Clostridioides difficile is a major healthcare-associated pathogen inducing infectious diarrhea, which is responsible for a wide spectrum of diseases, ranging from mild diarrhea to fulminant colitis and even death (1). Approximately 25–33% of patients with antibiotic-associated diarrhea and 90% of patients with pseudomembranous enteritis are due to C. difficile infection (CDI) (2). The CDI has been associated with increased morbidity and decreased quality of life in patients, accompanied by prolonged hospitalization (3–5).

Recently, the incidence of CDI has been reported to increase in various countries. Sweden, China, and several other countries have reported an incidence rate of 17.1/10,000 admission to hospitals (6). The incidence of CDI has largely increased due to the emergence of an epidemic ribotype (sequence type [ST]: 1/027/NAP1), and the development of more sensitive detection approaches (7). CDI has been recognized to initiate from unusual antibiotic exposure of intestinal microbiota. The most important risk factor for CDI is broad-spectrum antimicrobial drugs that induce intestinal microfloral dysbiosis (8).

The severity and consequences of CDI are influenced by multiple factors, namely, hypervirulent isolates, age, immune status, and underlying conditions of the patient (e.g., receipt of antimicrobial therapy) (9). A novel C. difficile isolate with binary toxin-positive (non-027, non-078), associated with severe diarrhea has been recently reported in our hospital (10). Furthermore, the emergence of resistance to antimicrobial agents has complicated the treatment for CDI patients (11). Therefore, exploring the prevalence of antimicrobial-resistant C. difficile in an institution can facilitate optimizing antimicrobial stewardship programs. In this study, important information on the incidence of CDI was provided, and antibiotic resistance of C. difficile among patients in intensive care units (ICUs) wards was conducted.

Patients with diarrhea are not routinely tested for C. difficile in China, therefore, CDI might be underestimated (16–18). In our study, we actively tested patients in the ICU ward, the isolation rate of C. difficile was 21.5% (184/857), which is similar to the study by Polage et al. (19), in which 21.0% (293 out of 1,416) of hospitalized adults were tested C. difficile positive by PCR. However, another study with an immunochromatographic test reported a 4.9% positive rate among the patients with diarrhea (20), which is much lower than our results. The difference might be due to the limitations and methods of the studies, or might be related to the size of the study population. Moreover, the ICU patients in our study almost received broad-spectrum antibiotics, which might have potentially induced the emergence of CDI (21).

The positive rate (14.8%, 115/774) of C. difficile toxin genes in this study is higher than our previous study (8%, 47/593) (9). The overall incidence rate of CDI (58.0/10,000 patient days) is also higher than our previous study (14.1/10,000 patient days) (4), and the highest incidence of CDI was found in GICU patients (82.0/10,000 patient days). This might be due to the wide administration of broad-spectrum antibiotics in recent years. In our study, among these 115 patients with CDI, 35 patients used two or more antibiotics in combination. Meanwhile, 32 patients used carbapenems, 31 patients used β-lactam mixture. In addition, the higher positive rate might be related to the use of the chromogenic medium, which could better detect C. difficile. A study by 17 hospitals in South Korea found that the CDI incidence increased from 17.0/100,000 adult inpatients in 2004 to 27.0/100,000 adult inpatients in 2008 (22). Moreover, the incidence rate reported in the United States during the same period was 87.5/100,000 hospitalized patients (23). The reasons for the increased incidence rate might be related to the advanced detection approaches.

Although A+B+ (87.0%) was the dominant C. difficile toxigenic type found in this study, the prevalence of A-B+ has been reported to increase in several other regions of the world (24). We found that only 12 (10.4%) isolates produce only one toxin (A-B+CDT-), which is lower than what has been reported previously in Beijing (23.3%) (17). Interestingly, infection with toxin-positive C. difficile isolates has been associated with higher mortality and recurrence rates (25). In this study, three (2.6%) isolates of C. difficile were positive for both the two toxin genes and binary toxin gene (A+B+CDT+), which is similar to that reported by another study in Shanghai (1.6%) (26). However, the overall prevalence of A+B+ C. difficile isolates in China is much lower than that in North America and Europe (27). Global variability in the prevalence of C. difficile strains with A+B+ could be due to the differences in testing methods, patient-related factors, surveillance sensitivity, and differential infection control practices and distribution of toxigenic C. difficile isolates (9).

Based on the MLST analysis, a total of 27 C. difficile STs were identified, which were divided into four main clades. The most common four STs (ST3, ST39, ST54, and ST26) were different from our previous study (9). Our previous study on CDI among patients with hospital-acquired pneumonia showed that the predominant STs of C. difficile were ST54 (20%), ST37 (15.6%), and ST3 (9.4%) (4). This might be related to the clonal spread of C. difficile isolates in the hospital. ST3 and ST54 were the main ST types in C. difficile isolates with tcdA-positive and tcdB-positive (A+B+CDT-). ST37 was the most common ST type in C. difficile isolates with only tcdB-positive (A-B+CDT-). In another study, ST54 was the most common ST type in C. difficile isolates with two toxin genes (tcdA+, tcdB+, cdtB-) (28). Meanwhile, ST37 belonged to C. difficile isolates with only tcdB-positive (A-B+CDT-) in another study in China (29). C. difficile isolates with binary (A+B+CDT+) belonging to ST5 and ST39. All the ST26 strains in this study were NTCD, which is supported by Couturier et al. (30).

Among these 115 toxigenic C. difficile isolates, 92 strains (80.0%) were resistant to at least three antibiotics, and multiple resistance to CHL, CLI, TE, AMP, LVX, and FSA (20 strains) were most common, followed by 12 strains were resistant to four antibiotics (CHL+CLI+AMP+FSA) (Figure 3). High-level resistance to AMP, CHL, CLI, and LVX was detected among the isolates with A-B+ and A+B+ in this study. The rates of CLI, TE, and AMP resistance among ST2 C. difficile isolates were lower than those in other STs. Previous data showed that resistance to CLI (8.3−100%), cephalosporins (51%), erythromycin (13–100%), and fluoroquinolones (47%) is commonly observed in C. difficile isolates within the past 15 years (2000–15) (31). Similar antibiotic resistance was also found in the C. difficile isolates investigated in this study. These data in our study suggest that antibiotic resistance of C. difficile remains prevailing. More worrisome, most of the C. difficile isolates investigated in this study showed resistance to multiple antibiotics.

Although several studies have reported the increasing MICs for MTZ and vancomycin in C. difficile, all isolates in the current study were susceptible to these two antibiotics. Both MTZ and vancomycin are the most commonly used antibiotics for mild-to-moderate and severe CDI (32), and they have been used for more than 30 years. In a multicenter study conducted in Taiwan, all C. difficile isolates were susceptible to MTZ, however, two isolates had reduced susceptibility to vancomycin (MIC = 4 μg/ml) (33). Furthermore, the MICs of tigecycline and fidaxomicin were low in our study. Fidaxomicin is associated with a significantly lower recurrence rate in CDI therapy, therefore, it is considered to have similar therapeutic efficacy as oral vancomycin (8, 12). In addition to vancomycin, MTZ, and fidaxomicin, tigecycline has been used in cases where severe adverse effects occurred following standard therapy (34). Although C. difficile isolates are resistant to multiple antibiotics in our study, antimicrobial therapy is still the first choice for CDI, and specific guideline recommendations of antimicrobial therapy should be based on the severity of the CDI.

In conclusion, C. difficile isolates from CDI patients in our hospital are dominated by those producing two toxins (A+B+CDT-). ST3 isolates are the most prevalent ST, and ST26 isolates are all NTCD. The higher positive rate of C. difficile might be due to the wide administration of broad-spectrum antibiotics in recent years. Although multidrug resistance is present in ≥50% of the C. difficile isolates, MTZ and vancomycin are still effective against C. difficile, serving as available treatment options for CDI patients.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Conceptualization and methodology by XM, XH, and AW. Software and formal analysis by ZP and CF. Data collection by CZ, YW, SL, and JD. Writing-original draft preparation by XM. Writing-review and editing by XM, CL, and AW. Funding acquisition by CL and AW. All authors have read and agreed to the published version of the manuscript.

This work was supported by the Key Research and Development Projects of Hunan Province (Nos. 2020SK3027 and 2020SK3028) and Natural Science funding of Hunan Province (No. 2021JJ31071).

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.

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