Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA.
Biomedical Engineering, University of Virginia, Charlottesville, VA, 22904, USA.
Biosens Bioelectron. 2020 Oct 15;166:112440. doi: 10.1016/j.bios.2020.112440. Epub 2020 Jul 21.
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.
摄入孢子的萌发通常是使细菌生长和宿主定植成为可能的必要第一步，例如艰难梭菌（C. difficile）感染。结肠中孢子的萌发率取决于微生物群落的组成及其因抗生素治疗而受到的破坏程度，因为共生菌的分泌物调节初级至次级胆盐水平以控制萌发。评估艰难梭菌孢子的萌发通常需要测量菌落形成单位，这是一项劳动密集型工作，至少需要 24 小时才能完成，但由于医院获得性抗生素相关腹泻的高复发率，因此经常需要进行。我们提出了一种快速评估孢子萌发的方法，该方法使用高通量单细胞阻抗细胞术（>300 个事件/秒）来定量活细菌细胞，通过对其特征电生理学与孢子进行门控，以便在培养 4 小时后即可评估萌发，检测限为每 50 μL 样本约 100 个活细胞。为了检测萌发的艰难梭菌细菌的表型，我们利用其与孢子聚集体和非存活艰难梭菌形式相比特征性更高的净电导率，这会导致在中等导电性介质（0.8 S/m）中产生独特的高频（10 MHz）阻抗相位分散。通过这种方式，我们可以在 4 小时内检测到体外初级胆盐水平增加和使用抗生素处理的小鼠模型中的粪便微生物群样本中孢子萌发率的显著差异，以评估对艰难梭菌感染的易感性。我们设想了一种快速诊断工具，用于评估宿主微生物群对关键抗生素治疗后细菌定植的敏感性。
