Culturomic and metagenomic methods optimization to detect multi-strain C. difficile colonization and infection - ABSTRACT Clostridioides difficile (Cd) infection (CDI) is a leading cause of healthcare-associated infections in the U.S., affecting ~450,000 people and costing ~$5.4B annually. Despite substantial advances in investigating Cd pathogenesis and transmission over the past 20 years, CDI remains a significant public health burden. Recent studies suggest multi-strain Cd infection is more common than previously recognized and may cause worse outcomes versus single strain infection. Another recent development is recognition of cryptic clade Cd strains that can cause CDI but may not be detected with current culture methods or diagnostic assays. A major impediment to studying multi-strain and cryptic clade Cd colonization and CDI is a lack of validated culture methods to optimize recovery of more multiple Cd strains or cryptic clade Cd strains. Typically, a single approach is used to isolate Cd from stool. However, growth of Cd in culture varies by strain, and is impacted by spore shock method (i.e. ethanol or heat shock), spore germinant (i.e. primary bile salt or lysozyme), and nutrients and selective antibiotics incorporated into the media. Another challenge to detecting multi-strain colonization and CDI is the need to type multiple colonies to detect the presence of more than one strain, which can significantly increase time and costs. In addition, the probability of detecting multiple strains will be dependent on the number of strains typed. We hypothesize that a combination of Cd culture conditions employed on the same stool specimen will enhance recovery of multiple and cryptic clade strains of Cd, and that metagenomic approaches can be used to detect multiple strains from culture and eliminate the need to type multiple colonies. The rationale for this proposal stems from the need for validated methods to detect the presence of multiple and cryptic clade strains prior to widespread study of multi-strain and cryptic clade strain Cd colonization and infection. Our central motivation is that whether multi-strain or cryptic clade strain Cd colonization and infection cause worse outcomes needs to be confirmed, as this will have a major impact in our understanding and approaches to Cd pathogenesis, diagnosis, treatment and prevention. We propose to develop methods to optimize detection of multi-strain and cryptic clade strain Cd colonization and CDI with the following aims: 1.1) Identify the combination of growth conditions and selective pressures to optimize recovery of multiple and cryptic clade strains of C. difficile from stool specimens that could be broadly employed to study C. difficile infection epidemiology, diagnosis, treatment, outcomes, and prevention., and 1.2) Identify the presence and relative abundance of multiple strains of C. difficile through culture-enriched metagenomic sequencing. Our proposal is significant because validated methods to detect multi-strain and cryptic clade strain Cd colonization and CDI is a necessary first step to study the prevalence and outcomes of multi-strain and cryptic clade strain Cd colonization and CDI. Our proposal is innovative in that it will change the current paradigm of Cd isolation and characterization from stool. The proposed work is impactful in its goal to develop methods that can be rapidly utilized by multiple investigators.
