Affiliation:
1. Kliniken der Stadt Köln gGmbH, Klinikum der Privaten Universität Witten-Herdecke mit Sitz in Köln, Institut für Pathologie, Cologne, Germany
Abstract
ABSTRACT
Although
Pneumocystis jirovecii
is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether
P. jirovecii
could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for
P. jirovecii
using PCR. Five days later, the cells and basal medium were harvested and tested for
P. jirovecii
using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated
P. jirovecii
from these
P. jirovecii
-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that
P. jirovecii
induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further
in vitro
studies of
P. jirovecii
, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants.
IMPORTANCE
This is the first report of the successful productive cultivation and propagation of
Pneumocystis jirovecii
, a human-pathogenic fungus of major clinical significance. These findings are groundbreaking because they will influence the field of diagnostic microbiology, facilitate the testing of antibiotics against
P. jirovecii
, and enable stability studies of this pathogen when exposed to the environmental factors and chemicals that hospitals are required to use for disinfection. Because productively culturing
P. jirovecii
has been attempted unsuccessfully for several decades, this study represents a breakthrough in this field.
Publisher
American Society for Microbiology
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