Quantifying bacterial spore germination by single-cell impedance cytometry for assessment of host microbiota susceptibility to Clostridioides difficile infection.

The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 h to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 h of culture at a detection limit of ~100 live cells per 50 μL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 h, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.