Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR

Abstract

A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10(-3) cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10(0) to 10(5) CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4',6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n = 38) at a level of 10(3) cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

FIG. 1.

Quantitative detection of bacteria by RT-qPCR and by qPCR. E. coli ATCC 11775^T (A), E. faecalis ATCC 19433^T (B), S. aureus ATCC 12600^T (C), C. perfringens JCM 1290^T (D), and P. aeruginosa ATCC 10145^T (E) were cultivated separately in BHI or MRS broth. RNA and DNA fractions were extracted from culture samples in the early stationary phase (18 h), and the bacterial counts were determined microscopically with DAPI staining. Based on the bacterial counts, 10-fold serial dilutions of RNA or DNA from 10⁵ to 10⁻³ bacteria were assessed by RT-qPCR (○) and qPCR (•) assays. The C_T values for triplicate samples obtained were plotted against the log₁₀ number of bacterial cells subjected to each reaction.

FIG. 2.

Comparison of bacterial counts in cultures determined by RT-qPCR and by the culture method. E. coli ATCC 11775^T (A), E. faecalis ATCC 19433^T (B), S. aureus ATCC 12600^T (C), C. perfringens JCM 1290^T (D), and P. aeruginosa ATCC 10145^T (E) were cultivated in BHI or MRS broth. RNA fractions were extracted from 10-fold serial dilutions of each bacterial culture (50 μl) in the range from 10⁰ to 10⁵ CFU. The number of bacteria in each sample was determined by RT-qPCR and then plotted against the CFU count for the same sample determined on BHI (for E. coli, E. faecalis, S. aureus,and P. aeruginosa) or GAM (for C. perfringens) agar plates; data for single samples from each of the three different cultures are shown for each dilution. For RT-qPCR, an analytical curve generated with the RNA dilution series for each target strain (Fig. 1) was used.

FIG. 3.

Effect of growth phase on bacterial counts determined by RT-qPCR. Throughout the growth phase in broth culture, the numbers of E. coli ATCC 11775^T (A), E. faecalis ATCC 19433^T (B), S. aureus ATCC 12600^T (C), and C. perfringens JCM 1290^T (D) cells were determined by RT-qPCR (○) and the culture method (•). The analytical curves generated with the RNA dilution series for each target strain in the stationary phase (18 h) (Fig. 1) were used to quantify the bacteria. The CFU counts were determined on BHI (for E. coli, E. faecalis, and S. aureus) or GAM (for C. perfringens) agar plates. The results are the means and standard deviations of triplicate samples.

FIG. 4.

Comparison of RT-qPCR counts and viable bacterial cell counts determined by a combination of DAPI staining and SYTO9-PI double staining of cultured bacteria. C. perfringens JCM 1290^T was incubated in MRS broth for 4 days and examined by RT-qPCR (○), SYTO9-PI double staining (▴), and the culture method (•) at 24, 48, 72, and 96 h. The viable bacterial count was calculated with the following equation: viable bacterial count = (number of cells labeled with SYTO9/number of cells labeled with both SYTO9 and PI) × (number of cells stained with DAPI). For RT-qPCR, the analytical curve generated with the dilution series of RNA extracted from C. perfringens cells at 18 h (Fig. 1D) was used to determine the bacterial number. The CFU count was determined by culturing samples on GAM agar plates for 24 h. The results are the means and standard deviations of triplicate samples.

FIG. 5.

Correlation between RT-qPCR counts and cultural counts in human feces. Total RNA fractions extracted from 38 human fecal homogenates were assessed by the RT-qPCR assay to determine the indigenous population levels of Enterobacteriaceae (A) and P. aeruginosa (B). The C_T values obtained were applied to the analytical curves for E. coli ATCC 11775^T and P. aeruginosa ATCC 10145^T (Fig. 1A and E) to determine the RT-qPCR counts. The CFU counts were determined by culturing the same fecal samples on DHL (A) or NAC (B) agar plates and then were plotted against the RT-qPCR counts.

FIG. 6.

Comparison of bacterial counts in human peripheral blood determined by RT-qPCR and the culture method. Human peripheral blood samples (0.5 ml) from three individuals were spiked with various amounts of live S. aureus ATCC 12600^T (A) or P. aeruginosa ATCC 10145^T (B) to obtain final concentrations ranging from 10⁰ to 10⁶ CFU per ml. RNA fractions extracted from each sample were then assessed by the RT-qPCR assay. The C_T values obtained were applied to the analytical curves for S. aureus ATCC 12600^T and P. aeruginosa ATCC 10145^T (Fig. 1C and E) to determine the RT-qPCR counts. The CFU counts were determined by culturing the same samples on BHI agar plates and then were plotted against the RT-qPCR counts; data for single samples from the three different donors are shown.