitals in Shandong Province, China. Infect Drug Resist. 2018;11:489–500.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Stoesser N, Crook DW, Fung R, Griffiths D, Harding RM, Kachrimanidou M, et al. Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection. J Clin Microbiol. 2011;49(11):3994–6.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Zhao L, Luo Y, Bian Q, Wang L, Ye J, Song X, et al. High-level resistance of toxigenic Clostridioides difficile genotype to macrolide-Lincosamide-Streptogramin B in community acquired patients in eastern China. Infect Drug Resist. 2020;13:171–81.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Chatedaki C, Voulgaridi I, Kachrimanidou M, Hrabak J, Papagiannitsis CC, Petinaki E. Antimicrobial susceptibility and mechanisms of resistance of Greek Clostridium difficile clinical isolates. J Glo Antimicrob Resist. 2019;16:53–8.

    Article  CAS  Google Scholar 

  53. Kachrimanidou M, Tzika E, Filioussis G. Clostridioides (Clostridium) difficile in food-producing animals, horses and household pets: a comprehensive review. Microorganisms. 2019;7(12):667.

    Article  PubMed Central  Google Scholar 

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This work was supported in part by a Key Research and Development Program of Zhejiang (2015C03048) and the Medical Health and Technology Plan of Zhejiang (2017KY571). The funding bodies had no role in study design, data collection, analysis, and interpretation or writing the manuscript.

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Authors and Affiliations



DZJ and JMJ: conceived the study, designed the experiments and revised the manuscript. QHH, ZYP and JW: collected samples and demographic data. HQS, QB, YL, XHZ, XJS and JLY: performed the experiments. QB, JW and YL: data analysis and data interpretation. HQS and QB: wrote the manuscript; All authors read and approved the final manuscript.

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Correspondence to Jianmin Jiang or Dazhi Jin.

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This study was approved by the institutional review boards of the XCCDC. All clinical information has been already collected in another project and doesn’t involve any identifiable private information. Thus, the informed consent requirement was waived.

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Shuai, H., Bian, Q., Luo, Y. et al. Molecular characteristics of Clostridium difficile in children with acute gastroenteritis from Zhejiang. BMC Infect Dis 20, 343 (2020).

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