Real-Time PCR for Detection and Differentiation of Gram-Positive and Gram-Negative Bacteria

Abstract

We developed a consensus real-time PCR protocol that enables us to detect spiked bacterial 16S DNA from specimens such as water, urine, plasma, and sputum. The technique allows an exact Gram stain classification of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes. All tested bacteria were identified correctly, and none gave a false-positive signal with the opposite Gram probe.

Currently, the early detection of bacterial DNA in the circulating blood of critically ill patients as a possible improvement of conventional microbiology culture diagnostic still is technically difficult (8). In particular, the contamination of PCR reagents with bacterial DNA, e.g., contamination of the bacterium-derived polymerases, is a major problem that prevents the high sensitivity provided by PCR technology (6, 20, 25, 29; E. C. Bottger, Letter, Clin. Chem. 36:1258-1259, 1990). Nevertheless, it has been shown that it is possible to detect bacterial DNA from clinical samples like blood (17), plasma (8), cerebrospinal fluid (21, 26), and other specimens (22, 26). In contrast to these new diagnostic approaches, blood cultures are time consuming and often yield false-negative results due to unacceptably low sensitivity, even though the patient has significant signs of systemic bacterial infection.

The early detection and adequate treatment of bacterial infections have a great impact on the outcome of patients with systemic infection. In practice, most infections are treated empirically with broad-spectrum antibiotics because of the usual delay of 24 to 48 h for routine microbiological processing of the clinical samples (19, 28). PCR has been successfully used to detect bacterial DNA in clinical samples and has improved the rate of microbial detection (5, 13, 14, 22).

To date, most efforts have centered on species-specific detection of bacterial DNA (3, 4, 9, 10, 15, 24, 31). Thus, it is necessary to develop a reliable broad-range detection system for bacterial DNA from clinical samples that is fast and easy to use and covers a wide range of clinically relevant microbes.

Therefore, we developed a rapid real-time PCR protocol for detection of bacterial DNA from biological fluids (water, plasma, urine, sputum) and exact Gram stain classification of intensive care unit (ICU)-relevant bacteria by means of fluorescence hybridization probes.

PCR on a real-time PCR instrument (LightCycler; Roche) was performed by employing broad-range primers for amplification of the highly conserved bacterial 16S DNA. The genomic DNA of 17 ICU-relevant bacteria was added to different biological fluids (water, plasma, urine, sputum), and DNA was extracted. Gram-positive bacteria included Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Staphylococcus epidermidis, and Staphylococcus aureus; gram-negative bacteria included Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Haemophilus influenzae, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Acinetobacter baumanii, Legionella pneumophila, and Stenotrophomonas maltophilia.

The PCR products were hybridized with two fluorescence dye-labeled hybridization probes binding specifically only to either gram-positive or gram-negative bacteria. The different fluorescence signals (640 and 705 nm) can be detected by the LightCycler and enable Gram stain differentiation of bacteria.

Oligonucleotide primers and hybridization probes.

Oligonucleotide primers were manufactured by Biosource Europe. The primers amplify specific parts of the 16S region of bacterial DNA. The primers PLK1 (5-TAC GGG AGG CAG CAG T-3) and PLK2 (5-TAT TAC CGC GGC TGC T-3) are highly conserved in different groups of eubacteria. A 187-bp fragment is synthesized by these primers. PLK2 is labeled with fluorescein internally.

The fluorescence dye-labeled hybridization probes ISN2 (5-CCG CAG AAT AAG CAC CGG CTA ACT CCG T-3) and ISP2 (5-CCT AAC CAG AAA GCC ACG GCT AAC TAC GTG-3) emit light at different wavelengths (640 and 705 nm) and can be used for detection and Gram stain differentiation of bacterial DNA by a fluorescence signal.

DNA extraction.

DNA extraction was performed with an automatic preparation device (MagnaPure; Roche) according to the manufacturer's preparation protocol. One hundred million CFU were added to sterile water, plasma, urine, and sputum, and afterwards bacterial DNA was extracted. The DNA was used for subsequent experiments.

PCR amplification.

PCR amplification was performed by using a real-time PCR system (LightCycler; Roche). The PCR mixture (20 μl) contained FastStart DNA Master SYBR Green I (ready-to-use Hot-Start reaction mixture for PCR containing FastStart Taq DNA polymerase, deoxynucleoside triphosphate mix with dUTP instead of dTTP, SYBR Green I dye, and 10 mM MgCl; Roche), 2.4 μl of MgCl (25 mM) stock solution per reaction mixture, 13.6 μl of sterile H₂O, and 2 μl of template. The PCR protocol was as follows: 1 cycle of denaturation at 95°C for 10 min (FastStart activation), 45 cycles of amplification (15 s of denaturation at 95°C, 8 s of annealing at 52°C, and 10 s of extension at 72°C). This step was followed by a melting curve analysis from 40 to 98°C and afterwards cooling to room temperature.

All 17 ICU-relevant bacteria were extracted from biological fluids (water, plasma, urine, and sputum) and detected by real-time PCR. The fluorescence probes enabled a correct Gram stain classification. The detection limit was <1 pg of bacterial DNA.

As depicted in Fig. 1, all gram-negative bacteria examined showed a fluorescence signal on channel F2 (640 nm, labeling dye of the gram-negative probe). In contrast, the gram-positive bacteria showed no fluorescence signal on channel F2. It made no difference from which biological fluid the bacteria had been prepared.

FIG. 1.

Fluorescence signal of the gram-negative hybridization probes on channel F2 (640 nm). The fluorescence scale on the ordinate is adapted automatically by the real-time PCR machine and indicates the fluorescence of the selected probe.

As depicted in Fig. 2, all gram-positive bacteria examined showed a fluorescence signal on channel F3 (705 nm, labeling dye of the gram-positive probe). The gram-negative bacteria showed no fluorescence signal on channel F3. It made no difference from which biological fluid the bacteria had been prepared.

FIG. 2.

Fluorescence signal of the gram-positive hybridization probes on channel F3 (705 nm). The fluorescence scale on the ordinate is adapted automatically by the real-time PCR machine and indicates the fluorescence of the selected probe.

When simulating mixed infections by adding P. aeruginosa and S. epidermidis in one sample, the PCR yielded a gram-negative signal (channel F2) and a gram-positive signal (channel F3).

An early and adequate treatment of infection is a major goal in ICU therapy, but this is often not achieved. One important reason for inadequate treatment with specific antibiotics is the delay in routine microbiological detection systems, which require at least 24 to 48 h for detection (19, 28). Yet, early detection and adequate treatment of bacterial infections have a great impact on the outcome for the patient (12). A faster and more sensitive method for the detection and specification of bacteria or bacterial DNA would allow earlier treatment with a specific antibiotic. Moreover, it has been shown that rapid bacterial identification reduces costs by decreasing the length of hospitalization and conserving hospital resources (2, 11). PCR has been successfully used to detect bacterial DNA in clinical samples and has improved the sensitivity of microbiological diagnostics (5, 13).

This study confirms that it is possible to detect bacterial DNA from different biological fluids and to achieve a fast and correct Gram stain classification in a one-run experiment.

Traditionally, Gram stain classification of, e.g., sputum has been used to support the decision for an initial antibiotic therapy. Kalin et al. found Gram-stained smears to be a valuable aid in the diagnosis of bacterial pneumonia; the results were in agreement with those of the culture for about 75% of the purulent samples (16). On the other hand, Reed et al. found in a meta-analysis a high variability in sensitivity (15 to 100%) and also a high variability in specificity (11 to 100%) (27). The authors concluded that conventional Gram stain classification may yield misleading results. Furthermore, Cooper et al. found an excessive variability when the same samples where examined by different technologists; so there is a substantial degree of subjectivity involved in reading Gram stains (7). Nagendra et al. found a similar variability when different microscopists examined the same Gram stains (23). This leads to the conclusion that conventional Gram stains, especially for sputa and tracheal aspirates, is of limited value in providing information for an efficient antibiotic therapy.

Other authors have performed Gram stain classification successfully by Gram stain-specific PCR (18), by nested PCR (5), and by PCR followed by probe hybridization (1, 13, 30), but all these methods are time consuming (because of the probe hybridization in blotting techniques) or needed more than one reaction vial per clinical sample (for specific primers and nested PCR).

The method introduced here enables analysis in less than 40 min by real-time PCR in a single-tube assay. An important issue is the amount of time required between the deposit of a clinical sample and a definite result. The hands-on time necessary for preparing DNA and performing and evaluating the PCR is less than 3 h.

Real-time PCR is a promising tool for the detection of bacterial DNA from biological fluids. Fluorescence hybridization probes allow a fast detection of low amounts of bacterial DNA and a correct Gram stain classification. This may accelerate therapeutic decisions and enable an earlier adequate antibiotic treatment. In the future, new approaches for an even more specific differentiation of microbes are conceivable.