Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

In vitro bioassays to monitor complex chemical mixtures at a carbon-based indirect potable reuse plant.

Potable water reuse technologies are used to treat wastewater to drinking water quality to help sustain a community's water resources. California has long led the adoption of potable water reuse technologies in the United States and more states are exploring these technologies as water resources decline. Reuse technologies also need to achieve adequate reductions in microbial and chemical contaminant risks to meet public health goals and secure public acceptance. In vitro bioassays are a useful tool for screening if reuse treatment processes adequately reduce toxicity associated with a range of chemical classes that are contaminants of concern. In this study, we used an aryl hydrocarbon receptor (AhR) and an estrogen receptor luciferase bioassay to detect the presence of dioxin-like and estrogenic compounds across a 3800 m3/d carbon-based indirect potable reuse plant that uses carbon-based treatment (SWIFT-RC). Our results demonstrate significant removal of dioxin-like compounds across the SWIFT-RC treatment train. Estrogenicity declined across the treatment train for some months but was extremely variable and low with many samples falling below the method quantification level; consequently, we were not able to reliably determine estrogenicity trends for SWIFT-RC. Comparing the bioanalytical equivalent concentrations detected in the SWIFT-RC water with established monitoring trigger levels from the state of California suggests that SWIFT-RC produced water that met the bioassay guidelines. The log total organic carbon concentration and AhR assay equivalent concentrations are weakly correlated when data across all SWIFT-RC processes are included. Overall, this research demonstrates the performance of in vitro bioassays at a demonstration-scale carbon-based IPR system and highlights both the potential utility and challenges associated with these methods for assessing system performance.