Culture-independent strain typing of Clostridioides difficile in fecal samples

Abstract Clostridioides difficile ( C. difficile ), the leading cause of healthcare-associated diarrhea, is a spore-forming obligate anaerobe that colonizes the intestinal tract and causes colitis in patients with compromised microbiome-mediated defenses. C. difficile is genomically diverse and is subtyped into ribotypes (RTs) and sequence types (STs) that vary in virulence and epidemic potential. Since secretion of the TcdB exotoxin is common to nearly all disease-associated C. difficile strains, PCR detection of TcdB has become a key clinical test used to diagnose C. difficile infection (CDI). This test and other available clinical laboratory tests, however, do not determine the prevalence of different C. difficile STs, making it impossible to identify transmission events or outbreaks in healthcare settings. Strain typing, because it requires technically demanding and time-consuming anaerobic fecal culturing of C. difficile , is not routinely performed, leaving healthcare epidemiologists unable to distinguish sporadic from epidemic occurrences of CDI. Herein, we optimized the established strain-level profiling tool, MetaPhlAn4 and StrainPhlAn, to distinguish C. difficile strains in the complex fecal microbiomes of hospitalized patients and demonstrate the utility of metagenomic short-read sequencing for culture-independent, rapid epidemiologic surveillance of CDI in healthcare environments.