Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide

Abstract

Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 microM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.

FIG. 1.

Effect of PMA on amplification of BacUni-UCD universal marker in viable and dead Bacteroides fragilis cells with different concentrations of solids. The contour lines represented ΔC_T values and were generated by the Origin Pro 8 software program. The mean cycle threshold differences (ΔC_T) were calculated by subtracting C_T values obtained without PMA treatment from C_T values obtained with PMA treatment. (A and B) ΔC_T for viable cells (A) or dead cells (B) in the absence of added solids. (C and D) ΔC_T for viable cells (C) or dead cells (D) at a solids concentration of 100 mg liter⁻¹. (E and F) ΔC_T for viable cells (E) or dead cells (F) at a solids concentration of 1,000 mg liter⁻¹.

FIG. 2.

Effect of PMA treatments at 100 μM and a 10-min light exposure on PCR amplification in human fecal samples containing defined ratios of fresh and heat-treated feces. The black squares (▪) denote a 1:10 dilution of fecal material, and the white circles (○) denote a 1:100 dilution of fecal material. The error bars represent standard deviations for three samples. (A) Least-squares linear regression between the concentration of BacHum-UCD marker and defined ratios of 10-fold-diluted fresh and heat-treated feces. (B) Least-squares linear regression between the concentration of the BacHum-UCD marker and defined ratios of 100-fold-diluted fresh and heat-treated feces.

FIG. 3.

Comparison of Bacteroidales gene copies determined using the BacHum-UCD assay in the presence and absence of PMA. Wastewater treatment influent, heat-treated influent, and effluent after UV disinfection were analyzed by quantitative PCR. The effluent was concentrated from 2 liters to 200 ml by hollow-fiber ultrafiltration (12), and DNA was extracted from the concentrated effluent and the influent samples. S_LOD, sample limit of detection.