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Applied and Environmental Microbiology, August 2005, p. 4214-4219, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4214-4219.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

Abu Sadeque Md. Selim,¹ Piyanuch Boonkumklao,³ Teruo Sone,¹ Apinya Assavanig,⁴ Masaru Wada,² and Atsushi Yokota^2*

Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan,¹ Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan,² Department of Biology, Faculty of Science, Maejo University, Sansai, Chiang Mai 50290, Thailand,³ Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand⁴

Received 24 September 2004/ Accepted 25 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345^T and Lactobacillus fermentum JCM 1173^T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 x 10³ cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10⁴ cells/g feces on day 4 and 10⁵ cells/g feces on days 11 to 18. However, cell populations of 10⁶ to 10⁷ cells/g feces were detected in the second trial. The total bacterial count, measured by 4',6-diamidino-2-phenylindole (DAPI) staining, was approximately 10¹¹ cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we isolated Lactobacillus thermotolerans, a novel species, from chicken feces collected in Thailand (9). The preference of this bacterium for the chicken intestine may be due to the body temperature of chickens, 42°C (2), which corresponds to the optimum temperature for growth of this bacterium (42°C), as determined by measurement of the specific growth rate (9).

Our current interest in L. thermotolerans is to characterize this bacterium ecologically in the chicken intestine, since no studies have been conducted to date on the ecology of this new organism. Studies on the distribution and colonization of L. thermotolerans in different organs of the gastrointestinal tract should provide new insights into chicken intestinal microbiology. For this an effective method for enumeration of this bacterium is required. Development of a molecular ecological enumeration method appears to be particularly valuable in the case of L. thermotolerans, since conventional culture methods are insufficient due to the relatively high temperature required for culturing this bacterium. Real-time PCR offers significant advantages over other molecular enumeration techniques in terms of the speed at which assays are performed and the ability to quantify the target microbial population. Real-time PCR has already been established as a promising tool for studies of the composition of microbial communities in the gastrointestinal tract or feces of humans (1, 4, 5, 12), as well as chickens (13). However, most studies that have focused on the chicken microbiota have been conducted using conventional culture methods (6, 8, 10, 11). To the best of our knowledge, there has not yet been a report focusing on real-time monitoring of chicken lactobacilli, which are commonly used probiotic organisms in the avian industry. The use of probiotics to promote health and nutrition has attracted a great deal of attention, and claims have been made in this context with regard to daily weight gain, improvement in feed conversion, and resistance to disease (3).

In this paper, conditions for a real-time PCR assay of L. thermotolerans and successful application of this assay for monitoring the population dynamics of this bacterium in chicken feces are described.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria and growth conditions.
The strains used in the current study are listed in Table 1. L. thermotolerans JCM 11425^T was cultured using mixed gas (N₂-H₂-CO₂, 8: 1: 1) and Difco lactobacillus MRS broth (Becton Dickinson and Company) at 42°C overnight, and the reference strains were grown under the same conditions, except that the culture temperature was 37°C.

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TABLE 1. Specificity of the primer-probe combination

Chicken maintenance and sample collection.
Two trials (trial 1 and trial 2) were conducted using five layer chicks (Boris Brown, 1 day old) in each trial at two different times. The chicks were obtained from the Hokuren Federation of Agricultural Cooperatives (Sapporo, Japan). They were kept in an individual section of a wooden box and were reared for 4 weeks for trial 1 and for 3 weeks for trial 2. The chickens were maintained and handled according to the recommendations of the ethics committee at our institute. The animals were given commercial feed (200 g/kg crude protein; energy, 2,950 kcal/kg) ad libitum once a day. Water was available during the entire experiment. Fresh feces were collected from individual chicks on days 4, 11, 18, and 27 in trial 1 and on days 1, 7, 14, and 21 in trial 2. A sterilized spatula was used for feces collection, and the samples were immediately transferred to the laboratory for analysis.

DNA extraction.
The genomic DNAs were isolated from the culture broth of L. thermotolerans and of reference strains used as negative controls and from feces using an UltraClean soil DNA kit (MO BIO Laboratories, Inc., Solana Beach, CA) by following the manufacturer's instructions. Before DNA extraction, samples (0.1 g feces or 0.1 ml culture) were incubated with 50 µl of lysozyme (5 mg/ml) and 15 µl of N-acetylmuramidase (10.2 U/µl) at 37°C for 15 min. The quality of the extracted DNA was analyzed by electrophoresis on a 1.5% agarose gel. The DNA concentrations were determined by absorbance at 260 nm with a Beckman DU 640 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA). The DNA was then used for the real-time PCR assay.

Design of primers and probe.
The PCR primers and probe were designed with Primer Express v2.0 (Applied Biosystems, Foster City, CA), and the specificity of the sequence was further checked against all the available data for 16S rRNA genes in the GenBank database. The probe was labeled with the fluorescent dye 6-carboxyfluorescein at the 5' end and with 6-carboxytetramethylrhodamine at the 3' end. The sequences were 5'-TGCACAGGATTGACGTTGGT-3', 5'-GGCAGGTTGCCTACGTGTTACT-3', and 5'-TCCCAACGAGTGGCGGACGG-3' for forward primer 92F, reverse primer 157R, and TaqMan probe 113T, respectively.

5' Nuclease PCR assay conditions.
The amplification reactions were carried out 50-µl (total volume) mixtures. These mixtures contained 1x TaqMan Universal PCR master mixture (Applied Biosystems), which contained each of the L. thermotolerans-specific primers at a concentration of 900 nM, 250 nM fluorescence-labeled L. thermotolerans-specific probe, and 20 ng of DNA for each treatment. The amplification reactions were performed with an ABI PRISM 7000 sequence detection system (Applied Biosystems), and the reactions were carried out by incubation for 2 min at 50°C (for activation of the uracil N-glycosylase) and for 10 min at 95°C (for activation of the AmpliTaq Gold DNA polymerase), followed by 15 s at 95°C (for denaturation) and 1 min at 63°C (for annealing and extension) for 40 cycles. Due to the positive signal of the closely related bacterium Lactobacillus mucosae (95% similarity), we increased the annealing temperature from 60 to 63°C. Data analysis was carried out using the ABI PRISM 7000 sequence detection system software (v1.0; Applied Biosystems). Each sample was analyzed in triplicate.

Specificity of the PCR assay.
The DNAs extracted from the pure culture of L. thermotolerans and the reference strains were used to test the specificity of the primer-probe set. The most closely related strains, L. mucosae DSM 13345^T and Lactobacillus fermentum JCM 1173^T, had two mismatches in the probe that was designed, which enabled discrimination between strains. Other bacterial strains were discriminated from L. thermotolerans by evaluation of a threshold cycle (C_t) value. The C_t was defined as the cycle at which the fluorescence was significantly different from the background. Therefore, the C_t value provided an accurate measure of the number of target molecules originally present in the sample. Samples of the purified DNA (20 ng) were used as templates in the real-time PCR assay, which was carried out by using the thermal cycling conditions and data analysis procedure described above. Sterilized distilled water (SDW) was used as a nontemplate control.

Accuracy of the assay.
In order to test the accuracy of the assay, 0.1-g samples of feces were mixed with various amounts (50, 100, or 150 µl) of a pure culture of L. thermotolerans (1.84 x 10⁷ cells/ml); the mixtures were then subjected to DNA extraction, and then the numbers of cells were determined by real-time PCR. The assay was repeated twice.

Total count for the bacterial population as determined with DAPI.
The total numbers of cells in the pure culture (L. thermotolerans cells/ml) and in the feces were determined after the samples were stained with a 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) n-hydrate solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 5 min at room temperature. The cells were examined with an Olympus BX50 microscope equipped with BX-FLA (Olympus Corporation, Tokyo, Japan). The DAPI signal was captured in 10 random microscopic fields.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Primer and probe design and specificity.
The specificity of the assay with the primer-probe combination that we developed for the detection of L. thermotolerans was assessed using purified genomic DNA from a target strain and the negative controls listed in Table 1. Real-time PCR-generated C_t values of 18.57 and 34.85 (averages) were obtained for our target strain and the negative controls, respectively. However, a C_t value of 34.50 was obtained for SDW, which corresponded to the value obtained for the negative controls (Table 1). Because of the hypothesis (5) that the C_t values of the negative controls and the SDW might have been due to Escherichia coli DNA contamination of the Taq polymerase, we repeated the assay after treatment of the master mixture with DNase I (Promega Corporation, Madison, WI). However, even after this treatment, we were unable to remove the signal obtained with the negative control and the SDW samples.

Calibration curve and sensitivity of the assay.
In order to construct a calibration curve and to determine the sensitivity of both primer-probe combinations, serial dilutions of DNA prepared from the pure culture of L. thermotolerans (1.84 x 10⁷ cells/ml) were used for the PCR assay (Fig. 1). The minimum detection limit of the assay was 1.84 x 10³ cells/ml of pure culture of L. thermotolerans (C_t, 32.22; SDW C_t, 34.50). The calibration curve was constructed by plotting the C_t values (Fig. 1) against known serial dilutions of L. thermotolerans DNA corresponding to cell concentrations between 1.84 x 10³ and 1.84 x 10⁷ cells/ml; the resulting curve is shown in Fig. 2. Each sample was analyzed in triplicate, and the variation in the C_t values of multiple replicate runs was found to be very low, as indicated by the standard deviation (Fig. 2). The efficiency of the curve was excellent (>94%), as the correlation coefficient was 0.9974, and a slope of –3.4652 was obtained. The efficiency was calculated on the basis of 100% efficiency corresponding to a slope of –3.32192 (14).

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FIG. 1. Amplification sensitivity of the 5' nuclease PCR assay for L. thermotolerans. DNA isolated from log-phase bacteria (1.84 x 107 cells/ml) was used in serial 10-fold dilutions. Delta Rn, fluorescence intensity after subtraction of the background signal. Line 1, signal of the original DNA sample corresponding to bacterial cells at a concentration of 1.84 x 107 cells/ml; line 2, 10-fold dilution of the original extracted DNA; line 3, 102-fold dilution of the original extracted DNA; line 4, 103-fold dilution of the original extracted DNA; line 5, 104-fold dilution of the original extracted DNA; line 6, nontemplate control.

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FIG. 2. Calibration curve. The Ct values obtained in Fig. 1 were plotted against known numbers of L. thermotolerans cells ranging from 1.84 x 103 to 1.84 x 107 cells/ml of pure culture. The R2 value was 0.9974. The Ct values shown are the averages of three replicates. The error bars indicate standard deviations.

Accuracy of the assay.
In order to test the accuracy of the assay, 50-, 100-, and 150-µl pure cultures of L. thermotolerans containing 9.2 x 10⁵, 1.8 x 10⁶, and 2.8 x 10⁶ cells (DAPI counts), respectively, were added to chicken feces (0.1 g, containing 2.1 x 10⁵ L. thermotolerans cells; real-time monitoring), and this was followed by DNA extraction and quantification by real-time PCR. All of the samples yielded values that were somewhat high, but the cell numbers were almost the same as the expected cell numbers (Fig. 3), thus demonstrating correlation regression (R², 0.9976). The results obtained suggest that the recovery of the cells from the samples was quite accurate; therefore, this newly developed PCR method can be used to examine the bacterial content of chicken feces.

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FIG. 3. Accuracy of real-time PCR assay. DNA from chicken feces samples (0.1 g) mixed with 50, 100, or 150 µl of a pure culture of L. thermotolerans (1.84 x 107 cells/ml) was extracted and detected by real-time PCR. The numbers of cells determined by real-time PCR () were compared to the expected values (•). Regression R2 values of 0.9976 and 0.9996 were obtained. The error bars indicate standard deviations.

Detection of L. thermotolerans in chicken feces.
Figure 4 shows the populations of L. thermotolerans in the chicken feces of individual chickens in two trials monitored by real-time PCR. In trial 1, the population of L. thermotolerans ranged from 1.8 x 10³ to 9.7 x 10⁹ cells/g feces. In general, the chickens showed a tendency toward higher numbers of L. thermotolerans cells until day 27. In trial 2, the total L. thermotolerans results also showed a tendency toward slow increases in some chickens from day 1 to day 21.

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FIG. 4. Changes in the number of L. thermotolerans cells at different times for individual chickens, as detected by real-time PCR. The results of trials 1 (left) and 2 (right) are shown. Each trial was conducted using five chickens, and the designations of the chickens are indicated on the graphs.

Table 2 shows the average total L. thermotolerans count for each trial, as obtained by real-time PCR performed with chicken feces samples. In trial 1, a higher number of cells (2.4 x 10⁹ cells/g feces) was observed on day 27 compared with the numbers observed from day 4 to day 18 (10⁴ to 10⁵ cells/g feces). In trial 2, the average cell populations increased 10-fold (10⁶ to 10⁷ cells/g feces) from day 1 to day 21. The total cell populations measured by DAPI staining were approximately 10¹¹ cells/g feces in both trials. Thus, L. thermotolerans cells accounted for 9.2 x 10^–6 to 8.5 x 10^–1% of the total cells in the feces in trial 1 and for 1.5 x 10^–3 to 4.5 x 10^–2% of the total cells in trial 2.

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TABLE 2. Mean numbers of total cells and L. thermotolerans cells in chicken feces as determined by DAPI staining (total cells) and by real-time PCR (L. thermotolerans)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here, we developed a real-time PCR assay that is rapid, specific, and sensitive for monitoring L. thermotolerans. The minimum sensitivity of our method was 1.84 x 10³ cells/ml of pure culture of L. thermotolerans, which is rather low compared to the sensitivities of other analyses, for which detection limits of 0 log₁₀ have been reported (5, 13). This was because of the unusual fluorescence signal expressed in the negative controls and SDW (Table 1), probably due to contamination by nucleotides present in the PCR master mixture, as previously reported (5). Although we attempted to eliminate the signal by using DNase I, the C_t value remained unchanged. The specificity of the primer-probe set used in the newly developed real-time PCR assay for L. thermotolerans detection was established by discrimination between intestinal bacteria and food bacteria (Table 1). The accuracy of this real-time PCR assay was nonetheless demonstrated in another quantitative experiment, in which feces were mixed with a known amount of a pure culture of L. thermotolerans for evaluation of the recovery achieved with the assay described here (Fig. 3). All of the samples yielded slightly higher numbers of cells than the numbers expected, which may have been due to an error in the DAPI counting of the added cells in L. thermotolerans pure cultures. It has been reported previously that phenolic compounds from plants may bind to DNA and thus can interfere with PCRs (7). The results shown in Fig. 3 clearly indicate that our new method is free from PCR inhibitors associated with the fecal samples. In fact, the possibility of inhibition of the PCR when fecal samples from chickens reared on commercial feed are used is thought to be lower than the possibility associated with PCR studies of herbivores, whose feed contains phenolic compounds originating from plant materials. Based on our results, we concluded that the present method enabled accurate and sensitive detection of L. thermotolerans cells in chicken feces. This new method thus provides a powerful tool for monitoring the distribution and abundance of this bacterium in a complex microbial community.

In general, most of the chickens used in trial 1 showed an increase in the L. thermotolerans population in the feces samples collected from day 4 to day 27 (Fig. 4). These results suggest that L. thermotolerans colonizes slowly (i.e., until the second week of life) and then rapidly proliferates until day 27. The average cell population of L. thermotolerans on day 27 (Table 2) was found to be 10-fold greater than that of Lactobacillus salivarius (10⁸ cells/g feces on day 40) reported previously (11) in chicken feces. In trial 2, the initial number of target cells on day 1 in chicks 1 and 3 was higher than the number of cells in chickens in trial 1. Moreover, the increase in the level of the target strain with time tended to be slower than the increase in the level of the target strain observed in trial 1. In the present study, the two trials were conducted at different times using different batches of chicks. Moreover, the microbiota of the chicks was not controlled by the suppliers. Therefore, based on our results, we concluded that L. thermotolerans is indeed a normal member of the microbiota of the chicken intestine. Since the total bacterial population in chicken feces is approximately 10¹¹ cells/g, our results demonstrated that L. thermotolerans accounts for a minor percentage of the bacterial diversity in chicken feces (Table 2).

In conclusion, we developed a highly sensitive and specific real-time PCR assay for detection of the L. thermotolerans present in chicken feces. This newly developed assay was successfully used to monitor the dynamics of this novel bacterium in chicken feces, thereby providing a powerful tool for studying the distribution and abundance of this bacterium in a complex microbial community. Our results also demonstrated that this novel bacterium is a normal member of the chicken intestinal microbiota.

We are currently developing a method involving fluorescent in situ hybridization coupled with confocal laser scanning microscopy to visualize epithelium surface colonization by L. thermotolerans in the chicken gastrointestinal tract. These studies, together with enumeration of this bacterium in the chicken intestine by real-time PCR, should provide much more comprehensive information about the ecology of L. thermotolerans in the chicken gastrointestinal tract.

 
This work was done by collaboration in a core university program between Yamaguchi University and Kasetsart University, which was supported by the Scientific Cooperation Program agreed to by the Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT). Abu Sadeque Md. Selim received a scholarship from the Ministry of Education, Culture, Sports, Science and Technology of Japan to support his studies in Japan.

We thank Yasuo Kobayashi and Kozo Asano, Graduate School of Agriculture, Hokkaido University, for critically reading the manuscript.

 
* Corresponding author. Mailing address: Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan. Phone: 81 11 706 2501. Fax: 81 11 706 4961. E-mail: yokota{at}chem.agr.hokudai.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, August 2005, p. 4214-4219, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4214-4219.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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• Martin, B., Jofre, A., Garriga, M., Pla, M., Aymerich, T. (2006). Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR. Appl. Environ. Microbiol. 72: 6040-6048 [Abstract] [Full Text]  

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