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Identifying PCR-ribotype 017-lineage isolates of Clostridioides difficile using MALDI-TOF MS.

Yumiko Matsuyama1, Mitsutoshi Senoh2, Hiroshi Miyamoto1

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Anaerobe
|November 15, 2025
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Summary

A new rapid method using MALDI-TOF MS can identify Clostridioides difficile strains like RT369, which are toxin A-negative, B-positive, and CDT-negative. This technique offers 100% sensitivity and specificity for detecting these outbreak-causing C. difficile strains.

Keywords:
Clostridioides difficileMALDI-TOF MSPCR ribotype 017 lineagePCR ribotype 369

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Area of Science:

  • Microbiology
  • Infectious Diseases
  • Diagnostic Technology

Background:

  • The Clostridioides difficile RT017-lineage, characterized as toxin A-negative, B-positive, and binary toxin-negative (A⁻B⁺CDT⁻), is prevalent in Asia.
  • In Japan, the multidrug-resistant RT369 strain has replaced RT017 within this lineage, causing significant outbreaks.

Purpose of the Study:

  • To develop and evaluate a rapid method for identifying A⁻B⁺C. difficile strains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • To specifically target the detection of the emerging RT369 strain responsible for outbreaks in Japan.

Main Methods:

  • Analysis of 79 toxigenic Clostridioides difficile isolates, comprising 37 A⁻B⁺CDT⁻ strains and 42 isolates with other toxin profiles.
  • Utilizing MALDI-TOF MS to detect specific mass-to-charge ratio (m/z) peaks indicative of the A⁻B⁺CDT⁻ toxin profile.

Main Results:

  • Distinct peaks at m/z 3289 and 3245 were consistently detected in all 37 A⁻B⁺CDT⁻ isolates, including 22 RT369 strains.
  • No such peaks were observed in the 42 non-A⁻B⁺CDT⁻ isolates.
  • The MALDI-TOF MS method demonstrated 100% sensitivity and specificity for identifying A⁻B⁺CDT⁻ C. difficile.

Conclusions:

  • MALDI-TOF MS provides a rapid and accurate method for identifying the RT017-lineage, including the outbreak-associated RT369 strain (A⁻B⁺CDT⁻).
  • This technique can be integrated into routine laboratory identification processes for improved C. difficile surveillance.
  • Culture media variations did not impact the reliability of MALDI-TOF MS results for C. difficile identification.