# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 2 Identification of Resistant and Viable Spores
In a recent study [57], researchers compared the differences between vegetative cells and spore counts as well as changes in the biomass of biofilms after exposure to disinfectants using optical density measurements, inverted light, and confocal laser scanning microscopy. In evaluating the effectiveness of hospital-based disinfectants on _C. difficile_ embedded in biofilms, it was found that no disinfectant could eliminate _C. difficile_. However, generic disinfectants, Clorox, OPA, and Virex, were the most effective at killing _C. difficile_ spores, irrespective of the age, ribotype, or wash condition. The germination of _C. difficile_ spores into vegetative form within the mammalian host (Figure 1, stage III) encompasses the major cellular structure, machinery, and morphology alterations, resulting in significant changes to the electrophysiological state of the spore. Assessment of _C. difficile_ spore germination usually involves measuring colony-forming units (CFUs), which is labor-intensive and takes at least 24 h but is necessary due to high recurrence rates of nosocomial antibiotic-associated diarrhea [58]. The structural transition (changes in interior conductivity and membrane resistance) from spores to the vegetative form can be monitored via dielectrophoresis (DEP). Dielectrophoresis involves the movement of polarized cells within a spatially non-uniform electric field and has been used to measure differences in bacteria based on cell walls [59]. Recently, a rapid method using high-throughput single-cell impedance cytometry (>300 events/s) was developed to quantify live bacterial cells by distinguishing their electrophysiology from spores in vivo (mouse model) and in vitro. This method allowed germination assessment after just 4 h of culture, with a detection limit of \(\sim\)100 live cells per 50 \(\mu\)L sample [60]. _C. difficile_ strains with increased sporulation are associated with increased transmission rates [42]. Methods quantifying sporulation characteristics vary greatly, relying on small sample sizes preventing standardizations [58]. [PERSON] et al. estimated that the sample size should employ at least seven strains of a given type (BI/NAP1/027), suggesting there is a lack of evidence to support the claim that epidemic _C. difficile_ types form spores with greater efficiency. In an effort to improve the consistency of further studies, the authors presented an experimental design capable of independently assessing four factors (vegetative cell growth rate, sporulation rate, total sporulation, and persistent vegetative cells) via a combination of methodologies. Such an approach was employed in the assessment of octahedron iron oxide nanocrystals to inhibit the germination and viability of two strains of _C. difficile_ (CCUG 37780 (tcdA\(-\), tcdB\(-\)) and CCUG 19126 (tcdA\(+\), tcdB\(+\))) in a mouse model [61]. The nanoparticle inhibited germination in a size/dose-dependent manner, although there was some strain variation. Coupled with the superior bioidal (comparable to bleach) activity, the authors suggested that the nanocrystals block the germinal receptors, altering the surface properties (change hydrophobicity) and thus preventing germination, which is a similar theory posited by similar authors in another report [62]. When the surface of a spore is defective (absence of exosporium, slow CaDPA release or mutation), established techniques may be insufficient; in such circumstances, an automated time-lapse microscopy-based germination assay may be better suited. In a recent study, a time-lapse microscopy (TLM) pipeline was used to observe morphological changes and stages of germination in real time using mutant spores [63]. Researchers concluded that Ca\({}^{2+}\) is not essential for initiating spore germination and that CaDPA can enhance the germination of nearby spores via a feedforward loop.