ABSTRACTClostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk ofC. difficileinfection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027C. difficileclinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 μg/mL, compared to the EUCAST breakpoint of 2 μg/mL) and contained avanRCd343A>G mutation resulting in a Thr115Ala substitution in the VanRCdresponse regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 μg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additionalC. difficileisolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributedC. difficileribotypes worldwide. Considering that the VanRCdThr115Ala mutation was also independently reported in sevenC. difficileclinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance.IMPORTANCEThe perpetually evolving antimicrobial resistance (AMR) ofC. difficileis an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanRCd343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates ofC. difficileobtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets.