Conditioning chemotherapy exposure is associated with epigenetic modifications in <i>Clostridioides difficile</i> isolates from stem cell transplant recipients

<i>Clostridioides difficile</i> is a highly methylated organism within the gut microbiome that is responsible for <i>Clostridioides difficile</i> infection (CDI), a common disease that is mediated by toxins production from the bacterium. <i>C. difficile</i> infection is ten times more common in chemotherapy patients than the average patient, but the reasons for this disparity are unclear. Conditioning chemotherapy (CC), an integral part of cancer treatments, has the ability to induce methylation changes in many cell types. We posit that CC induces methylation changes within <i>C. difficile</i> that may promote toxin production and consequently CDI. To test our hypothesis, we sought to identify the epigenetic changes, particularly methylation changes, within <i>C. difficile</i> isolates before and after chemotherapy and within isolates that express toxin and isolates that do not. After stool sampling, we isolated <i>C. difficile</i> by culture then sequenced and created a hybrid assembly of each isolate using nanopore long read sequencing and Illumina short read sequencing. Bioinformatics tools such as Dorado and Samtools were used to basecall and determine methylation states, while Unicycler was used for genome assembly. Methylartist was then used for data visualization. Genome-wide methylation profiling revealed distinct epigenetic signatures in <i>Clostridioides difficile</i> associated with toxin expression and chemotherapy exposure. Whole-genome 6mA analysis demonstrated significant differences between toxin-positive and toxin-negative isolates, with prominent methylation changes in <i>tcdA</i> and <i>tcdE</i>, while selected sporulation genes were unmethylated in toxin-negative strains. Chemotherapy was associated with a significant shift in global 6mA methylation patterns. Targeted 5mC analysis of the pathogenicity locus revealed reduced methylation around <i>tcdB</i> and across multiple toxin genes following chemotherapy, whereas sporulation genes remained unaffected. These findings indicate chemotherapy-associated epigenetic remodeling of toxin-associated loci in <i>C. difficile</i>.