| Summary: | <i>Pneumocystis jirovecii</i>, a fungus causing severe <i>Pneumocystis</i> pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with <i>P. jirovecii</i> and a small number of experiments involving animal-derived <i>Pneumocystis</i> species have been published to date. However, <i>P. jirovecii</i> culture conditions may differ significantly from those of animal-derived <i>Pneumocystis</i>, as there are major genotypic and phenotypic differences between them. Establishing a well-performing <i>P. jirovecii</i> cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for <i>Pneumocystis jirovecii</i>. To identify promising approaches for cultivation, a literature survey encompassing animal-derived <i>Pneumocystis</i> cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for <i>P. jirovecii</i> culture. This allowed us to develop a medium that produced a 42.6-fold increase in <i>P. jirovecii</i> qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing <i>Pneumocystis</i> clusters in the final medium, DMEM-O3. <i>P. jirovecii</i> doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated <i>P. jirovecii</i> under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.
|
|---|