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J Clin Microbiol. Jul 2008; 46(7): 2305–2313.
Published online Apr 30, 2008. doi:  10.1128/JCM.02171-07
PMCID: PMC2446937

Method To Detect Only Live Bacteria during PCR Amplification [down-pointing small open triangle]

Abstract

Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 108 heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 108 heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 103 to 107 antibiotic-treated L. monocytogenes cells but could detect 102 live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 103 to 107 heat-treated cells but could detect 101 live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.

PCR is widely used as an effective tool to detect bacteria in foods and clinical samples. The disadvantage of PCR is that it cannot discriminate dead from live bacteria. To overcome this disadvantage, reverse transcriptase PCR that targets mRNA has been used. The mRNA derived from high levels of dead bacteria (104 to 107/ml), however, cannot be removed from samples, and subsequently the reverse transcriptase PCR becomes positive (31, 35). Measuring the RNA/DNA molar ratio is not sensitive enough to detect low levels of live bacteria in samples containing high levels of dead bacteria.

To discriminate live and dead bacteria by PCR, cross-linking agents such as psoralen, a methylisopsoralen derivative (4′-aminomethyl-4,5′-dimethylisopsoralen[4′-AMDMIP]), and ethidium monoazide (EMA) have been used (4, 5, 20, 23, 25, 27-29). They selectively permeate the cell walls of dead bacteria and irreversibly bind to chromosomal DNA by covalent attachment (20, 23, 27-29). It has been reported that EMA could cross-link to DNA at the rate of 1 agent per 10 to 80 bp in vitro (17). The PCR amplification of DNA from dead bacteria was inhibited by cross-linking action (23, 27-29), and the PCR signal from dead bacteria was reduced to 1/300 to 1/1,000 (23, 27, 29). It has been reported that pasteurized milk contains 105 to 107 cells/ml of injured/dead bacteria (1, 30). When these methods are applied to the pasteurized samples, the PCR products from injured/dead bacteria are amplified. It is very difficult to judge whether the PCR product is derived only from live bacteria in test samples.

DNase was added to cleave the chromosomal DNA of dead bacteria (21), and PCR signal intensity from dead bacteria decreased to 1/10. External DNase, however, could not completely suppress PCR products from dead bacteria, because DNase could not penetrate the cell membranes of dead bacteria due to its high molecular weight.

Developing rapid PCR methods to substitute for the culture method is a pressing matter in clinical and food hygiene tests. Most clinical samples are derived from patients administered antibiotics. Various foods have been pasteurized to kill bacteria while minimizing the denaturation of food components such as proteins. Therefore, the bacteria present in clinical samples and foods may be injured. Hyperthermophilic enzymes are reported to be maintained in the bacteria of pasteurized milk (9, 11, 36). The activities of bacterial DNA gyrase and topoisomerase IV (16) are likely to be maintained. Here, we focused on active bacterial DNA gyrase and topoisomerase IV retained in heat-treated bacteria. By utilizing the enzyme activity, we could completely suppress the PCR end products of heat-injured bacteria. There have been no reports of the inhibition of PCR products from heat-treated bacteria using DNA gyrase/topoisomerase IV poison (e.g., fluoroquinolones) and/or mammalian topoisomerase poisons (T-poisons). We employed EMA, which cleaves DNA after photoactivation (32), DNA gyrase/topoisomerase IV poison (8, 15, 24, 37), and mammalian topoisomerase (I and II) poisons (3, 7, 14, 18, 19, 34). We examined whether the DNA of injured/dead bacteria are cleaved by EMA with the aid of ciprofloxacin (CIN) or T-poisons and whether PCR final products from the bacteria could be completely suppressed. We used the Listeria monocytogenes strain, because this bacterium is important in both clinical and food hygiene applications.

MATERIALS AND METHODS

Reagents.

EMA (Sigma, St. Louis, MO) was used for DNA cross-linking and the DNA cleavage of bacteria. CIN, purchased from Fluka Chemie GmbH (Buchs, Germany), was dissolved in physiological saline. Ampicillin (AMP) and gentamicin (GEN) were from Sigma. Camptothecin (CAM), etoposide (ETP), ellipticine (ELP), mitoxantrone (MIT), and amsacrine (m-AMSA) were purchased from Sigma and were dissolved in dimethyl sulfoxide (DMSO).

Bacteria and culture.

Listeria monocytogenes JCM 2873 was cultured at 30°C in brain heart infusion (BHI) broth (Eiken Kagaku, Tokyo, Japan). To prepare live bacterial suspensions, bacteria in the logarithmic growth phase were suspended in physiological saline. The number of live bacteria was counted by plating the bacterial suspension on Luria (L) agar after the appropriate dilution.

Preparation of heat-treated L. monocytogenes.

The live bacterial suspension (1 ml) was transferred to a 1.5-ml microtube (Eppendorf, Tokyo, Japan), and the tube was soaked in a boiling water bath for 50 s. Thereafter, it was immediately chilled in an ice-water bath. This treatment simulated high-temperature, short-time pasteurization and avoided denaturing the DNA gyrase/topoisomerase IV of bacteria. The temperature of the contents was measured by a thermal sensor (TX 10; Yokogawa M C Corp., Musashino, Japan).

Preparation of antibiotic-treated L. monocytogenes.

L. monocytogenes (3.0 × 106 cells/ml) was treated in L broth with AMP and GEN (the final concentrations were 500 and 200 μg/ml, respectively). The suspension was incubated at 30°C for 3 weeks to bring L. monocytogenes completely to the injured/dead state and/or to examine whether DNA gyrase/topoisomerase IV is retained during long-term administration. The cell counts for the antibiotic-treated L. monocytogenes were done by a standard curve made from live bacterial counts and its optical density at 600 nm (OD600).

EMA treatment and visible light irradiation (EMA + Light).

EMA was dissolved in sterile water at the concentration of 1 mg/ml and filtrated through a 0.20-μm microfilter (Minisart-plus; Sartorius AG, Gottingen, Germany). After EMA was added at the concentration of 10 μg/ml to each heat- or antibiotic-treated and live bacterial suspension, it was kept at 4°C for 5 min in the dark. The suspension was then set in an ice-water bath and irradiated for 5 min with visible light (Flood PRF; 100 V, 500 W; Iwasaki Electric Co., Ltd., Tokyo, Japan) set 20 cm from the solution. The concentration of EMA was set at 10 μg/ml, because >10 μg/ml of EMA could penetrate live L. monocytogenes as well.

Use of T-poisons together with EMA + Light.

After EMA + Light treatments, bacteria were washed by centrifugation. T-poisons were used to make DNA more degraded by interfering with the breakage reunion function of DNA gyrase/topoisomerase IV that is retained in the heat-treated cells. T-poisons were added to 1 ml of bacterial suspensions at volumes and concentrations (in parentheses) as follows: 8 μl of CIN (0.5 mg/ml), 10 μl of CAM (1 mg/ml), 10 μl of ETP (1 mg/ml), 5 μl of ELP (0.1 mg/ml), 10 μl of MIT (0.1 mg/ml), and 10 μl of m-AMSA (1 mg/ml). The bacterial suspensions were then incubated at 30°C for 30 min.

CIN treatment to confirm DNA gyrase and topoisomerase IV activities retained in heat-treated L. monocytogenes.

Live and heat-treated L. monocytogenes cells were suspended in fresh BHI broth, and CIN was added at a final concentration of 20 μg/ml. They were then incubated at 30°C for 1.5, 3.5, 5, and 72 h. Simultaneously, the heat-treated bacterial suspension not treated with CIN was prepared as a control to examine the influence of DNase retained in heat-treated L. monocytogenes on chromosomal DNA. It was incubated for 72 h.

Treatment of L. monocytogenes-added human blood.

Heparinized blood from a healthy human was cooled beforehand at 4°C. Live and antibiotic-treated L. monocytogenes was inoculated to the heparinized blood at concentrations of 1.8 × 100 to 1.8 × 107 cells/ml. After the sample was diluted twofold with physiological saline, 1 ml was slowly overlaid on 1.0 ml of Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in a sterilized microtube (2 ml of volume). The layers were then subjected to centrifugation at 100 × g for 5 min at 4°C, and the blood plasma containing microorganism was collected. EMA + Light treatment along with the washing of the bacteria were carried out as mentioned above. When T-poison was added, the bacterial suspensions were incubated for 45 min at 30°C, followed by the same washing step. As a control, sterilized water and 0.5% (vol/vol) DMSO were used to replace EMA and T-poisons, respectively.

Treatment of commercial milk spiked with L. monocytogenes.

Live and heat-treated L. monocytogenes (2.2 × 100 to 2.2 × 107 cells/ml) was spiked into pasteurized milk (125°C, 2 s) in which no live L. monocytogenes was detected beforehand by culture and no L. monocytogenes was detected by PCR without EMA as described below (detection limit for live L. monocytogenes in milk, 2.2 × 101 cells/ml). T-poisons initially were added to 1 ml of milk inoculated with L. monocytogenes and then incubated at 30°C for 3 h. One milliliter of 1% Triton X-100-2 mM EDTA solution (pH 8.0) was added and centrifuged at 3,000 × g for 5 min at room temperature. After the lipid and supernatant were removed, the washing step (at 15,000 × g for 10 min at room temperature) was done with 2 ml of physiological saline, and then 1 ml of physiological saline was added. EMA + Light treatment and the washing of bacteria were performed as mentioned above. As a control, 0.5% DMSO and sterile water were added instead of T-poisons and EMA, respectively.

DNA extraction from bacteria.

After 0.5 ml of 5 mM EDTA was added to bacterial pellets in a microtube (2 ml), 20 μl of achromopeptidase (Wako Pure Chemical Industries, Ltd., Osaka, Japan), dissolved at 5 mg/ml in 10 mM NaCl, was added and incubated at 50°C for 30 min. At this point, 0.5 ml of 10 mM Tris-HCl (pH 8.0), 20 μl of 1,250-U/ml proteinase K (Sigma, St. Louis, MO), and 400 μl of 10% (wt/vol) lauryl sulfate sodium salt solution were added one after another. The solution was incubated at 50°C overnight. The chromosomal DNA was purified by the usual phenol-chloroform extraction and ethanol precipitation. TE buffer (150 μl; 10 mM Tris-HCl, 1 mM disodium EDTA) was added to the purified DNA. The concentration of DNA was calculated from the OD260, and purity was evaluated from the ratio of OD260/OD280. Furthermore, the usual RNase treatment was carried out successively for PCR.

Gel electrophoresis of chromosomal DNA.

Seakem GTG agarose (FMC BioProducts, Rockland, ME) was dissolved in 0.8% Tris-acetate-EDTA (TAE) buffer. After 1 μg of purified DNA was applied to wells, electrophoresis was performed at 100 V. The λ-EcoT14 I digest and/or a 100-bp DNA ladder (Takara-Bio, Ohtsu, Japan) were used as DNA markers. After the gel was stained with 1 μg/ml ethidium bromide, the result was visualized with a UV transilluminator at 254 nm of UV light and recorded on Polaroid film (type 667; Nippon Polaroid, Tokyo, Japan).

Real-time PCR.

Reactions were performed in the real-time PCR system (iCycler iQ; Bio-Rad, Hercules, CA). The fluorescence threshold was set at a value of 10× the standard deviation calculated from the fluorescence values, from 0 to 10 cycles. The first cycle for which the signals of real-time PCR amplification were above the threshold fluorescence value was set as the threshold cycle (CT) value.

Targeting genes and primers used for PCR.

The 23S rRNA gene primers to precisely discriminate live and heat-treated L. monocytogenes were 23S-MF (5′-ACCAGGATTTTGGCTTAGAAG-3′) and 23S-MR (5′-CACTTACCCCGACAAGGAAT-3′) (12). The length of the PCR product was 894 bp.

The listeriolysin O gene (hly) also was targeted to discriminate live L. monocytogenes from heat- and antibiotic-treated L. monocytogenes. The hly primers were hly-F (5′-TGCAAGTCCTAAGACGCCA-3′) and hly-R (5′-CACTGCATCTCCGTGGTATACTAA-3′) (22). The length of the PCR product was 113 bp.

Amplification of 23S rRNA gene and hly by real-time PCR.

Fifty microliters of PCR master mix was prepared and contained 150 ng of template DNA, 5 μl of 10× Ex-Taq buffer (Takara-Bio), 200 μM each deoxynucleoside triphosphate (Takara-Bio), 0.25 μM of 23S rRNA gene or hly gene primers (Takara-Bio), 0.4× SYBR green (BMA, Rockland, ME), and 1.25 U of Ex-Taq polymerase (Takara-Bio).

The PCR protocol for the 23S rRNA gene of L. monocytogenes was 1 cycle at 4°C for 3 min, 1 cycle at 94°C for 30 s, and 40 cycles at 94°C for 20 s, 46°C for 30 s, and 72°C for 1 min. After PCR, the Tm (melting-point measurement) pattern analysis of PCR product was carried out with 1 cycle at 95°C for 3 min, followed by being cooled at 60°C and heated to 95°C at the rate of 0.75°C per min.

The PCR protocol for hly was 1 cycle at 4°C for 3 min, 1 cycle at 94°C for 30 s, and 40 cycles at 95°C for 20 s, followed by 60°C for 1 min. After PCR, the Tm pattern analysis of the PCR product was performed with the same procedures as that for the 23S rRNA gene.

In an experimental procedure applied to blood and milk, a direct PCR cocktail (G & g Science, Fukushima, Japan) was added to the bacterial pellet suspended in sterilized water (10 μl) after treatment with a combination of EMA + Light and T-poison (EMA + Light + T-poison) and successive washing, taking into consideration the simplification of DNA extraction. That is, 5 μl of bacterial suspension treated by EMA + Light + T-poison was added to 50.5 μl of PCR cocktail. The thermal cycle profile, which was the same as that of the PCR protocol for hly, was utilized.

Electrophoresis of PCR final products amplified by real-time PCR.

A 0.8 or 3% agarose gel was made from the Seakem GTG agarose and TAE buffer for PCR final products from 23S rRNA gene and hly, respectively. The λ-EcoT14 I digest and 100-bp DNA ladder (Takara-Bio) were used as DNA markers. After 10 μl of PCR product was applied to the wells, it was separated at 100 V.

RESULTS

Time course of temperature and number of CFU of bacteria when bacterial suspension was inserted in boiling bath.

The relationship between the time of insertion into a boiling bath and the temperature of the contents was the following: 0 s, 25.0 ± 0.15°C; 27 s, 65.0 ± 0.20°C; 34 s, 70.0 ± 0.10°C; 47 s, 80.0 ± 0.15°C; 70 s, 90.0 ± 0.25°C; 90 s, 93.8 ± 0.60°C; and 120 s, 99.0 ± 0.45°C (n = 3). The relation of the immersion time and the viable cell counts (in log10 CFU/milliliter) of L. monocytogenes JCM 2873 was the following: 0 s, 8.1 ± 0.20 counts; 10 s, 7.5 ± 0.10 counts; 20 s, 6.1 ± 0.10 counts; 30 s, 4.7 ± 0.15 counts; 40 s, 2.4 ± 0.10 counts; and 50 s, no counts (n = 3). The detection limit was 5 CFU/ml. Insertion for 50 s offered a condition similar to that of high-temperature, short-time pasteurization; that is, 72 to 75°C for 15 to 16 s.

Effects of EMA on the cleavage of chromosomal DNA of heat-treated and live L. monocytogenes.

Live and heat-treated cells of L. monocytogenes were treated with EMA + Light (4°C for 5 min in the dark; irradiation time, 5 min), and chromosomal DNA was purified. The gel electrophoresis patterns are shown in Fig. Fig.1.1. When EMA + Light was performed on live bacteria, long fragments near 19,329 bp (derived from chromosomal DNA, because the band is fuzzy on the lower site, i.e., the anode side of the gel) were detected. As for the heat-treated L. monocytogenes, the long fragments did not appear, but smear bands were detectable at a range of less than 1,489 bp when EMA + Light treatment was done.

FIG. 1.
Gel electrophoresis patterns of chromosomal DNA purified from live and heat-treated L. monocytogenes before and after EMA + Light treatment. N, no EMA + Light treatment; E, EMA + Light treatment (at 4°C in the dark for ...

Discrimination by PCR of live and heat-treated L. monocytogenes after EMA + Light using the 23S rRNA gene.

PCR was performed to target the 23S rRNA gene of L. monocytogenes after being treated with EMA + Light. The PCR band from live L. monocytogenes was observed after EMA + Light treatment, but that from heat-treated cells was not observed by EMA + Light (Fig. (Fig.2).2). Evidently, the discrimination of live from heat-treated L. monocytogenes could be done.

FIG. 2.
Amplification results of 23S rRNA gene PCR after no treatment or EMA + Light treatment using live and heat-treated L. monocytogenes. The amplification results for the 23S rRNA gene (894 bp) of L. monocytogenes are represented. N, no EMA + ...

Discrimination of live and heat-treated L. monocytogenes by targeting short DNA.

When PCR targets pathogenic bacteria, short genes specific for the organism often are amplified. Our method tested whether the hly gene (113 bp) can be used for discrimination between live and heat-treated L. monocytogenes. The PCR final product from heat-treated cells was not suppressed after processing by EMA + Light; thus, discrimination between live and heat-treated L. monocytogenes was not successful (Fig. (Fig.3,3, lanes N and E). Therefore, combination methods (EMA + Light + T-poison) were performed (Fig. (Fig.3).3). When ETP, MIT, and m-AMSA were treated at 30°C for 30 min after EMA + Light treatment, the PCR final products from heat-treated cells were greatly inhibited, although those from live cells were not.

FIG. 3.
Amplification results of PCR that targeted the hly gene after the combination treatment of EMA + Light and CIN/mammalian topoisomerase (I and II) poisons on live and heat-treated L. monocytogenes. The targeted hly gene was 113 bp. T-poison treatments ...

Involvement of DNA gyrase/topoisomerase IV in DNA cleavage.

T-poisons impair DNA activity by accelerating the forward rate (breakage) and inhibiting the reunion of the breakage reunion activity of topoisomerases. CIN is one T-poison. The effect of CIN on the cleavage of chromosomal DNA was examined using live and heat-treated L. monocytogenes. The results for live and heat-treated L. monocytogenes are shown in Fig. Fig.44 in CIN (+) lanes. When live L. monocytogenes was treated with CIN, the level of long fragments decreased time dependently during 0 to 3.5 h of incubation. The intensity of long fragments increased at 5 h but decreased to near the detection limit at 72 h. Heat-treated L. monocytogenes next was incubated at 30°C for 72 h with and without CIN. With CIN, the band intensity of long fragments decreased in a time-dependent manner and was near the detection limit at 72 h. Without CIN, the band intensity of long fragments (near 19,329 bp) did not decrease. These results show that DNA gyrase/topoisomerase IV is active in heat-treated L. monocytogenes, because T-poisons work only when topoisomerase is active. The results also imply that the influence of DNase retained in heat-treated L. monocytogenes on DNA cleavage is minimal.

FIG. 4.
Effects of CIN on the retaining activities of DNA gyrase and topoisomerase IV in heat-treated L. monocytogenes. The influence of CIN on the chromosomal DNA of live and heat-treated L. monocytogenes and the effect of DNase retained in heat-treated bacteria ...

Discrimination of live and heat-treated L. monocytogenes by CIN using real-time PCR that targeted the 23S rRNA gene.

In a comparison of the thickness of bands in gels (Fig. (Fig.3),3), we cannot see the additive effect of CIN on EMA + Light treatment. To make the slight difference in discrimination power more distinct, the effect of CIN on the degree of PCR suppression was evaluated by the CT value of real-time PCR. The higher the CT value, the greater the PCR inhibition. ΔCT means the degree of PCR suppression, which is represented as CT (at treatment time) minus CT (at 0 h). For live cells, the ΔCT (at 0 to 72 h) was 0.0 ± 0.00 (at 0 h), 0.0 ± 0.00 (at 1.5 h), 1.6 ± 0.10 (at 3.5 h), 1.0 ± 0.08 (at 5 h), and 2.9 ± 0.10 (at 72 h) (means ± standard deviation; n = 3). As for heat-treated L. monocytogenes, the ΔCT (at 0 to 72 h) was 0.0 ± 0.00 (at 0 h), 0.0 ± 0.00 (at 1.5 h), 1.0 ± 0.10 (at 3.5 h), 1.4 ± 0.10 (at 5 h), and 6.1 ± 0.15 (at 72 h).

The ΔCT value of 2.9 ± 0.01 after 72 h of CIN treatment of live organisms was significantly different from the ΔCT value (6.1 ± 0.15) of heat-treated cells (P < 0.05 by the t test). Hence, the degree of PCR suppression was greater for heat-treated cells than for live L. monocytogenes by CIN treatment for 72 h.

Detection limit of live L. monocytogenes in human blood with PCR that targeted the hly gene.

Figure Figure5A5A shows the results of detection for live L. monocytogenes inoculated into healthy human blood using PCR that targeted the hly gene. The PCR bands stemming from 1.8 × 102 to 1.8 × 107 cells/ml of L. monocytogenes in blood were clear but were not detectable from L. monocytogenes at concentrations of 1.8 × 100 to 1.8 × 101 cells/ml.

FIG. 5.
Detection limit of L. monocytogenes and the discrimination of the live or antibiotic-treated L. monocytogenes cells in human blood by PCR that targeted the hly gene. (A) Live L. monocytogenes (1.8 × 100 to 1.8 × 107 cells/ml) inoculated ...

PCR after EMA + Light + T-poison on live and antibiotic-treated L. monocytogenes inoculated into healthy human blood.

Live L. monocytogenes was treated with 500 μg/ml of AMP plus 200 μg/ml of GEN. Live and antibiotic-treated L. monocytogenes cells were inoculated into healthy human blood at concentrations of 1.8 × 107, 2.9 × 104, and 2.9 × 103 cells/ml. Figure 5B to D presents the amplified hly gene by PCR after EMA + Light + T-poisons (CAM, ETP, ELP, and m-AMSA) treatment. In the case of no treatment and EMA + Light (1.8 × 107 cells/ml) (Fig. (Fig.5B),5B), the bands of live and antibiotic-treated L. monocytogenes were clearly detected. However, the intensity of the bands of antibiotic-treated L. monocytogenes decreased to under or near the detection limit after adding CAM, ETP, or m-AMSA. The effect of ELP treatment was weak.

When 2.9 × 104 cells/ml of L. monocytogenes were mixed in blood (Fig. (Fig.5C),5C), the PCR bands of live L. monocytogenes clearly appeared without treatment (lane N), after EMA + Light treatment (lane E), and after EMA + Light + T-poison treatment. In the case of antibiotic-treated cells, the effects of T-poisons were clear; that is, PCR bands did not appear after EMA + Light + T-poison treatments.

Even when the L. monocytogenes dose was lowered to 2.9 × 103 cells/ml in blood (Fig. (Fig.5D),5D), the effects of T-poisons were almost the same as those shown in Fig. Fig.5C5C.

Detection limit of live L. monocytogenes-spiked commercially available milk with PCR that targeted the hly gene.

Figure Figure6A6A shows the results of the detection of live L. monocytogenes inoculated into pasteurized commercial milk using PCR that targeted the hly gene. The PCR bands derived from 2.2 × 101 to 2.2 × 107 cells/ml of L. monocytogenes in milk were detectable. No band of L. monocytogenes for 2.2 × 100 cells/ml was detected, which means that the detection limit is between 2.2 × 101 and 2.2 × 100 cells/ml.

FIG. 6.
Detection limit of L. monocytogenes and discrimination of live or heat-treated cells spiked into pasteurized milk with PCR that targeted the hly gene. (A) Live L. monocytogenes spiked into pasteurized (125°C for 2 s) commercial milk (2.2 × ...

Amplifications of PCR targeting hly gene after EMA + Light + T-poison to live and heat-treated L. monocytogenes-spiked commercial milk.

L. monocytogenes was inoculated into milk at concentrations of 2.2 × 107 (Fig. (Fig.6B)6B) and 2.2 × 103 cells/ml (Fig. (Fig.6C).6C). Figures 6B and C indicate the final products (hly) amplified by PCR, which was performed after EMA + Light + T-poison (CAM, ETP, ELP, and m-AMSA) treatment on live and heat-treated L. monocytogenes. In the case of 2.2 × 107 cells/ml (Fig. (Fig.6B),6B), bands of live and heat-treated L. monocytogenes apparently were detected both without treatment (lane N) and after EMA + Light (lane E) treatment. As for CAM + EMA + Light and m-AMSA + EMA + Light, the bands of heat-treated L. monocytogenes were near or under the detection limit, although the bands of live cells were clearly detected and the intensity was near that of nontreated live cells. In ETP or ELP + EMA + Light treatment, PCR bands of live and heat-treated bacteria appeared; thus, the effectiveness of the discrimination of ETP and ELP was weak when milk was used.

For 2.2 × 103 cells/ml of L. monocytogenes in milk (Fig. (Fig.6C),6C), the PCR bands of live L. monocytogenes obviously appeared in no treatment, EMA + Light treatment, and EMA + Light + T-poison (four kinds of agents) treatment. However, PCR bands did not appear after EMA + Light and EMA + Light + T-poisons (four agents).

DISCUSSION

Rapid PCR methods that detect only viable and culturable bacteria are required in food hygiene and clinical tests in place of the culture method. Rudi et al. (27, 29) applied EMA as a cross-linking agent to discriminate between live and dead pathogens and targeted 85-bp DNA for Campylobacter jejuni and the 113-bp DNA of the hly gene of L. monocytogenes. PCR signals from dead, but not live, bacteria were suppressed to 1/1,000 in dead cell counts, which means that the CT value of dead bacteria would increase by 10 cycles compared to values from no use of cross-linking EMA. Rudi et al., however, could not suppress real-time PCR final products from dead pathogens. In the case of food or clinical samples, in which the concentrations of live and dead pathogens are unknown, it is very difficult to judge whether CT values of test samples are derived from live or dead bacteria. No end product of PCR from dead cells is necessary in the factory or bedside, and the FDA requests the complete inhibition of PCR product from dead bacteria.

We have completely suppressed PCR signals from 107 to 108 cells of heat- and antibiotic-treated bacteria that were considered background signals in many food and clinical samples. Furthermore, PCR products derived from 102 cells/ml (in blood) and 101 cells/ml (in pasteurized milk) of live bacteria were detected. It is a very important consequence in clinical diagnostics and food testing that a high level of heat- and antibiotic-treated bacteria was not detected, but a low level of live bacteria was detectable by our PCR method. Although gel electrophoresis was used as the detection step for PCR-amplified genes in the present study, the automated Tm analysis (using a real-time PCR apparatus) for PCR products described in Materials and Methods may be used, considering the rapidity, simplicity, and sensitivity to the dose of antibiotic used of live bacteria during the detection steps. If EMA + Light or EMA + Light + T-poison was combined with Tm analysis after real-time PCR, 102 and 101 cells/ml of live bacteria in blood or milk, respectively, could be detected within 3 to 6 h.

We recently demonstrated that 1 to 10 μg/ml of EMA has the direct DNA cleavage function without mediating enzymes in bacteria, and that 50 to 100 ng/ml of EMA has the function of single-stranded breaks (32). For heat-treated L. monocytogenes, PCR that targeted long DNA (894 bp in the 23S rRNA gene) was suppressed by EMA + Light more than that of short DNA (113 bp in hly) (Fig. (Fig.22 and and3).3). This may be because there are more cleavage sites in long DNA. It was reported that the nanograms-per-milliliter level of EMA as a topoisomerase II poison could be cross-linked to DNA at the rate of 1 agent per 10 to 80 bp in vitro (17). In the present study, which used 10 μg/ml of EMA, direct DNA cleavage was not induced in the hly region of heat-treated L. monocytogenes (Fig. (Fig.3).3). We suppose that Rudi et al. (27) did not notice that PCR inhibition by EMA was not satisfactory when short DNA had been targeted. As seen above, the main cause of the failure to suppress the PCR product is that the PCR target gene was short. Even if PCR was targeted to short DNA such as that of hly, live L. monocytogenes could clearly be discriminated from heat-treated L. monocytogenes (108 cells/ml) by combining EMA + Light together with T-poisons (Fig. (Fig.33).

The mechanism of our method for discriminating live from dead bacteria is shown in Fig. Fig.7.7. After EMA (dark red bar) penetrates heat- and antibiotic-treated cells and intercalates to the chromosomal DNA (23, 27-29), the cleavage of DNA is greatly induced by the irradiation of visible light (32). The cleavage sites are seen in double-stranded DNA. When the targeted gene is as short as hly, DNA cleavage sites are not likely to be contained in every bacterial cell. The cell membranes of heat- and antibiotic-treated L. monocytogenes are physiologically injured. Therefore, when a T-poison such as m-AMSA is added, m-AMSA randomly cross-links to chromosomal DNA and DNA cleavage is accelerated by inhibiting the reunion in breakage-reunion by DNA gyrase and/or topoisomerase IV retained in heat- and antibiotic-treated cells. As EMA and m-AMSA randomly cross-link, more cleavage is induced by the combination of EMA + Light + m-AMSA (Fig. (Fig.7).7). Hence, the disappearance of bands in Fig. Fig.55 and and66 is thought to be due mainly to the poisoning effects of T-poisons.

FIG. 7.
(Left) Scheme for PCR suppression by EMA, psoralen, and a methylisopsoralen derivative (4′-AMDMIP) as a DNA cross-linking agent (current scheme). (Right) Scheme for PCR suppression through DNA cleavage by a new function of EMA and T-poisons containing ...

The effectiveness of EMA + Light + T-poison (mainly CAM, ETP, and m-AMSA) was demonstrated in a model of bacteremia (Fig. (Fig.5).5). In adult bacteremia patients, the numbers of microorganisms present in blood are fewer than 10 cells/ml, and 30 ml of blood is used for culture to maximize microbial recovery. A 30-ml volume of blood could be concentrated to approximately 1 ml for PCR testing. Thus, the concentration of live bacteria would be approximately 3 × 102 cells/ml (26). In the case of bacteremia in infants, the number of bacteria existing in blood often is more than 1.0 × 103 cells/ml, but only 1 to 4.5 ml of blood should be cultured, taking into consideration the weight of the infant (13). Bacteria injured or killed by antibiotics are supposed to exist in blood together with live bacteria. In the present study, therefore, the live and antibiotic-treated L. monocytogenes cells were spiked into healthy human blood at the concentrations of 2.9 × 104 (Fig. (Fig.5C)5C) and 2.9 × 103 cells/ml (Fig. (Fig.5D).5D). On the other hand, live and antibiotic-treated L. monocytogenes cells were inoculated into blood at a concentration of 1.8 × 107 cells/ml, considering the presence of high levels of injured/dead bacteria in urine from urinary tract infection and sputa of tuberculosis patients given anti-tuberculosis agents (6). EMA + Light + T-poison may be effective to rapidly discriminate live from injured/dead pathogen.

Pasteurized milk contains 105 to 107 cells/ml of injured/dead bacteria, and approximately half of the bacteria are gram positive (1, 30). If L. monocytogenes was estimated to be the major contaminant, only live L. monocytogenes should be detected by PCR in a high level of background injured/dead L. monocytogenes. The effectiveness of EMA + Light and EMA + Light + T-poison (mainly m-AMSA and CAM) was tested in food hygiene tests of dairy products. Hence, as shown in Fig. Fig.6B,6B, live and heat-treated L. monocytogenes (2.2 × 107 cells/ml) cells were inoculated into pasteurized milk. It is conceivable that the discriminating power of EMA + Light + T-poisons was inferior in milk compared to that in blood (Fig. (Fig.5B),5B), except for the case of m-AMSA + EMA + Light. It has been reported that 2.4 to 7.5% of raw milk is contaminated by live L. monocytogenes (10), and live L. monocytogenes exists at a concentration of 2.0 × 102 cells/g in raw milk cheese (2). Hence, live and heat-treated L. monocytogenes (2.2 × 103 cells/ml) also was inoculated into pasteurized milk (Fig. (Fig.6C).6C). When L. monocytogenes in milk is of low concentration (live and heat-treated cells, 2.2 × 103 cells/ml), EMA + Light without T-poisons could discriminate live from heat-treated L. monocytogenes as well (Fig. (Fig.6C6C).

The verification of active DNA gyrase/topoisomerase IV retained in heat-treated bacteria is speculated as follows. As shown in Fig. Fig.4,4, when live and heat-treated L. monocytogenes cells are treated with CIN for 72 h, the levels of long fragments derived from chromosomal DNA (close to 19,329 bp) obviously decrease. Fluoroquinolones, such as CIN, cause the inhibition of DNA synthesis and trigger cell killing by interfering with breakage reunion that is mediated by DNA gyrase (33). The reason for this phenomenon is thought to be that DNA scission is stimulated by inhibiting the reunion of breakage reunion or enhancing the forward DNA breakage rate by DNA gyrase and/or topoisomerase IV that is persistent in heat-treated and live bacteria.

Finally, our EMA + Light + T-poison method will be applied to boiled foods. When foods are treated by higher temperatures and for longer periods, the foods contain mainly dead bacteria in which no activity of DNA gyrase/topoisomerase IV is retained. In such cases, at least CAM and m-AMSA, among the T-poisons, would cross-link to chromosomal DNA and might specifically suppress PCR final products from dead bacteria (7). In this case, however, the PCR suppression is due to the cross-linking effect but not poisoning activity (7). EMA could function as a random and direct cleavage agent of chromosomal DNA with the irradiation of visible light, even if DNA gyrase and/or topoisomerase IV are completely denatured in dead cells (32).

Acknowledgments

We are grateful to Hiroaki Nakayama for expertise and advice regarding the mechanism of DNA gyrase poison and topoisomerase IV poisons.

Footnotes

[down-pointing small open triangle]Published ahead of print on 30 April 2008.

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