Longitudinal surveillance of antibiotic resistance and virulence evolution in Clostridioides difficile : a 4-year retrospective study of hospitalized patients in a tertiary hospital in China

Articles
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Junjie Lao, Li Zhang, Xinghan Huang, Guangzhi Du, Wenjie Yang, et al. (2026).
Longitudinal surveillance of antibiotic resistance and virulence evolution in Clostridioides difficile : a 4-year retrospective study of hospitalized patients in a tertiary hospital in China.
Microbiology Spectrum. 10.1128/spectrum.00494-26

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Longitudinal surveillance of C. difficile resistance and virulence in a Chinese tertiary hospital (2021–2024)

Vancomycin and metronidazole remain effective against C. difficile in this Chinese cohort, but MICs are creeping upward year over year and toxin-gene carriage is rising — a warning signal worth tracking despite no frank resistance yet.

Background

Clostridioides difficile is the leading cause of nosocomial infectious diarrhea and pseudomembranous colitis. Chinese treatment guidelines rely on vancomycin (VA) and metronidazole (MTZ) as first-line therapy, but sporadic resistance to both has been reported globally. Long-term surveillance data from Chinese hospitals are sparse.

The authors asked: over a 4-year window in a single tertiary hospital, how have VA/MTZ susceptibility and virulence-gene carriage in C. difficile changed, and is there evidence of the hypervirulent RT027 lineage or nosocomial outbreak spread?

Methods

  • Setting/samples: Random stratified sampling of 114 fecal samples from inpatients at Affiliated Hangzhou First People’s Hospital (2021–2024).
  • Workflow: Antigen/toxin protein detection → strain isolation and identification → antimicrobial susceptibility testing (MIC for VA and MTZ) → whole-genome sequencing.
  • Bioinformatics: Resistance-gene and toxin-gene profiling; pan-genome–based phylogenetic tree construction to assess clonal spread and RT027 emergence.
  • Statistics: Kruskal–Wallis (H) across years for MIC differences; Spearman correlation between calendar year and MIC; χ² for proportion of toxigenic strains.

Key findings

Susceptibility trends (no resistance, but drifting up):

  • No VA- or MTZ-resistant isolates were identified over 4 years.
  • Mean VA MIC increased significantly year-on-year (H = 33.208, P < 0.05).
  • Mean MTZ MIC peaked in 2022 and declined in 2023–2024 (H = 41.990, P < 0.05), but one 2024 isolate reached the resistance breakpoint (MIC = 2.00 μg/mL).
  • Year of isolation was positively correlated with MIC for both drugs: VA r = 0.528 (P < 0.05); MTZ r = 0.377 (P < 0.05).

Virulence trends:

  • The proportion of toxin-producing strains rose each year, reaching 100% in 2024 (χ² = 11.75, P < 0.05).

Epidemiology:

  • No hospital outbreaks were detected via phylogenetic analysis.
  • (RT027 monitoring was a stated objective; the abstract does not report positive detection.)

Limitations and open questions

  • Single-center, retrospective design with only 114 samples over 4 years limits generalizability and statistical power for rare resistance events.
  • The positive year–MIC correlations are modest (especially MTZ, r = 0.377) and MTZ trends were non-monotonic, complicating interpretation of a true directional drift.
  • One isolate at the MTZ resistance breakpoint warrants confirmation and mechanistic follow-up (resistance-gene correlates were profiled but not detailed in the abstract).
  • Whether rising toxigenic carriage reflects true strain evolution, sampling shifts, or changes in antibiotic pressure in the hospital remains open.
Original abstract

ABSTRACT Clostridioides difficile ( C. difficile ) is the primary pathogen responsible for nosocomial infectious diarrhea and pseudomembranous colitis. In China, metronidazole and vancomycin are the preferred treatments for C. difficile infection (CDI). This study aimed to investigate the evolution of vancomycin (VA) and metronidazole (MTZ) resistance, as well as the longitudinal changes in virulence over time, using next-generation sequencing, drug susceptibility tests, and analysis of resistance and virulence genes. Additionally, we monitored the emergence of the highly virulent C. difficile strain RT027 and the spread and potential outbreak of C. difficile in the hospital setting. A random stratified sampling method was used to select 114 fecal samples from inpatients at Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, between 2021 and 2024. Clinical data from the enrolled patients were also collected. We conducted antigen and toxin protein detection for C. difficile , strain isolation and identification, drug sensitivity tests, whole genome sequencing, and bioinformatics analysis. This included comparisons of drug resistance genes, detection of toxin genes, and the construction of phylogenetic trees based on pan-genome analysis to investigate the resistance and toxin gene variations in C. difficile . Among the 114 samples collected from Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, no vancomycin- or metronidazole-resistant strains were identified. However, the average minimum inhibitory concentration (MIC) of C. difficile to vancomycin increased annually ( H = 33.208, P < 0.05). The average MIC of C. difficile to metronidazole was highest in 2022 but decreased in 2023 and 2024 ( H = 41.990, P < 0.05). Notably, in 2024, one C. difficile strain exhibited an MIC for metronidazole at the resistance threshold (2.00 μg/mL). Further Spearman correlation analysis of the strain years with drug sensitivity results revealed a positive correlation between strain years and the MIC levels of vancomycin and metronidazole ( r = 0.528, P < 0.05; r = 0.377, P < 0.05). The proportion of toxin-producing strains increased annually, with 100% of strains in 2024 producing toxins, representing the highest proportion compared to the previous three years ( X ² =11.75, P < 0.05). Both vancomycin and metronidazole remain effective for the treatment of CDI in clinical practice. However, the sensitivity of C. difficile to these two drugs is gradually decreasing, and the rate of toxin gene carriage is also rising in clinical cases. No hospital outbreaks of C. difficile infections were identified in this study. IMPORTANCE Clostridioides difficile has developed resistance to multiple antibiotics, including cephalosporins, clindamycin, and fluoroquinolones. This has exacerbated the global antibiotic resistance crisis. In China, according to current treatment guidelines, vancomycin and metronidazole are the preferred first-line drugs for treating C. difficile infections. However, there are reports indicating the emergence of new resistance to both vancomycin and metronidazole. Although there is extensive research on the long-term antibiotic resistance of C. difficile abroad, research on the continuous monitoring of antibiotic resistance and potential outbreaks of C. difficile in China is relatively limited. To fill this gap, we studied positive C. difficile strains from a tertiary general hospital in China. Through Next-Generation Sequencing (NGS), drug sensitivity testing, and analysis of drug resistance and virulence genes, we revealed the evolution of C. difficile’s resistance to vancomycin and metronidazole, as well as changes in virulence, and monitored the spread within the hospital and potential outbreaks of C. difficile .

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