Toxin A-negative, toxin B-positive (A−B+) Clostridioides difficile has been found worldwide, particularly in Asia [[1], [2], [3], [4], [5], [6], [7], [8]]. Most clinical A−B+ isolates are negative for the binary toxin (CDT), belong to clade 4, and have been reported as PCR ribotypes (RTs) 017, 369, or 368 in Japan [1,2,5,9]. Among epidemic A−B+CDT− isolates in Japan, RT017 has been replaced by RT369, which was reported to cause outbreaks in Japanese healthcare facilities since the early 2000s [1,2,5,[10], [11], [12]]. The RT369 isolates from Japan are resistant to other antimicrobial agents such as fluoroquinolones [1,2,10] in addition to the previously high resistance to clindamycin of RT017 [3,4,7,13].

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been widely used for rapidly and accurately identifying microorganisms, such as C. difficile. MALDI-TOF MS has been reported to be capable of identifying specific C. difficile RTs, such as C. difficile RTs 027, 176, 078, and 001 [[14], [15], [16]]. A report from China documented the identification of multilocus sequence type (ST) 37 by MALDI-TOF MS, which mainly corresponded to RT017 [17].

We report the use of MALDI-TOF MS for identifying RT017-lineage C. difficile, such as RT369, a clinically important RT in Japan. Furthermore, this technique was assessed as an additional tool to be implemented concurrently with C. difficile identification in clinical laboratories.