Antimicrobial resistance (AMR) is one of the biggest threats to public health. Without a focused global effort, it may cause ten million deaths per year by 2050 [1]. In this context, Clostridioides difficile (prev. Clostridium difficile) is a major threat to healthcare and a relevant AMR genes reservoir [2,3]. Moreover, recent studies have suggested that C. difficile is a potential zoonotic pathogen, highlighting the importance of “One Health” approach for its control [4,5].

AMR plays a significant role in CDI pathogenesis and spread [6,7], as antimicrobial treatment is the main risk factor for CDI development, which facilitates the development of multidrug-resistant C. difficile isolates [7]. Simultaneously, AMR seems to be a strong factor driving the emergence of new C. difficile strains, including the so-called epidemic isolates [[7], [8], [9]]. The most well-known example is the spread of the C. difficile ribotype (RT) 027, which caused outbreaks in several countries and was linked to the increased fluoroquinolone resistance [10,11]. In addition to RT027, several other AMR strains have been associated with outbreaks worldwide, including those resistant to lincosamides and rifampicin [6,12,13]. Other recent studies have suggested that intensive tetracycline use in farm animals might be linked to RT078 spread and increased community-acquired CDI rate, including in non-elderly patients [9].

Despite the known importance of AMR in C. difficile epidemiology and treatment, antimicrobial susceptibility testing for this pathogen is still based on the determination of the minimal inhibitory concentration (MIC) by the agar dilution method, which is technically demanding and labor-intensive [14]. Some studies have tested the disk diffusion method for this purpose, which is an inexpensive and simple technique that can be easily performed in a routine laboratory [14,15]. Studies comparing MIC results with the disk diffusion method are limited mostly to moxifloxacin, metronidazole, and vancomycin [[14], [15], [16], [17]]. Till date, very few studies have focused on other antimicrobials relevant in CDI pathogenesis and epidemiology. Thus, this study aimed to evaluate disk diffusion for C. difficile susceptibility testing by comparing disk diffusion results with the MICs for erythromycin, rifampicin, and tetracycline.