Evaluating microbial viability differently: impedance cytometry versus culture

Introduction
Developing analytical methods for the quality control of fecal transplants (FTs) is a major challenge for preparation units. A previous study demonstrated that it is possible to estimate the total microbial load and viability in FTs using flow cytometry. However, this technology is still costly and complex to implement. Impedance flow cytometry (IFC) is a simpler and more economical alternative. It counts and analyses cells by measuring variations in electrical current as they pass individually through an electromagnetic field. This study aims to compare the results obtained by IFC with those of the reference method (culture), in order to assess its relevance for FTs control.

Materials and methods
Analyses were performed on fresh or frozen stool samples from healthy donors (HD), validated or not, and diarrhoeic stool samples from patients (DD), using the IFC method and culture. First, the samples were homogenised in a 0.9% NaCl solution and then vortexed in presence of glass beads to break up particles. After filtration, fecal suspension was diluted in PBS to obtain a concentration between 10³ and 5 x 10⁶ particles/mL, which is necessary for IFC measurement. Each sample was analysed using IFC, rediluted and then seeded onto Columbia agar containing 5% sheep blood (Bio-Rad). The agar was then incubated aerobically for 48 hours and anaerobically for five days. The results obtained by the two methods were statistically compared using a Student’s t-test (α = 5%).

Results
The analysis covered 20 samples, 10 of which were from HD group and 10 from DD group. Of these, 15 were fresh stools (including 10 from the DD group), two were frozen and two were thawed frozen (TF). Statistical comparison showed no significant difference between the two counting methods (8.6 ± 1.6 log/mL by IFC versus 8.06 ± 1.3 log/mL by culture; p-value = 0.175). The average cell viability measured by IFC was 31.6% ± 18.5%. Samples from HD showed significantly higher average viability (41.8% ± 10.9%) than samples from DD (21.5% ± 19.4%). Moreover, there was a significant difference in viability between fresh and frozen samples from HD group (48.6% vs. 34.9%).

Discussion
IFC appears to be a quick and easy method of estimating total bacterial concentration and viability without the need for specific markers. Our results demonstrate a high degree of concordance between quantifications obtained by IFC and by culture, confirming the robustness of the method. However, unlike other fecal microbiota analysis techniques, IFC cannot identify bacterial species, limiting the scope of the results.

Key words : fecal transplant – microbiological control – impedance cytometry