Abstract
Chlamydia trachomatis is an obligate intracellular pathogenic bacterium with a biphasic developmental cycle manifesting two distinct morphological forms: infectious elementary bodies (EBs) and replicative intracellular reticulate bodies (RBs). Current standard protocols for quantification of the isolates assess infectious particles by titering inclusion-forming units, using permissive cell lines, and analyzing via immunofluorescence. Enumeration of total particle counts is achieved by counting labeled EBs/RBs using a fluorescence microscope. Both methods are time-consuming with a high risk of observer bias. For a better assessment of C. trachomatis preparations, we developed a simple and time-saving flow cytometry-based workflow for quantifying small particles, such as EBs with a size of 300 nm. This included optimization of gain and threshold settings with the addition of a neutral density filter for small-particle discrimination. The nucleic acid dye SYBR® Green I (SGI) was used together with propidium iodide and 5(6)-carboxyfluorescein diacetate to enumerate and discriminate between live and dead bacteria. We found no significant differences between the direct particle count of SGI-stained C. trachomatis preparations measured by microscopy or flow cytometry (p > 0.05). Furthermore, we completed our results by introducing a cell culture-independent viability assay. Our measurements showed very good reproducibility and comparability to the existing state-of-the-art methods, indicating that the evaluation of C. trachomatis preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of C. trachomatis preparations for downstream applications.
Highlights
The infection caused by C. trachomatis manifests a broad spectrum of distinct clinical symptoms, which are conditioned by a strict tissue tropism determined by different serovars
The ultrastructural analysis of preparations showed the presence of elementary bodies (EBs) characterized by small size (∼300 nm diameter) and highly condensed chromatin, as well as larger reticulate bodies (RBs) with relaxed chromatin (Figure 1)
Because the EB-enriched fraction fraction collected collected at atthe the44/54%
Summary
Chlamydia trachomatis remains the most commonly reported sexually transmitted bacterial pathogen in the world [1]. The WHO estimated that there were 131 million new cases of chlamydial infection globally among adults in 2012 [2]. In the United States alone, where prevention programs benefit from strong data provided by the Centers for Disease Control and Prevention, nearly two million cases have been reported annually, with an increasing rate since the year 2000 [3]. The infection caused by C. trachomatis manifests a broad spectrum of distinct clinical symptoms, which are conditioned by a strict tissue tropism determined by different serovars. Serovars D to K represent the most prevalent sexually transmitted organism in high-income countries [5]. Serovars L1 to L3 cause invasive urogenital and anorectal infections with an increasing incidence among
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