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Discrimination of viable from non-viable gram-negative bacterial pathogens in airborne particles using propidium
Rajni Kaushik1, Rajasekhar Balasubramanian
1Singapore-Delft Water Alliance, National University of Singapore, Singapore 117576, Singapore.
Abstract:
The presence of bacterial pathogens in airborne particulate matter (PM) has been of considerable concern from the public health standpoint. Conventional culture-based methods are tedious, time consuming and are unable to quantify stressed viable but non-culturable (VBNC) populations of these pathogens. This study reports the optimization, validation and application of a new and rapid quantitative method for enumeration of four live potential Gram-negative bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila) in PM of biomass burning origin. This method makes use of an intercalating dye (propidium monoazide, PMA) in conjunction with real-time PCR (qPCR) analysis following DNA extraction from PM samples for distinguishing viable from non-viable potential bacterial pathogens. This method was not affected by the complex matrix of the environmental samples, nor by any PCR inhibition effects. The number of viable pathogens ranged from 0 to 8×10(4) gene copies/m(3) in PM. With the exception of A. hydrophilia, all the three pathogens were found to be present in PM. The correlation between the counts obtained using the PMA-qPCR (modified qPCR) and those from the culture-based method was very high with R(2)~1.0 and p value<0.0001.
Insights
A new method using propidium monoazide (PMA) and real-time PCR (qPCR) rapidly quantifies live bacterial pathogens in airborne particulate matter (PM). This technique accurately detects viable bacteria, including Escherichia coli and Pseudomonas aeruginosa, crucial for public health assessments.
Area of Science:
- Environmental microbiology
- Public health
- Molecular biology
Background:
- Airborne particulate matter (PM) poses public health risks due to potential bacterial pathogens.
- Conventional culture methods are slow and fail to detect viable but non-culturable (VBNC) bacteria.
- Accurate quantification of live airborne bacteria is essential for risk assessment.
Purpose of the Study:
- To develop and validate a rapid, quantitative method for enumerating live Gram-negative bacterial pathogens in PM.
- To distinguish viable from non-viable bacteria in environmental samples.
- To assess the presence and levels of specific pathogens like Escherichia coli and Pseudomonas aeruginosa in biomass burning PM.
Main Methods:
- DNA extraction from PM samples.
- Propidium monoazide (PMA) treatment to exclude DNA from dead cells.
- Quantitative real-time PCR (qPCR) for sensitive and specific pathogen detection.
- Validation against conventional culture-based methods.
Main Results:
- The PMA-qPCR method successfully quantified live bacterial pathogens in PM.
- Viable pathogen levels ranged from 0 to 8x10^4 gene copies/m^3.
- Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were detected in PM.
- The PMA-qPCR results showed a very high correlation (R²~1.0) with culture-based counts.
- The method demonstrated robustness against complex environmental matrices and PCR inhibition.
Conclusions:
- PMA-qPCR is a rapid, reliable, and accurate method for quantifying viable bacterial pathogens in airborne PM.
- This technique overcomes limitations of traditional culture methods, including the detection of VBNC bacteria.
- The findings highlight the presence of viable pathogens in PM, emphasizing the need for effective air quality monitoring and public health interventions.
