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Keywords:

  • Bifidobacterium breve;
  • human faeces;
  • probiotic strain;
  • propidium monoazide;
  • quantitative real-time PCR;
  • strain specific

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Aims:  To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces.

Methods and Results:  The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 105–109 cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g−1 [mean (±SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide–carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g.

Conclusions:  Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately.

Significance and Impact of the Study:  Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The importance of a healthy lifestyle has created a great interest in probiotics. In the human gastrointestinal tract, bifidobacteria are a numerically important group of micro-organisms that are considered to exert positive influences on biological activities related to host health (Collins and Gibson 1999; Kleerebezem and Vaughan 2009; Leahy et al. 2005). Bifidobacterium breve strain Yakult (BbrY) has been used in fermented milk products for many years and is one of the most intensively studied probiotics. Some of the various benefits of BbrY include improving the balance of intestinal microbes by increasing the numbers of beneficial bacteria and decreasing the numbers of harmful bacteria, increasing the total amount of volatile fatty acids in the gastrointestinal environment, decreasing urinary mutagenicity, activating the immune system and anti-infectious activity, and acting as an anti-inflammatory bowel disease adjunct (Asahara et al. 1999, 2004; Imaoka and Umesaki 2009; Ishikawa et al. 2003; Kanamori et al. 2009; Kanazawa et al. 2005; Kato et al. 2004; Matsumoto et al. 2001; Shimakawa et al. 2003; Sugawara et al. 2006).

The basic requirement for probiotic bacteria to exert expected positive effects is to be alive (Fuller 1989); therefore, appropriate quantification methods are crucial (Klaenhammer and Kullen 1999). To determine the effectiveness of probiotics, it is therefore essential to establish a specific method to identify them and measure their numbers.

The current method for detecting and identifying BbrY in faecal samples after ingestion of BbrY involves using both transgalactosylated oligosaccharide–carbenicillin (T-CBPC) selective agar medium for BbrY and strain-specific identification by randomly amplified polymorphism DNA fingerprinting (RAPD; Williams et al. 1990) or by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (Shimakawa et al. 2003). These methods, however, require considerable time, labour, experience and skill for isolating BbrY. Recently, a PCR-based method for strain-specific identification using a strain-specific primer has been reported (Ahlroos and Tynkkynen 2009; Bunte et al. 2000; Fujimoto et al. 2008; Maruo et al. 2006). This method is a powerful tool for identifying and enumerating specific strains from various kinds of materials.

We developed a strain-specific primer set for BbrY (pBbrY) by using a specific RAPD band sequence from BbrY. Using quantitative real-time PCR (qPCR) with pBbrY, we found that the number of BbrY in the faeces after ingestion of BbrY was about ten times higher than the number of living bacteria detected by the culture method. However, the number determined by the former method included both viable and dead cells. Although bifidobacteria have beneficial effects in both the live and dead state (López et al. 2010; Yasui et al. 1999; Young et al. 2004), live cells can proliferate and secrete beneficial factors. It is therefore important to be able to discriminate viable cells from dead cells to characterize the usefulness of BbrY as a probiotic.

Recently, differentiation of viable from dead cells in samples with several types of bacteria has been accomplished by using a PCR-based method with propidium monoazide (PMA) treatment, which selectively penetrates dead cells, which have compromised membrane integrity, but not viable cells with intact cell membranes (Bae and Wuertz 2009; García-Cayuela et al. 2009; Nocker et al. 2007). PMA is a DNA-intercalating dye that enables covalent binding to DNA under bright visible light; this makes DNA insoluble and strongly inhibits PCR amplification. Subjecting a bacterial population to PMA treatment before PCR therefore results in selective amplification of DNA from viable cells with intact membranes.

In this study, we developed a procedure for the detection and quantification of viable BbrY in faeces by a PCR-based method. The procedure was based on the combined use of the DNA-intercalating agent PMA and qPCR, using strain-specific primers derived from RAPD analysis. We also demonstrated the application of this technique for enumerating viable BbrY in faeces after the ingestion of fermented milk containing BbrY.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Reference strains and culture conditions

The 112 bacterial strains (30 strains of Bif. breve and 82 other strains of bacteria commonly isolated from human faeces; Table 1) were obtained from the Culture Collection of the Yakult Central Institute (YIT and Y; Tokyo, Japan). Anaerobic bacteria were cultured at 37°C for 1 or 2 days in GAM broth, Modified ‘Nissui’ (code 05433; Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) supplemented with 0·5% glucose. Lactic acid bacteria were cultured in MRS broth (Becton Dickinson, Sparks, MD, USA) at 37°C for 1 day. For a quantitative PCR standard, the number of BbrY was counted after the bacteria had been stained with 4′,6-diamino-2-phenylindole (DAPI). Briefly, 500 μl of bacterial cells was collected and resuspended with 450 μl of PBS. Then, 50 μl of formaldehyde (3·7% final concentration) was added to the bacterial suspension to fix the cells. The suspension was left overnight at 4°C. Then, 10 μl of fixed bacterial suspension was dropped onto each 1 × 1 cm well of an MAS-coated glass slide (Matsunami Glass Ind., Ltd, Osaka, Japan). The glass slide was left overnight at RT and washed once with 99·5% ethanol. After being air-dried, bacterial cells were stained with 5 μl of DAPI and embedded in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Microscopic counts and image acquisition from glass slides were performed using a Q-550 FW system (Leica DM RXA2 and Q-Fluoro, Wetzlar, Germany). Ten fields were used, and a minimum of 100 cells were counted per field.

Table 1.   Bacterial strains used in this study
  1. All strains were obtained from the Yakult Central Institute (YIT and Y; Tokyo, Japan). The identification number for each strain other than type strains is given in parentheses. ATCC, American Type Culture Collection (USA); DSM, German Collection of Microorganisms and Cell Cultures (German); JCM, Japan Collection of Microorganisms (Japan).

Bacteroides (Bact.) spp.Bact. distasonis YIT 6162T, Bact. fragilis YIT 6158T, Bact. ovatus YIT 6161T, Bact. thetaiotaomicron YIT 6163T, Bact. uniformis YIT 6164T, Bact. vulgatus YIT 6159T
Bifidobacterium (Bif.) spp. (except Bif. breve)Bif. adolescentis YIT 4011T, Bif. angulatum YIT 4012T, Bif. animalis YIT 4044T, YIT 4121 (DSM 10140), Bif. asteroides YIT 4033T, Bif. bifidum YIT 4039T, Bif. boum YIT 4091T, Bif. catenulatum YIT 4016T, Bif. choerinum YIT 4067T, Bif. coryneforme YIT 4092T, Bif. cuniculi YIT 4093T, Bif. dentium YIT 4017T, Bif. gallicum YIT 4085T, Bif. gallinarum YIT 4094T, Bif. indicum YIT 4083T, Bif. longum YIT 4018 (ATCC 15697), YIT 4021T, YIT 4082 (JCM 1269), Bif. magnum YIT 4098T, Bif. merycicum YIT 4095T, Bif. minimum YIT 4097T, Bif. pseudocatenulatum YIT 4072T, Bif. pseudolongum ssp. globosum YIT 4101T, Bif. pseudolongum ssp. pseudolongum YIT 4102T, Bif. pullorum YIT 4104T, Bif. ruminantium YIT 4105T, Bif. saeculare YIT 4111T, Bif. subtile YIT 4116T, Bif. thermophilum YIT 4073T
Bif. breveBif. breve YIT 4014T, YIT 4015 (ATCC 15698), YIT 4023, YIT 4024, YIT 4043, YIT 4049 (ATCC 15701), YIT 4063, YIT 4064, YIT 4079, YIT 11016, YIT 11043, YIT 11044, YIT 11045, YIT 11046, YIT 11047, YIT 11049, YIT 11062, YIT 11063, YIT 11064, YIT 11065, YIT 11066, YIT 11067, YIT 11068, YIT 11069, YIT 11888, Y 91010, Y 91023, Y 94016, Y 94028, strain Yakult (YIT 12272)
Clostridium (Cl.) spp.Cl. celatum YIT 6056T, Cl. perfringens YIT 6050T
Collinsella aerofaciensC. aerofaciens YIT 10235T
Enterococcus (Ent.) spp.Ent. faecalis YIT 2031T, Ent. faecium YIT 2032T
Escherichia coliE. coli YIT 6044T
Eubacterium (Eu.) spp.Eu. biforme YIT 6076T, Eu. rectale YIT 6082T
Lactobacillus (Lact.) spp.Lact. acidophilus YIT 0070T, Lact. amylophilus YIT 0255T, Lact. amylovorus YIT 0211T, Lact. bifermentans YIT 0260T, Lact. brevis YIT 0076T, Lact. buchneri YIT 0077T, Lact. casei YIT 0180T, YIT 9029, Lact. coryniformis ssp. coryniformis YIT 0237T, Lact. crispatus YIT 0212T, Lact. delbrueckii ssp. delbrueckii YIT 0080T, Lact. delbrueckii ssp. lactis YIT 0086T, Lact. delbrueckii ssp. bulgaricus YIT 0181T, Lact. fermentum YIT 0081T, Lact. gallinarum YIT 0218T, Lact. gasseri YIT 0192T, Lact. helveticus YIT 0083T, Lact. johnsonii YIT 0219T, Lact. malefermentans YIT 0271T, Lact. oris YIT 0277T, Lact. parabuchneri YIT 0272T, Lact. paraplantarum YIT 0445T, Lact. pentosus YIT 0238T, Lact. plantarum YIT 0102T, Lact. pontis YIT 0273T, Lact. reuteri YIT 0197T, Lact. rhamnosus YIT 0105T, Lact. sakei YIT 0247T, Lact. salivarius ssp. salivarius YIT 0104T, Lact. sharpeae YIT 0274T, Lact. vaginalis YIT 0276T, Lact. zeae YIT 0078 (ATCC 393)
Lactococcus (L.) spp.L. garviae YIT 2071T, L. lactis ssp. cremoris YIT 2007T, L. lactis ssp. lactis YIT 2008T, L. lactis ssp. hordiniae YIT 2060T, L. plantarum YIT 2061T, L. raffinolactis YIT 2062T
Propionibacterium acnesP. acnes YIT 6165T
Ruminococcus (R.) spp.R. bromii YIT 6078T, R. lactaris YIT 6084T, R. productus YIT 6141T
Streptococcus thermophilusStrep. thermophilus YIT 2001, YIT 2021, YIT 2037T

RAPD-PCR analysis

Bacterial DNA for RAPD analysis was extracted by physical destruction and benzyl chloride purification, as previously described (Fujimoto et al. 2008). RAPD-PCR amplification was performed using 27 RAPD primers, as described by Fujimoto et al. (2008). RAPD products were electrophoresed at 50 V in a 1·5% agarose gel.

Cloning and sequence analysis of RAPD products specific to BbrY

Potential strain-specific RAPD markers were extracted from agarose gels with a Suprec-01 gel extraction kit (Takara, Shiga, Japan). The collected amplification products were cloned using a TA cloning kit with pCR 2.1 vector (Invitrogen, Leek, the Netherlands). The nucleic acid sequences of eight clones of each potential strain-specific RAPD marker were determined with an ABI model 373A DNA sequencer with a Dye Terminator sequencing kit (Applied Biosystems, Foster, CA, USA).

Specificity of RAPD-derived primers

The specificity of the BbrY-specific primer set (pBbrY) was confirmed by PCR using DNA from 112 bacterial strains (Table 1). PCR amplifications were performed in a DNA Engine PTC-200 (MJ Research, Waltham, MA, USA). Each reaction mixture (20 μl) contained 10 mmol Tris–HCl (pH 8·3), 50 mmol KCl, 1·5 mmol MgCl2, 200 μmol of each dNTP, 1·5 U Taq DNA polymerase (Takara), 0·3 μmol primers and 10 ng template DNA. The amplification programme consisted of one cycle of 94°C for 2 min; 32 cycles of 94°C for 20 s, 60°C for 10 s and 72°C for 20 s; and finally one cycle of 72°C for 3 min. PCR products were electrophoresed at 100 V in a 1·5% agarose gel.

PMA treatment

Pure culture of BbrY or faecal samples were treated with PMA, as described by Nocker et al. (2007). PMA (Biotium, Inc, CA, USA) was dissolved in 20% dimethyl sulfoxide to create a 20 mmol l−1 PMA stock solution and stored at −20°C in the dark. An adequate amount of PMA stock solution was added to 500 μl of pure culture of BbrY or 10-times-diluted faecal solutions to make final concentrations of 5, 50 and 150 μmol l−1. Following incubation at room temperature for 5 min in the dark with occasional mixing, triplicate samples were light-exposed for 1, 2 and 5 min at a distance of about 20 cm from two 500-W halogen light sources. After photo-induced cross-linking, cells were pelleted at 20 000 g for 4 min to remove the supernatant. PMA-treated samples were preserved at −80°C until the DNA was extracted.

Quantification of added BbrY in faecal samples by quantitative PCR

Various concentrations of viable or heat-killed (incubated at 80°C for 10 min) BbrY (104–109 cells per g of faeces) were added to three faecal samples containing no BbrY (confirmed by the culture method and qPCR with pBbrY). The total concentration of intestinal micro-organisms by DAPI was 10·7 (±0·4) log cells per g [mean (±SD)]. Faecal samples were collected in individual sterile Faeces Containers (Sarstedt, Nümbrecht, Germany), refrigerated and taken to the laboratory within 4 h. The DNA was extracted from these mixed faeces and subjected to qPCR analysis using pBbrY.

Examination of faecal samples after ingestion

Informed consent was obtained from the volunteers who provided the faecal samples used. The Ethical Committee of the Yakult Central Institute provided ethical clearance for this microbiological research study in accordance with the Helsinki Declaration. The study population comprised 11 healthy volunteers (age range, 23–59 years; mean ± SD, 32·8 ± 10·3 years) who ingested a commercially available fermented milk product (Bifine S™; Yakult Hansha Co. Ltd, Tokyo, Japan), containing 10·7 log CFU BbrY, once daily for 10 days. Faeces excreted before and after drinking the fermented milk product were collected in individual sterile Faeces Containers (Sarstedt), refrigerated and taken to the laboratory within 4 h. No subject ingested probiotic products, including the study product, during the 3 weeks before drinking the BbrY-containing fermented milk product for this study.

Enumeration of BbrY by the standard culture method

Counts (in CFU) of BbrY were determined using strain-specific T-CBPC selective agar medium. T-CBPC selective agar medium consists mainly of transgalactosylated oligosaccharide (TOS) as a growth factor, and carbenicillin (CBPC) and streptomycin as selective agents of Bif. breve strain Yakult (BbrY). We used commercially available TOS propionate agar medium (Yakult Pharmaceutical Industry Co., Ltd, Tokyo, Japan) supplemented with CBPC and streptomycin as a strain-specific selective culture medium. The medium contained (per litre) 10 g of trypticase, 1 g of yeast extract, 3 g of KH2PO4, 4·8 g of K2HPO4, 3 g of (NH4)2SO4, 0·2 g of MgSO4, 0·5 g of l-cysteine, 15 g of sodium propionate, 10 g of TOS, 15 g of powdered agar, 5000 000 units of streptomycin sulfate (Sigma Chemical, St. Louis, MO, USA) and 1000 μg of CBPC disodium salt (Sigma). Aliquots (0·1 ml) of tenfold serial dilutions of faeces (starting sample, 0·5 g) in 0·85% NaCl were spread on T-CBPC agar and incubated anaerobically at 37°C for 72 h. For each faecal sample, we collected all colonies from the T-CBPC plate inoculated with the highest dilution that yielded growth and subjected these colonies to real-time PCR analysis using pBbrY. The number of BbrY per gram of faeces (wet weight) was estimated from the number of colonies that were identified as containing BbrY by PCR with pBbrY. All isolates were also analysed by RAPD analysis.

Extraction of DNA from faecal samples

The DNA from faecal samples was extracted by using a Stool Mini kit (Qiagen, Valencia, CA, USA), as previously described (Fujimoto et al. 2008), with slight modification. The tenfold-diluted faecal solution (200 μl) was pelleted by centrifugation at 20 000 g for 5 min, washed three times with 1·0 ml phosphate-buffered saline to remove PCR inhibitors and suspended in 600 μl Buffer ASL (Qiagen). The faecal suspension was heated to 70°C for 5 min. The suspension was vortexed with glass beads (700 mg; 0·1 mm in diameter) and 500 μl buffer-saturated phenol using a FastPrep Fp120 (Bio 101, Irvine, CA, USA) at a speed setting of 6·5 m s−1 for 30 s. We added 100 μl of 3 mol l−1 sodium acetate (pH 4·8) to the suspension, which was kept on ice for 5 min and then centrifuged at 20 000 g for 8 min. The supernatant (700 μl) was mixed with 700 μl Buffer ASL and an InhibitEX tablet (Qiagen). After centrifugation of the mixture at 20 000 g for 2 min, 550 μl of supernatant was mixed with 550 μl of Buffer AL (Qiagen) and 550 μl of 100% ethanol. The lysate was trapped on a QIAamp spin column (Qiagen). After the column was washed with washing buffer (AW1 and AW2; Qiagen), DNA was eluted in 100 μl of Buffer AE (Qiagen).

Real-time PCR analysis

PCR amplification and detection of all or live BbrY were performed in an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems), as previously described (Fujimoto et al. 2008), with slight modification. The reaction mixture (20 μl) contained 10 mmol l−1 Tris–HCl (pH 8·3), 50 mmol l−1 KCl, 1·5 mmol l−1 MgCl2, 200 μmol l−1 of each dNTP, 500 μg ml−1 bovine serum albumin (Takara), a 1 : 75 000 dilution of SYBR Green I (Invitrogen), 0·4 U Taq DNA polymerase Hot Start version (Takara), 0·3 μmol of each of the specific primers and 5 μl of template DNA diluted 10-, 102-, or 103-fold. The amplification programme consisted of an initial heating step at 94°C for 5 min; 40 cycles of 94°C for 20 s, 60°C for 10 s and 72°C for 20 s; and a final extension step at 72°C for 3 min. Fluorescence intensities were detected during the last step of each cycle. To distinguish the targeted PCR product from the nontargeted PCR products (Ririe et al. 1997), melting curves were obtained after amplification by slow heating from 60 to 95°C in increments of 0·2°C s−1 with continuous fluorescence collection.

Statistical methods

Pearson’s correlation coefficients were used to determine the correlations between the number of added BbrY and the counts obtained with qPCR in the experiment with added BbrY. Simple linear regression was used to develop regression equations for statistically significant relationships. Mean differences in the concentrations of BbrY using qPCR were analysed by the paired t-test. The data obtained to optimize the PMA treatment test were statistically analysed by the variance analysis, and the means were separated according to Tukey’s HSD test with a significance level (P) of 0·05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Screening for strain-specific RAPD markers

To identify a strain-specific PCR product for BbrY, we tested a total of 27 RAPD primers on 30 Bif. breve strains. Primer p1285 (5′ AGC CAG TTT C 3′) generated a 1·1-kb BbrY-specific band (arrows, Fig. 1). We determined the sequence of this BbrY-specific PCR product (accession no. AB568490). As a result of the FASTA analysis in the DDBJ/GenBank/EMBL DNA database, we found that the partial sequence of BbrY-specific PCR product (position 106–860) had 80·3% similarity to the SalX-type ABC antimicrobial peptide transport system ATPase component of Bifidobacterium longum DJO10A (CP000605, position 1925211–1926284).

image

Figure 1.  RAPD patterns obtained from ten Bifidobacterium breve strains using p1258 primer. Lane 1, 100-bp DNA size marker; lanes 2 and 12, Bif. breve strain Yakult (BbrY); lanes 3 through 11, other strains of BbrY. Arrows indicate potential strain-specific bands of BbrY. RAPD, randomly amplified polymorphism DNA.

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Design of a specific RAPD-derived primer pair

To amplify a BbrY-specific PCR product, we designed a candidate strain-specific primer set p1285-1F (AGC CAG TTT CGA GGT ATG GC) and p1285-1151R (AGC CAG TTT CCG AAG TTA CC) and used this primer set for PCR on 30 strains of Bif. breve. Five strains (YIT 4063, YIT 4064, YIT 4079, YIT 11888 and BbrY) yielded PCR products. These products were sequenced and aligned so that a new, more specific, target sequence for BbrY could be identified. We then designed a new set of BbrY-specific primers (pBbrY: pBbrY-F, ATG GCA AAA CCG GGC TGA A, and pBbrY-R, GCG GAT GAG AGG TGG G) and tested its specificity against DNA extracted from 112 bacterial strains, including the 30 strains of Bif. breve (Table 1). These primers exclusively supported PCR amplification of BbrY template DNA, with no cross-reaction against nontarget micro-organisms. The amplification product was 313 bp long and had a melting temperature of 89·8°C.

Quantitative PCR detection of viable or heat-killed BbrY with PMA treatment

To optimize the PMA treatments, viable and heat-killed BbrY cells were exposed to different concentrations of PMA (5, 50 or 150 μmol l−1) and different photoactivation times (1, 2 or 5 min). Using qPCR did not result in significant differences in enumeration of viable BbrY under any conditions (all 9·6 log cells per ml). In the case of heat-killed BbrY, using qPCR without PMA treatment resulted in a reduced count of BbrY (8·6 log cells per ml). The number of BbrY was significantly higher at a low PMA concentration (5 μmol l−1) than at PMA concentrations of 50 and 150 μmol l−1 (< 0·05). Changes in photoactivation time did not influence the enumeration of heat-killed BbrY when qPCR with PMA treatment at 50 or 150 μmol l−1 was used. Moreover, there was no significant difference between PMA treatments with 50 and 150 μmol l−1 in the number of heat-killed BbrY. PMA treatment at 5 μmol l−1 was not sufficient to significantly decrease the number of heat-killed BbrY compared with no PMA treatment.

We confirmed that the number of heat-killed BbrY using qPCR with PMA treatment in 50 μmol l−1 PMA solution and with 2-min photoactivation (4·7 (±0·3) log cells per ml) was significantly decreased (< 0·05) than that without PMA treatment [8·6 (±0·1) log cells per ml] (Fig. 2).

image

Figure 2.  Amplification by quantitative real-time PCR of DNA from heat-killed (80°C, 10 min) or unheated bacterial cells from pure culture of Bifidobacterium breve strain Yakult (BbrY) treated with different concentrations of PMA (5, 50 or 150 μmol l−1) and with photoactivation times (1, 2 or 5 min). (inline image) 0 min; (inline image) 1 min; (inline image) 2 min and (inline image) 5 min. PMA, propidium monoazide.

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Quantitative PCR detection of viable or heat-killed BbrY, with PMA treatment, added to faeces

We confirmed that PMA treatment worked to reduce the numbers of dead cells for quantifying viable BbrY in the faecal samples. We added 10·5 log cells per g−1 heat-killed BbrY directly to faecal samples. Following PMA/no PMA treatment, we extracted DNA and quantified BbrY by using qPCR with pBbrY. With heating, the number of BbrY by qPCR using pBbrY without PMA treatment was reduced slightly to 9·8 (±0·1) log cells per g, whereas the number of BbrY with PMA treatment was significantly reduced to 5·5 (±0·5) log cells per g (< 0·001).

We next added viable BbrY directly to faecal samples in amounts from 104 to 109 g−1. After PMA treatment, we analysed the correlation between the number of added BbrY and the value obtained with qPCR. We also determined the lower limit of detection of BbrY in the faeces by qPCR with pBbrY and PMA treatment. When 105–109 viable BbrY was added per gram of faeces, the qPCR gave accurate results (r2 = 0·9983, < 0·001; Fig. 3a). Faecal samples not treated with PMA had the same detection limit and correlation between added BbrY and the value obtained with qPCR (r2 = 0·9996, < 0·001; Fig. 3b).

image

Figure 3.  Correlation between the number of viable Bifidobacterium breve strain Yakult added to faecal samples and that determined by quantitative real-time PCR (qPCR) with PMA treatment (a) and without PMA treatment (b). The regression line was made between 105 cells per g and 109 cells per g, as detected by qPCR. The regression line was calculated with an intercept of 0. Error bars represent standard deviations from three independent tests. PMA, propidium monoazide.

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Quantitative detection of ingested BbrY in faeces

We used our quantitative PCR method with PMA treatment (with 50 μmol l−1 PMA solution and 2-min photoactivation) to measure the number of BbrY in the faeces of subjects who drank a fermented milk product containing BbrY. Before ingestion, the number of BbrY in the faeces was below the detection limit (qPCR, <105 cells per g; CFU, <102 CFU g−1), indicating that we used both the strain-specific qPCR method and the conventional culturing method in T-CBPC medium. After ingestion, BbrY was detected in all subjects at 8·1 (±0·8) log cells per g (mean ± SD) by qPCR without PMA treatment and was detected in 10 of 11 subjects at 7·5 (±1·0) log cells per g by qPCR with PMA treatment. In addition, BbrY was isolated from all subjects at 6·9 (±1·5) log CFU g−1 using a T-CBPC agar plate (Table 2).

Table 2.   Number of Bifidobacterium breve strain Yakult in the faeces of 11 volunteers who ingested a fermented milk product for 10 days
SubjectLog cells or CFU g−1 of faeces
Before ingestionAfter ingestion
qPCRCFUqPCRCFU
Without PMAWith PMAWithout PMAWith PMA
  1. PMA, propidium monoazide; qPCR, quantitative real-time PCR.

  2. *The lower limits of detection of quantitative real-time PCR (qPCR) and the culture method were 105 cells per g faeces and 102 CFU g−1 faeces, respectively.

  3. †Mean and ±SD were calculated by using the detection limit of qPCR (105 cells per g faeces) for subject b.

a<5·0*<5·0<2·0*8·58·47·9
b<5·0<5·0<2·06·7<5·03·3
c<5·0<5·0<2·08·58·37·4
d<5·0<5·0<2·08·88·47·9
e<5·0<5·0<2·06·45·34·8
f<5·0<5·0<2·08·47·97·6
g<5·0<5·0<2·08·47·76·9
h<5·0<5·0<2·09·18·88·3
I<5·0<5·0<2·08·27·16·8
j<5·0<5·0<2·08·28·07·2
k<5·0<5·0<2·08·58·17·5
Mean†<5·0<5·0<2·08·17·56·9
SD†0·81·01·5

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Numerous reports of PCR-based methods for strain-specific detection and enumeration have been recently published. Investigators designing strain-specific primer sets have used RAPD (Ahlroos and Tynkkynen 2009; Fujimoto et al. 2008; Maruo et al. 2006; Tilsala-Timisjarvi and Alatossava 1998), subtraction hybridization techniques (Bunte et al. 2000), sequence analysis of AFLP markers (Sisto et al. 2009), sequence data from the 16S–23S rRNA intergenic spacer regions, phage-related sequences and the S-layer gene (Brandt and Alatossava 2003; Flint and Angert 2005; Saito et al. 2004). In this study, we confirmed the RAPD technique to be one of the best methods of developing strain-specific primers because, as noted by Briczinski et al. (2009); it can compare the whole genome of many strains in detail to easily and rapidly find a strain-specific sequence.

Using both pBbrY and T-CBPC medium, we confirmed that BbrY and other strains with sequences identical to those of the target site of specific primer in their genomes did not exist in the volunteers’ faeces before they drank the fermented milk product containing BbrY.

We also quantified the number of BbrY by using the standard method of culture on T-CBPC selective agar medium. T-CBPC is a strain-specific selective medium. We then used PCR with pBbrY to identify some colonies that appeared on the T-CBPC agar plate at the highest faecal dilution that yielded growth. DNA extracted directly from 68 colonies that appeared on the T-CBPC agar plates was subjected to PCR analysis. Forty-eight isolates were identified as BbrY by using pBbrY and RAPD analysis, and the remaining 20 isolates were confirmed as non-BbrY by both methods. We have confirmed that PCR using pBbrY enabled us to identify the colonies on the T-CBPC medium efficiently and accurately. It also enabled us to reduce the need for the laborious process of identification that we had to use previously, including isolation and purification of isolates, RAPD or immunological methods using monoclonal antibody.

A DNA-intercalating dye, such as PMA, can be covalently linked to DNA by photoactivation and enables conventional PCR amplification of target DNA from viable but not dead cells. We attempted ethidium monoazide (EMA) treatment (Soejima et al. 2008) to enumerate viable BbrY using qPCR, but the number of viable BbrY with EMA treatment exhibited a clear declining trend about ten times lower (data not shown). EMA has been suggested as being toxic to some viable cells (Nocker et al. 2006; Pan and Breidt 2007); we therefore chose to use PMA treatment with qPCR to accurately enumerate viable BbrY. We confirmed the optimal conditions of PMA treatment (50 μmol l−1 PMA, 5-min incubation, 2-min photoactivation; Nocker et al. 2007) for enumeration of viable BbrY by varying the conditions and examining the results. PMA treatment did not affect the numbers of viable BbrY cells detected, but when compared with non-PMA treatment, it reduced the numbers of heat-killed cells detected (by about 1/10 000) (Fig. 2).

We have previously reported on a method of DNA extraction from faeces for qPCR (Fujimoto et al. 2008). The method includes destruction of glass beads, phenol extraction, degradation of RNA using RNase and purification using the Stool Mini kit (Qiagen). This extraction method can remove a large amount of existing rRNA by processing RNase, and consequently the background fluorescence in qPCR is less. We have also confirmed that qPCR with a strain-specific primer using DNA extracted by this method can detect Lactobacillus casei strain Shirota at 4·7 log cells per g faeces. In the present study, we reconfirmed that the detection limit of BbrY by qPCR without RNase treatment (105 cells per g faeces) was not significantly different from our previous results.

We confirmed that the counts of BbrY in faeces by qPCR with or without PMA treatment were highly and significantly correlated with the numbers of viable BbrY (Fig. 3). We also confirmed that heating at 80°C for 10 min was effective in killing all the viable cells of BbrY. The number of heat-killed BbrY detected in the faeces by qPCR with PMA treatment was 1/10 000 that detected without PMA treatment. Moreover, the number of BbrY detected by qPCR with PMA treatment did not change in faecal samples that had been supplemented with viable BbrY and preserved at −80°C for 3 months.

By qPCR without PMA treatment, the total number of BbrY detected in the faeces after ingestion of BbrY [8·1 (±0·8) log cells per g] was more than ten times the number of colony-formable bacteria detected by the culture-dependent method [6·9 (±1·5) log CFU g−1]. On the other hand, by qPCR with PMA treatment, the number of viable BbrY detected in the faeces after ingestion of BbrY was 7·5 (±1·0) log cells per g. Consequently, 40% of the total BbrY in the faeces after ingestion was viable (as judged by the integrity of the cell membrane in terms of its permeability to PMA entry), and the number of viable BbrY was four times the number of viable cells counted by the culture-dependent method. It is possible that culture on T-CBPC agar underestimates the number of viable BbrY cells because it has the inherent disadvantage of using selective culture media supplemented with antibiotics [even if the minimum inhibition concentrations of CBPC and streptomycin against BbrY were 6·25 μg ml−1 and >5000 U ml−1, respectively (data not shown)].

Precisely, how the bifidobacteria function as beneficial probiotics is not yet resolved. However, it has been suggested that viable bifidobacteria exert their health-promoting effects on the host via their metabolism and metabolites. In contrast, cell components of Bifidobacterium unrelated to whether the cells are viable or dead have been proposed to stimulate immune function (Leahy et al. 2005; López et al. 2010; Yasui et al. 1999). Consequently, there is a need to separately enumerate viable and dead cells if we are to understand the role of BbrY as a probiotic.

In conclusion, the BbrY-specific PCR primer set that we developed enabled efficient and accurate identification of the colonies that formed on T-CBPC medium. We confirmed that the use of a combination of quantitative PCR with PMA treatment and our BbrY-specific primers quickly and accurately analysed the number of viable BbrY in faecal samples. In the light of the increasing public interest in probiotics, we need to demonstrate the efficacy of our method in experiments with large numbers of test subjects. However, we believe that the strain-specific primers with PMA treatment we have described here will be powerful tools for understanding BbrY as probiotics in future studies.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • Ahlroos, T. and Tynkkynen, S. (2009) Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR. J Appl Microbiol 106, 506514.
  • Asahara, T., Shimizu, K., Ohashi, Y., Matsuki, T., Matsumoto, K., Takada, T., Yuki, N., Takayama, H. et al. (1999) The effects of bifidobacteria-fermented milk on human urinary mutagenicity, which increases following ingestion of cooked ground beef. J Intest Microbiol 12, 8996.
  • Asahara, T., Shimizu, K., Nomoto, K., Hamabata, T., Ozawa, A. and Takeda, Y. (2004) Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7. Infect Immun 72, 22402247.
  • Bae, S. and Wuertz, S. (2009) Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl Environ Microbiol 75, 29402944.
  • Brandt, K. and Alatossava, T. (2003) Specific identification of certain probiotic Lactobacillus rhamnosus strains with PCR primers based on phage-related sequences. Int J Food Microbiol 84, 189196.
  • Briczinski, E.P., Loquasto, J.R., Barrangou, R., Dudley, E.G., Roberts, A.M. and Roberts, R.F. (2009) Strain-specific genotyping of Bifidobacterium animalis subsp. lactis by using single-nucleotide polymorphisms, insertions, and deletions. Appl Environ Microbiol 75, 75017508.
  • Bunte, C., Hertei, C. and Hammes, W.P. (2000) Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract. Syst Appl Microbiol 23, 260266.
  • Collins, M.D. and Gibson, G.R. (1999) Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am J Clin Nutr 69, 1052S1057S.
  • Flint, J.F. and Angert, E.R. (2005) Development of a strain-specific assay for detection of viable Lactobacillus sp. HOFG1 after application to cattle feed. J Microbiol Methods 61, 235243.
  • Fujimoto, J., Matsuki, T., Sasamoto, M., Tomii, Y. and Watanabe, K. (2008) Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA. Int J Food Microbiol 126, 210215.
  • Fuller, R. (1989) Probiotics in man and animals. J Appl Bacteriol 66, 365378.
  • García-Cayuela, T., Tabasco, R., Peláez, C. and Requena, T. (2009) Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int Dairy J 19, 405409.
  • Imaoka, A. and Umesaki, Y. (2009) Rationale for using Bifidobacterium probiotic strains-fermented milk against colitis based on animal experiments and clinical trials. Probiotics Antimicrob Proteins 1, 814.
  • Ishikawa, H., Akedo, I., Umesaki, Y., Tanaka, R., Imaoka, A. and Otani, T. (2003) Randomized controlled trial of the effect of bifidobacteria-fermented milk on ulcerative colitis. J Am Coll Nutr 22, 5663.
  • Kanamori, Y., Iwanaka, T., Sugiyama, M., Komura, M., Takahashi, T., Yuki, N., Morotomi, M. and Tanaka, R. (2009) Early use of probiotics is an important therapy in infants with severe congenital anomaly. Pediatr Int 52, 362367.
  • Kanazawa, H., Nagino, M., Kamiya, S., Komatsu, S., Mayumi, T., Takagi, K., Asahara, T., Nomoto, K. et al. (2005) Synbiotics reduce postoperative infectious complications: a randomized controlled trial in biliary cancer patients undergoing hepatectomy. Langenbecks Arch Surg 390, 104113.
  • Kato, K., Mizuno, S., Umesaki, Y., Ishii, Y., Sugitani, M., Imaoka, A., Otsuka, M., Hasunuma, O. et al. (2004) Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis. Aliment Pharmacol Ther 20, 11331141.
  • Klaenhammer, T.R. and Kullen, M.J. (1999) Selection and design of probiotics. Int J Food Microbiol 50, 4557.
  • Kleerebezem, M. and Vaughan, E.E. (2009) Probiotic and gut lactobacilli and bifidobacteria: molecular approaches to study diversity and activity. Annu Rev Microbiol 63, 269290.
  • Leahy, S.C., Higgins, D.G., Fitzgerald, G.F. and Van Sinderen, D. (2005) Getting better with bifidobacteria. J Appl Microbiol 98, 13031315.
  • López, P., Gueimonde, M., Margolles, A. and Suárez, A. (2010) Distinct Bifidobacterium strains drive different immune responses in vitro. Int J Food Microbiol 138, 157165.
  • Maruo, T., Sakamoto, M., Toda, T. and Benno, Y. (2006) Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique. Int J Food Microbiol 110, 6976.
  • Matsumoto, S., Watanabe, N., Imaoka, A. and Okabe, Y. (2001) Preventive effects of Bifidobacterium- and Lactobacillus-fermented milk on the development of inflammatory bowel disease in senescence-accelerated mouse P1/Yit strain mice. Digestion 64, 9299.
  • Nocker, A., Cheung, C.Y. and Camper, A.K. (2006) Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67, 310320.
  • Nocker, A., Sossa-Fernandez, P., Burr, M.D. and Camper, A.K. (2007) Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73, 51115117.
  • Pan, Y. and Breidt, F. Jr (2007) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73, 80288031.
  • Ririe, K.M., Rasmussen, R.P. and Wittwer, C.T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245, 154160.
  • Saito, Y., Sakamoto, M., Takizawa, S. and Benno, Y. (2004) Monitoring the cell number and viability of Lactobacillus helveticus GCL1001 in human feces by PCR methods. FEMS Microbiol Lett 231, 125130.
  • Shimakawa, Y., Matsubara, S., Yuki, N., Ikeda, M. and Ishikawa, F. (2003) Evaluation of Bifidobacterium breve strain Yakult-fermented soymilk as a probiotic food. Int J Food Microbiol 81, 131136.
  • Sisto, A., De Bellis, P., Visconti, A., Morelli, L. and Lavermicocca, P. (2009) Development of a PCR assay for the strain-specific identification of probiotic strain Lactobacillus paracasei IMPC2.1. Int J Food Microbiol 136, 5965.
  • Soejima, T., Iida, K., Qin, T., Taniai, H., Seki, M. and Yoshida, S. (2008) Method to detect only live bacteria during PCR amplification. J Clin Microbiol 46, 23052313.
  • Sugawara, G., Nagino, M., Nishio, H., Ebata, T., Takagi, K., Asahara, T., Nomoto, K. and Nimura, Y. (2006) Perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery: a randomized controlled trial. Ann Surg 244, 706714.
  • Tilsala-Timisjarvi, A. and Alatossava, T. (1998) Strain-specific identification of probiotic Lactobacillus rhamnosus with randomly amplified polymorphic DNA-derived PCR primers. Appl Environ Microbiol 64, 48164819.
  • Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18, 65316535.
  • Yasui, H., Kiyoshima, J., Hori, T. and Shida, K. (1999) Protection against influenza virus infection of mice fed Bifidobacterium breve YIT 4064. Clin Diagn Lab Immunol 6, 186192.
  • Young, S.L., Simon, M.A., Baird, M.A., Tannock, G.W., Bibiloni, R., Spencely, K., Lane, J.M., Fitzharris, P. et al. (2004) Bifidobacterial species differentially affect expression of cell surface markers and cytokines of dendritic cells harvested from cord blood. Clin Diagn Lab Immunol 11, 684690.