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Optimization of PMA-qPCR for Staphylococcus aureus and determination of viable bacteria in indoor air
1Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan.
Abstract:
Staphylococcus aureus may cause infections in humans from mild skin disorders to lethal pneumonia. Rapid and accurate monitoring of viable S. aureus is essential to characterize human exposure. This study evaluated quantitative PCR (qPCR) with propidium monoazide (PMA) to quantify S. aureus. The results showed comparable S. aureus counts between exclusively live cells and mixtures of live/dead cells by qPCR with 1.5 or 2.3 μg/mL PMA (P>.05), illustrating the ability of PMA-qPCR to detect DNA exclusively from viable cells. Moreover, qPCR with 1.5 or 2.3 μg/mL PMA performed optimally with linearity over 103 -108 CFU/mL (R2 ≥0.9), whereas qPCR with 10, 23 or 46 μg/mL PMA significantly underestimated viable counts. Staphylococcus aureus and total viable bacteria were further determined with PMA-qPCR (1.5 μg/mL) from 48 samples from a public library and two university dormitories and four from outside. Viable bacteria averaged 1.9×104 cells/m3 , and S. aureus were detected in 22 (42%) samples with a mean of 4.4×103 cells/m3 . The number of S. aureus and viable bacteria were positively correlated (r=.61, P<.005), and percentages of S. aureus relative to viable bacteria averaged 12-44%. The results of field samples suggest that PMA-qPCR can be used to quantify viable S. aureus cells.
Insights
Propidium monoazide quantitative PCR (PMA-qPCR) accurately quantifies viable Staphylococcus aureus. This method effectively distinguishes live bacteria, crucial for monitoring human exposure to S. aureus in various environments.
Area of Science:
- Microbiology
- Molecular Biology
- Environmental Health
Background:
- Staphylococcus aureus is a significant human pathogen, causing a range of infections from skin conditions to severe pneumonia.
- Accurate enumeration of viable S. aureus is critical for understanding human exposure and infection risk.
- Existing methods may not reliably differentiate between live and dead bacterial cells, potentially skewing exposure assessments.
Purpose of the Study:
- To evaluate the efficacy of quantitative PCR (qPCR) combined with propidium monoazide (PMA) for the specific quantification of viable Staphylococcus aureus.
- To determine optimal PMA concentrations for accurate viable S. aureus detection.
- To assess the applicability of PMA-qPCR for quantifying viable S. aureus in real-world environmental samples.
Main Methods:
- Quantitative PCR (qPCR) was employed in conjunction with varying concentrations of propidium monoazide (PMA).
- Experiments involved mixtures of live and dead S. aureus cells to test PMA's ability to exclude dead cell DNA.
- The linearity and optimal range of PMA-qPCR were assessed using known concentrations of S. aureus (CFU/mL).
- Environmental samples from public spaces (library, dormitories) were analyzed for viable bacteria and S. aureus using PMA-qPCR.
Main Results:
- PMA-qPCR with 1.5 or 2.3 μg/mL PMA accurately quantified viable S. aureus in mixed cell populations (P > .05).
- Optimal linearity (R² ≥ 0.9) for PMA-qPCR was observed between 10³ -10⁸ CFU/mL with 1.5 or 2.3 μg/mL PMA.
- Higher PMA concentrations (10-46 μg/mL) significantly underestimated viable bacterial counts.
- In environmental samples, viable bacteria averaged 1.9×10⁴ cells/m³, with S. aureus detected in 42% of samples (mean 4.4×10³ cells/m³).
- A significant positive correlation (r=.61, P<.005) was found between S. aureus and total viable bacteria counts.
Conclusions:
- Propidium monoazide quantitative PCR (PMA-qPCR) is a reliable method for exclusively quantifying viable Staphylococcus aureus.
- The optimal PMA concentration for accurate detection is between 1.5 and 2.3 μg/mL.
- PMA-qPCR is suitable for assessing viable S. aureus contamination in environmental settings, providing valuable data for public health monitoring.
