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Chlamydia pneumoniae in vitro and in vivo: a critical evaluation of in situ detection methods
A Meijer1, P J Roholl, S K Gielis-Proper
1Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands. Adam.Meijer@rivm.nl
Aims:
There is a considerable discrepancy between data from the detection of Chlamydia pneumoniae in atherosclerotic lesions obtained by means of immunocytochemistry and data obtained using the polymerase chain reaction. This study evaluated methods for the in situ detection and assessment of the viability of C pneumoniae bacteria.
Methods:
Chlamydia pneumoniae membrane protein, heat shock protein 60, and lipopolysaccharide were detected by immunocytochemistry, and genomic DNA and 16S rRNA by in situ hybridisation in paraffin wax embedded sections of cultured HEp2 cells infected with C pneumoniae and of lungs from mice infected intranasally with C pneumoniae.
Results:
Inclusions reactive for all three antigens, DNA, and 16S rRNA were seen in infected HEp2 cells, in all positive bronchus and alveolar epithelial cells, and in some of the positive infiltrate cells in the lungs of mice up to seven days after infection. In all alveolar macrophages and in the infiltrate cells positive for antigens only, the staining pattern was granularly dispersed throughout the cytoplasm up to seven days after infection. At 21 days after infection, only this granular staining pattern was seen for antigens in infiltrate cells and macrophages in the alveoli and bronchus associated lymphoid tissue. At this time point, DNA or 16S rRNA were detected sporadically, but always as inclusion-like staining.
Conclusions:
Because antigens with an inclusion-like staining were detected only together with DNA and 16S rRNA, this type of staining pattern suggested the presence of viable bacteria. Thus, the granular staining pattern of antigens in the absence of staining for DNA and 16S is most likely caused by non-viable bacteria. In conclusion, these methods are suitable for the in situ detection of C pneumoniae and the assessment of its viability.
Insights
This study developed methods to detect Chlamydia pneumoniae in situ and assess bacterial viability. Inclusion-like staining indicates viable bacteria, while granular staining suggests non-viable Chlamydia pneumoniae.
Area of Science:
- Microbiology
- Pathology
- Immunology
Background:
- Discrepancies exist in detecting Chlamydia pneumoniae in atherosclerotic lesions using immunocytochemistry versus PCR.
- Accurate detection and viability assessment of C. pneumoniae are crucial for understanding its role in disease.
Purpose of the Study:
- To evaluate methods for in situ detection of Chlamydia pneumoniae.
- To assess the viability of C. pneumoniae within infected cells and tissues.
Main Methods:
- Immunocytochemistry was used to detect C. pneumoniae antigens (membrane protein, HSP60, LPS).
- In situ hybridization detected genomic DNA and 16S rRNA.
- Experiments were conducted on infected HEp2 cells and mouse lungs.
Main Results:
- Inclusions reactive for antigens, DNA, and 16S rRNA indicated viable C. pneumoniae in HEp2 cells and mouse lungs up to 7 days post-infection.
- Granular antigen staining alone, without DNA/16S rRNA detection, was observed in macrophages and infiltrate cells at 21 days, suggesting non-viable bacteria.
- Sporadic DNA/16S rRNA detection with inclusion-like staining occurred at 21 days.
Conclusions:
- Inclusion-like staining for antigens, DNA, and 16S rRNA is indicative of viable Chlamydia pneumoniae.
- Granular antigen staining without nucleic acid detection suggests non-viable bacteria.
- The developed methods are suitable for in situ detection and viability assessment of C. pneumoniae.
