Clostridioides difficile infection (CDI) is the most common healthcare acquired infection in the USA and the most common cause of death due to gastroenteritis with total healthcare costs exceeding 4.8 billion U.S. dollars/year [1]. C. difficile is a Gram-positive, spore-forming, obligate anaerobe. CDI is caused by ingestion of C. difficile spores in a susceptible host allowing for germination and secretion of two large homologous toxins, toxin A (TcdA) and toxin B (TcdB) that cause disease [2,3]. These toxins inactivate epithelial cell GTPases resulting in colonocyte death, loss of intestinal barrier integrity, neutrophilic colitis, and colonic inflammation [4,5]. Some C. difficile strains can produce a third type of potent transferase toxin called the binary toxin that enhances bacterial virulence [6].

While significant advances have been achieved in understanding the pathogenesis of C. difficile and developing therapies, it remains a major clinical challenge, especially for emerging hypervirulent and antimicrobial-resistant strains [7]. Mammalian models such as mouse, rat, and hamster are commonly used to study the pathogenesis of CDI [[8], [9], [10]]. However, simple nonmammalian models such as invertebrate animals (nematode, fruit fly, and the greater wax moth larvae) and vertebrate animal (zebrafish) have been used to study the microbial pathogenesis of CDI including C. difficile toxin expression [11], colonization [12], and screening novel therapy candidates [13,14]. However, a systematic review about the utilization of nonmammalian model to study CDI has not been done. This review focuses on four common nonmammalian models (nematode, fruit fly, greater wax moth larvae, and zebrafish) and provides a comprehensive summary of these nonmammalian models used to study CDI. Translational outcomes and strength and weakness of each nonmammalian mode are discussed.