Development of Strain-Specific PCR Primers for Quantitative Detection of Bacillus mesentericus Strain TO-A in Human Feces

Abstract

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10⁸ colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10^4.8 and 10^5.8 cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Probiotics are live microbial food supplements that beneficially affect the host by improving its microbial balance.^1,2) Probiotics have been isolated from both human and animal intestinal tracts.³⁾ Extensive studies describing the beneficial effects of probiotics on human health have been reported.^4–10)

The probiotic formulation BIO-THREE has been administered as a human and animal drug for over 50 years. Ingestion of BIO-THREE is believed to lead to refinement of gastrointestinal function through enhancement of the intestinal microbiota, thereby reducing the risk of infection. For example, studies have revealed that BIO-THREE ingestion leads to improvement in acute infectious diarrhea, inflammatory bowel disease (IBD), schizophrenia, and aids in recovery from pancreaticoduodenectomy.^11–14)

Bacillus mesentericus TO-A (abbreviated as BM TO-A) is one of the component strains of BIO-THREE. Based upon sequencing of the 16S ribosomal RNA (rRNA) gene (deposited in the GenBank/EMBL/DDBJ data library under accession no. AB687550) and the gyrA, groEL, polC, purH, and rpoB genes (accession nos. AB817068 to AB817072) according to the methods of Rooney et al.,¹⁵⁾ we recently determined that BM TO-A is closely related to B. subtilis. Bacillus subtilis is widely found in the environment, and in addition to its application in probiotics, B. subtilis has been extensively used in research and biotechnology.¹⁶⁾ Due to the high degree of genetic homology between various members of the B. subtilis group and related species, it can be difficult to identify the organism at the strain-specific level. Therefore, in this study we developed BM TO-A strain-specific primers using randomly amplified polymorphic DNA (RAPD). The level of BM TO-A in the feces of healthy volunteers was determined quantitatively using real-time quantitative polymerase chain reaction (qPCR) with the strain-specific primers before and after ingestion of a preparation containing the organism, and the results were compared to those obtained using standard culture methods.

MATERIALS AND METHODS

Bacterial Strains

BM TO-A was acquired from TOA Pharmaceutical Co., Ltd. (Tokyo, Japan), and 20 B. subtilis strains and strains of the related species B. amyloliquefaciens (IFO3034 and JCM20197) and B. atrophaeus (JCM9070 and JCM20230) were obtained from various culture collections (Fig. 1). All strains were grown on Tryptic Soy Agar (TSA, Eiken, Tokyo, Japan) for 24 h at 37°C.

DNA Extraction and Purification

DNA was extracted from bacterial cultures or feces using the method of Saito and Miura,¹⁷⁾ with some modifications. Briefly, appropriate aliquots of bacterial culture or 10-mg samples of feces were suspended in lysis buffer. Bacteria were homogenized using a FastPrep FP120 disruptor (MP Biomedicals, Santa Ana, CA, U.S.A.) operated at 6.5 m/s for 20–60 s, and the resulting lysate was subjected to phenol extraction and ethanol precipitation of DNA. DNA was purified using a Monofas DNA purification kit (GL Sciences, Tokyo, Japan), according to the manufacturer’s instructions. Finally, the DNA was eluted in TE buffer (10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) [pH 8.0]).

RAPD Analysis and Sequencing

The RAPD method used was based on that of Williams and used Ready-to-Go RAPD Analysis Beads (GE Healthcare, Buckinghamshire, U.K.).¹⁸⁾ RAPD amplification was carried out using a KOD-Plus (Toyobo, Osaka, Japan) with RAPD-primer 6 (5′-CCC GTC AGC A-3′). Aliquots of the RAPD-amplified products were electrophoresed at 50 V on a 3% agarose gel, which was then stained with SYBR Green. RAPD fingerprints and the associated dendrogram were analyzed using FPQuest (Bio-Rad, Hercules, CA, U.S.A.).

A potential BM TO-A strain-specific RAPD band was cloned using TArget Clone Plus (Toyobo) and Competent high DH5-alpha, viz. competent cells (Toyobo). The sequence of the RAPD band from the resulting clones was determined using a cycle sequencing method and a Big Dye Terminator cycle sequencing kit (Life Technologies, Carlsbad, CA, U.S.A.) and ABI PRISM 3100 Genetic Analyzer (Life Technologies).

Inverse PCR

Inverse PCR of B. mesentericus TO-A genomic DNA was based on the method of Ochman.¹⁹⁾ Briefly, genomic DNA was digested with DdeI (New England Biolabs, U.S.A.), and circularization and ligation were carried out using T4 DNA ligase. Inverse PCR was performed with the BM-invPCR primer set (5′-GGC TTT CAA TAT AGG TAC GGA GAG-3′, and 5′-CGT ATG CGT AAT TTG ATT CTT AAT G-3′), which was derived from the sequence of the B. mesentericus TO-A strain-specific RAPD band using KOD -Plus-. The amplification program consisted of 1 cycle of 94°C for 2 min, 30 cycles of 98°C for 10 s, 64°C for 30 s, and 68°C for 60 s, and finally, 1 cycle of 68°C for 5 min. Sequence analysis of the resulting products was carried out as described above.

Design of the BM TO-A Strain-Specific Primers

The sequences obtained from the potential BM TO-A strain-specific RAPD band and inverse PCR were analyzed for potential sequence similarity using BLAST searching of the GenBank database. Based on the RAPD band sequence, two BM TO-A strain-specific PCR primer sets were designed, which were designated BM-177 and BM-987. For more strain-specific and quantitative detection, BM-qPCR primers based on the inverse PCR sequence were designed for use in qPCR.

Validation of the Specificity of the Primers Designed for BM TO-A at the Strain Level

To ascertain the reliability of the DNA extraction procedure and the specificity of the BM TO-A strain-specific primer sets, multiplex PCR analysis was carried out using these primer sets along with a 16S universal primer set. The 16S universal primer set (named 16S-uni) was constructed by modification of primers reported by Hiraishi^20,21) (forward: 5′-CTG GCT CAG GAC GAA CG-3′; reverse: 5′-GTG ACG GGC GGT GTG TA-3′). The multiplex PCR used DNA from 25 bacterial strains, and the conditions were the same as those described above for inverse PCR, except that the final concentration of the 16S-uni primers was 60 nM.

Subject Recruitment and BM TO-A Ingestion Trial

This portion of the study was conducted based on the principles of the Declaration of Helsinki (Ethical Principles for Medical Research Involving Human Subjects). In choosing subjects for the BM TO-A ingestion trial, 24 participants were randomly selected from a pool of individuals who had answered a questionnaire and were confirmed to be healthy adults. Informed consent was obtained from each volunteer prior to the procedure, and the protocol was approved by the institutional review board of TOA Pharmaceutical. The study group consisted of 12 males and 12 females between the ages of 21 and 67 years (mean age [S.D.]: 42.8 [11.9] years).

The ingestion trial consisted of two consecutive 7-d periods: administration and withdrawal. During the administration period, subjects ingested 9 BM TO-A tablets/d, which was equivalent to 3×10⁸ colony forming unit (CFU) of BM TO-A/d.

Feces excreted on Day 0 (just before the start of the study), Day 3, and Day 7 of each period were collected in containers, refrigerated, and cultured within 8 h of collection. Fecal samples to be used for DNA extraction were suspended in lysis buffer and stored at −50°C until use.

No subject ingested other probiotic products including BM TO-A, such as BIO-THREE (TOA Pharmaceutical) during this study.

Examination of the Detection Limit and Linearity of BM TO-A in Feces Using qPCR

The addition of various concentrations of BM TO-A (10^4.3–10^9.8 per gram of feces) to 24 fecal samples collected on Day 0 negated the detectability of BM TO-A by both culture methods and qPCR using the BM-qPCR primer set. DNA was extracted from these mixed feces samples and subjected to qPCR analysis using the BM-qPCR primer set with EXPRESS SYBR GreenER (Life Technologies) and a CFX96 Real-Time System (BioRad). The amplification program consisted of 1 cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 67°C for 20 s, and 72°C for 30 s.

BM TO-A was quantified based on common methods.^22–24) That is, standard DNA was extracted from a dried powder of a known concentration of BM TO-A CFUs per gram. The extraction method was the same as that used for unknown samples. Standard DNA, diluted standard DNA, and unknown sample DNA were simultaneously analyzed by qPCR. The standard curve for quantification was constructed using the CT values of the standard DNA and the diluted standard DNA samples. Quantification of BM TO-A in the unknown sample was calculated based upon comparison of the corresponding CT values with the standard curve.

Quantitative Detection in Feces of Subjects That Had Ingested BM TO-A

The number of BM TO-A cells in feces was determined by culture using TSA with 3% NaCl. Aliquots (0.1 mL) of 10-fold serial dilutions of feces (starting sample, 0.5 g) in saline were spread onto agar plates and incubated aerobically at 51°C for 18 h.

The number of BM TO-A in feces was also determined using real-time PCR with the BM-qPCR primers with EXPRESS SYBR GreenER and a CFX96 Real-Time System. The amplification program was as described in the previous section.

Statistical Analysis

The Pearson’s correlation coefficient was used to determine the correlation between the number of added B. mesentericus TO-A CFUs and the number determined using qPCR. Statistical analyses were conducted using SPSS ver. 19 (IBM, Armonk, NY, U.S.A.). Differences were considered significant at a p value less than 0.05.

RESULTS

RAPD-PCR and Cloning of the PCR Product

In order to detect only BM TO-A and not other B. subtilis strains in feces, we employed the RAPD technique to develop strain-specific primers. The RAPD fingerprints and associated dendrogram obtained using RAPD-primer 6 are presented in Fig. 1. The dendrogram indicates that almost all of the B. subtilis strains examined formed a large cluster that includes BM TO-A, and two related species, B. amyloliquefaciens (strains IFO3034 and JCM20197) and B. atrophaeus (strains JCM9070 and JCM20230) were also connected.

The size of the PCR products obtained with RAPD-primer 6 varied from about 250 bp to 1.8 kb for BM TO-A. A potentially strain-specific RAPD product band of approximately 1 kb was isolated from the gel, cloned, and sequenced. The 991-bp DNA sequence was deposited in the GenBank/EMBL/DDBJ data library under accession no. AB642166. The reproducibility of the RAPD patterns was evaluated by repeating the experiment three times under the same RAPD conditions.

Nucleotide Sequence of the Specific RAPD Marker and Subsequent Primer Design

The 991-bp DNA sequence of the BM TO-A strain-distinctive region obtained by RAPD was analyzed for nucleotide sequence similarity in the coding region as well as predicted protein sequence similarity. At the DNA level, the RAPD sequence had identity (ranging from about 70 to 90% in 150–990 nucleotide [nt] overlaps) with Bacillus subtilis ssp. spizizenii str. W23 (accession no. CP002183) and several Bacillus licheniformis strains. The RAPD sequence had 73% identity with bacteriophage phi-105 DNA (accession no. AB016282) in each of the matched sequence regions. Avoiding this similarity region, we designed the BM TO-A strain-specific primer sets BM-177 (forward: 5′-TCC TCA AAT CAC CGT CTA GGG-3′; reverse: 5′-CAG ATA ATA CAA ACG CTA CGA T-3′) and BM-987 (forward: 5′-GTC AGC ATT AAG AAT CAA ATT ACG CAT AC-3′; reverse: 5′-GTC AGC AGA AGA AGA GAA TTT TGG AG-3′).

To acquire a more specific sequence, we used inverse PCR with the BM-invPCR primer set. Inverse PCR of the 991-bp sequence described above resulted in the identification of a more distinctive sequence for which very few matches were obtained in a BLAST search of the GenBank/EMBL/DDBJ DNA databases. The BM-qPCR primer (forward: 5′-CAC CGC GAA CAA CAT AGA CAC C-3′; reverse: 5′-CGG CCC GAA GCG ATT ATG AAA GCC G-3′) was designed based on this sequence.

Validation of the Specificity of the Primers Designed for BM TO-A at the Strain Level

Although the 1-kb RAPD band was also observed in B. amyloliquefaciens and some of the other B. subtilis strains, the designed BM TO-A strain-specific primer sets (i.e., BM-177, BM-987, and BM-qPCR) amplified only DNA associated with the target strain (Figs. 1, 2). The PCR products produced by the multiplex PCR were sufficient to identify BM TO-A. Double band positivity was characteristic for BM TO-A. Analysis of all other B. subtilis strains and two related Bacillus species using the 16S universal primers resulted in the amplification of only a 1.4-kbp band (Fig. 2).

Detection Limit and Linearity of BM TO-A in Feces as Determined Using qPCR

BM TO-A was added directly to fecal samples, and the correlation between the number of added CFUs and the value obtained using qPCR was determined in addition to the lower limit of BM TO-A detection in feces. A high degree of linearity was observed (y=0.976x; r²=0.909; Fig. 2) in qPCR analyses when 10^4.3–10^9.8 BM TO-A were added per gram of feces. Addition of 10^4.8 BM TO-A per gram of feces yielded amplification products in 22 of 24 fecal samples, whereas addition of 10^4.3 per gram of feces resulted in amplification in only half of the 24 samples examined.

Quantitative Detection of Ingested BM TO-A in Feces

Next, we used qPCR to determine the number of BM TO-A present in the feces of subjects who had ingested tablets containing this strain, and compared the results to those obtained using a culture method (Table 1). The number of BM TO-A detected in feces reached its near maximal level by Day 3 of administration, and this level was maintained for the 7-d administration period in >90% of samples. BM TO-A was still detected in the feces of subjects 3 d after cessation of ingestion. Although similar results were obtained using both the qPCR and culture methods, the highest counts and frequency of occurrence were observed using the qPCR method.

DISCUSSION

The term probiotics, popularized by Fuller,^1,2) has been defined as “living microorganisms, which upon ingestion in certain numbers exert health benefits beyond inherent general nutrition.”²⁵⁾ Therefore, to exert health benefits the probiotic strains must survive through the gastrointestinal tract. In order to evaluate this property of target probiotic strains, culture methods have been used to re-isolate ingested strains from feces.^3,26) However, it is difficult to apply culture methods when there are no suitable selective media for the target species. Furthermore, when bacteria of the same species as the target probiotic organism inhabit the gastrointestinal tract, a specific method for the isolation and identification of the ingested probiotic strain must be developed (e.g., selective media containing antibiotics and additives).²⁷⁾

Rapid progress in the development of molecular biology techniques has enabled researchers to utilize characteristic DNA regions of target bacterial strains for purposes of specific detection. A number of useful polymorphic techniques, such as RAPD, repetitive-sequence-based PCR (rep-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), and amplified fragment length polymorphism (AFLP), have been used for the detection of organism-specific distinctive DNA sequences.^28–34)

Although only a few reports describe specific genetic detection methods applicable at the strain level, Fujimoto et al.,³⁵⁾ Maruo et al.,³⁶⁾ and Tilsala-Timisjärvi & Alatossava³⁷⁾ reported the use of RAPD for strain-specific detection of Lactococcus lactis ssp. cremoris FC, Lactobacillus casei Shirota, and Lactobacillus rhamnosus Lc 1/3, respectively. Polymorphic markers previously unused for specific detection (i.e., 16S rRNA and internal transcribed spacers) have now been exploited as strain-specific detection markers for probiotic organisms such as Bifidobacterium animalis ssp. lactis GCL2505 and Lactobacillus sp. HOFG1.^38,39) These methods allow for the direct detection of target strains in a variety of contaminated sample types without the use of culture methods. Methods involving qPCR with strain-specific primers can now be used to trace a probiotic strain from the gastrointestinal tract.

Therefore, we utilized the RAPD technique to obtain a DNA sequence that was potentially BM TO-A strain-specific. The predicted amino acid sequence resulting from translation of the DNA sequence of this RAPD fragment showed a high degree of sequence similarity with a hypothetical phage-like protein. Evans et al. and Werning et al. discussed and defined in detail the gene structure of strain-distinctive DNA regions.^40,41) However, we were not concerned with the genetic architecture of the BM TO-A-specific region, since the purpose of this study was simply to establish a method for quantitative detection of BM TO-A in human feces. A more extensive study of this region of the BM TO-A genome was thus outside the scope of this research.

BM TO-A, B. subtilis JCM 20096, and B. amyloliquefaciens IFO 3034 have all been designated B. mesentericus; however, in more recent taxonomy these organisms are not referred to by this scientific name, and instead, these strains are identified as B. subtilis or related Bacillus species. Until now, BM TO-A was identified as B. subtilis ssp. subtilis based upon the sequences of its 16S rRNA, gyrA, groEL, polC, purH, and rpoB genes as well as DNA–DNA hybridization homology. The results of the present RAPD fingerprint analyses support the previous identification of BM TO-A.

From among 25 Bacillus strains examined, including those thus far called B. mesentericus, the designed BM TO-A strain-specific primer sets detected only BM TO-A (Fig. 2). The BM-987 primer set was designed to use nested PCR^42–44) for the BM-177 primer set. However, use of the BM-177 or BM-987 primer sets is sensitive enough to allow direct detection of BM TO-A. From the above discussion, it can be concluded that these primer sets (i.e., BM-177, BM-987, and BM-qPCR) are sufficient for the detection of BM TO-A at the strain level, at least with respect to DNA extracted from pure culture.

After developing these potential BM TO-A-specific primer sets, we evaluated the specificity of the BM-qPCR primer set for quantitative detection of BM TO-A in human feces. Although the total number of fecal bacteria is typically on the order of 10¹¹ cells per gram of feces,^45–47) a specific PCR product was detected only in feces samples to which BM TO-A had been added. The qPCR results showed satisfactory linear regression in fecal samples amended with BM TO-A (Fig. 2). Furthermore, the frequency of detection was 100% when more than 10^5.3 BM TO-A per gram of feces were added. The frequency declined to 92% and 50% when the amount of BM TO-A added was 10^4.8 and 10^4.3 per gram of feces, respectively. Fujimoto et al. indicated that qPCR produced a highly linear relationship when 10^4.6–10^9.6 of target bacteria were added per gram of feces.³⁵⁾ Addition of 10^4.1 of target bacteria per gram of feces resulted in detectable amplification product in only 3 of 6 fecal samples tested, and consequently they defined the detection limit of their strain and method as less than 10^4.6 per gram of feces. Collectively, these results suggest that the BM TO-A strain-specific primer set we developed can be used for the specific detection of this strain in feces using qPCR. It seems reasonable to assume that highly specific and quantitative detection of BM TO-A in feces can be achieved when the number of BM TO-A in feces is not less than 10^4.8 per gram. The results also suggest that at least 10^4.3 BM TO-A per gram of feces must be present for qualitative detection.

In the BM TO-A ingestion trial, the results obtained using both the qPCR and culture methods were in general agreement at the corresponding periods (Table 1). However, on Day 3 of the withdrawal period, we detected BM TO-A in the feces of 11 of 24 subjects at a level of 10^5.3±0.8 per gram (mean±S.D., n=11) using the qPCR approach, while BM TO-A was detected in the feces of 6 of the 24 subjects at a level of 10^3.9±0.8 per gram (n=6) using the culture method. During the entire study, including the duration of both the administration and withdrawal periods, the number of BM TO-A detected in the feces using the qPCR method was significantly higher than that obtained by counting the number of CFUs on culture plates. Previous reports also indicate that both the detection frequency and number of cells detected in feces by qPCR are substantially higher than the corresponding values obtained using culture methods.^26,48,49) The results of Fujimoto et al.³⁵⁾ were similar to ours in that the number of ingested bacteria detected by qPCR was tens of times higher than the number detected using the culture method. Essentially all bacteria with intact chromosomal DNA can be detected using the PCR method, whether they are alive or dead. Hence, it is probable that the total number of ingested BM TO-A detected in the feces by qPCR was 10-fold higher than the number of bacteria detected using the culture method because the latter detects only living bacteria.

In this study, we developed BM TO-A strain-specific primers using RAPD and inverse PCR. The resulting primers were then used to monitor the passage of this strain through the gastrointestinal tract of healthy volunteers. The scheme described here is applicable to the development of strain-specific primers for other species. The primers we developed may be useful for monitoring the passage of ingested BM TO-A through the gastrointestinal tract when species related to this strain are also present or in cases when it is difficult to re-isolate strains from feces using culture methods. While we acknowledge the need to demonstrate the efficacy of our method in experiments involving a larger number of test subjects, we believe that the strain-specific primers we have described here will be powerful tools for future studies.

Fig. 1. RAPD Fingerprints Generated Using RAPD-Primer 6, and the Associated Dendrogram for B. subtilis (21 Strains, Including B. mesentericus TO-A) and Two Related Bacillus Species (4 Strains)

The B. mesentericus TO-A strain-specific RAPD band is indicated by arrowheads. Abbreviations: ATCC=American Type Culture Collection; JCM=Japan Collection of Microorganisms; NBRC=National Institute of Technology and Evaluation Biological Resource Center; IAM=Institute of Applied Microbiology Culture Collection; IFO=Institute for Fermentation. The strain Indicated “*” was former name “Bacillus mesentericus.”

Fig. 2. Validation of the Specificity of Primers (A) BM-177, (B) BM-987, and (C) BM-qPCR for BM TO-A at the Strain Level

Lane M shows the 100 bp ladder (TaKaRa Bio, Shiga, Japan). Lanes 1–25 are identical to those described in Fig. 1. Lane N represents the negative control (DNA extraction performed on a culture containing no bacteria).

Fig. 3. Correlation between the Number of BM TO-A Added to Fecal Samples and the Number of BM TO-A Determined Using qPCR

The regression line was constructed between values for 104.3 and 109.8 BM TO-A added per gram of feces because amplification products were not always detected in samples in which 104.3 or fewer BM TO-A per gram were added. The regression line was calculated with an intercept of 0. Error bars indicate S.D.

Table 1. Quantification of B. mesentericus TO-A in Fecal Samples

	Before ingestion	Administration		Withdrawal
	Day 0	Day 3	Day 7	Day 3	Day 7
Culture method	<3.5	4.87±0.65a)	4.90±0.26	3.92±0.84
	(0%)b)	(58%)	(79%)	(25%)	(0%)
Quantitative PCR	<4.3	5.84±0.41	5.87±0.44	5.29±0.82	5.11
	(0%)	(100%)	(96%)	(46%)	(4%)

a) Data expressed as mean±S.D. of log₁₀ number per gram feces. b) Figures in parenthesis are frequency of occurrence (%).

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