Antibiotic-associated diarrhea, particularly in the hospital setting, is frequently a result of infection with the toxigenic Gram-positive bacterium Clostridioides difficile. Neutrophil-predominant leukocytosis is a hallmark of C. difficile infection (CDI) and is predictive of clinical outcomes [1,2]. As revealed by animal models, rapid neutrophil infiltration of the colon occurs in response to toxin -mediated epithelial barrier disruption and consequent mucosal invasion by colonic microbiota [3,4]. Neutrophils play a primary role in defense against infection but are also responsible for aggravating mucosal damage with release of reactive oxygen radicals, NETs, and chemokines and cytokines that attract additional proinflammatory cells [5]. CDI also promotes neutrophil activation and release of granule contents in the infected gut as evidenced by the increased levels of granule components calprotectin and lipocalin-2 in stool of CDI patients [[6], [7], [8]].

Activation and degranulation is typically accompanied by concomitant changes in neutrophil surface marker staining and gene expression [5,[9], [10], [11]]. Infectious stimuli and other stressors can also promote emergency granulopoiesis with release of immature neutrophils from bone marrow into circulation [12]. Being highly reactive, neutrophils are typically studied in fresh samples and are not considered suitable for storage for analysis at later times. We here have explored flow cytometry with fixed blood cells to capture peripheral blood neutrophil status in CDI patients at diagnosis, allowing us to identify CD10− neutrophils as a characteristic of early acute CDI. We also identified, in parallel, an enrichment of neutrophil degranulation markers in plasma that predict disease severity and mortality.