The Efficient Method for Simultaneous Monitoring of the Culturable as Well as Nonculturable Airborne Microorganisms

<div><p>Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of bioaerosol monitoring.</p></div

Total DNA mass extracted after bacterial spiking.

<p>Linear regression between total DNA mass extracted with developed protocol from spiked agarose matrices and total DNA mass extracted directly from bacterial cells (R2 = 0.76, slope coeficient 0.68, intercept 38.2). 95% confidence interval for fitted line is presented with grey area.</p

Rarefaction curves from clone libraries and from isolates.

<p>Rarefaction analysis of 16S rRNA genes from clone libraries and from isolates, both obtained from alpine air samples. 95% confidence intervals are shown.</p

Primers and amplification conditions for the detection of <i>mbt</i>A or 16S rRNA gene.

<p>Primers and amplification conditions for the detection of <i>mbt</i>A or 16S rRNA gene.</p

Scheme of the developed protocol.

<p>Scheme of the developed protocol.</p

CFU per 2³ air sample size (CFU±SD) and <i>H</i> index determined in clones and isolates from alpine area. Values in brackets are high and low 95% confidence interval.

<p>CFU per 2³ air sample size (CFU±SD) and <i>H</i> index determined in clones and isolates from alpine area. Values in brackets are high and low 95% confidence interval.</p

Total DNA mass, copy numbers of 16S rRNA gene and <i>mbtA</i> gene per m³ of sampled air at three locations inside WWTP.

<p>Total DNA mass, copy numbers of 16S rRNA gene and <i>mbtA</i> gene per m³ of sampled air at three locations inside WWTP.</p

Ratio of total DNA extracted either from retentate or filtrate.

<p>Ratio of total DNA extracted either from PES membrane filter (retentate) – (•) or concentrated with ultrafiltration (filtrate) – (▴), after bacterial spiking of agarose followed by enzymatic hydrolysis in relation to the sum of DNA extracted from each individual sample (abbreviated as total extracted DNA mass). The sum of the • and ▴ percentages in each vertical line is 100%, which is represented on the total extracted DNA mass axis. Black lines represent linear trend: filtrate (R² = 0.331, slope coefficient −0.03 and intercept 89.7) and retentate (R² = 0.331, slope coefficient 0.03 and intercept 10.3). 95% confidence intervals for both fitted lines are presented with grey area.</p