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Detection of Staphylococci in mouse phagocytic cells by in situ hybridization using biotinylated DNA probes
A Matsuhisa1, Y Saito, H Ueyama
1Department of Microbiology (I), Jikei University School of Medicine, Tokyo, Japan.
Abstract:
In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.
Insights
This study introduces a novel in situ hybridization method to detect intracellular Staphylococcus bacteria within mouse phagocytic cells. This technique accurately identifies Staphylococcus aureus and Staphylococcus epidermidis, regardless of their viability, aiding in bacterial infection diagnosis.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Intracellular bacteria pose diagnostic challenges due to their location within host cells.
- Accurate detection of Staphylococcus species is crucial for effective treatment of infections.
Purpose of the Study:
- To develop and validate a sensitive method for detecting intracellular Staphylococcus aureus and Staphylococcus epidermidis.
- To assess the utility of in situ hybridization in identifying these bacteria within phagocytic cells.
Main Methods:
- Experimental infection of C3H mice with Staphylococcus species via abdominal or intravenous routes.
- Phagocyte smear technique using bacteriolytic enzymes to preserve cell morphology.
- In situ hybridization utilizing strain-specific biotinylated DNA probes and immunocytochemistry for signal detection.
Main Results:
- Intracellular Staphylococcus aureus and S. epidermidis were successfully visualized within mouse phagocytic cells.
- The DNA probes demonstrated high strain specificity, with no cross-hybridization observed.
- The technique proved effective in detecting bacteria regardless of their viability.
Conclusions:
- In situ hybridization with phagocyte smears is a reliable method for detecting intracellular Staphylococcus species.
- This technique offers a valuable diagnostic tool for intracellular bacterial infections.
- The method's ability to detect non-viable bacteria expands its diagnostic applicability.