BMC Microbiology BioMed Central Methodology article

Background The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. Results A real-time polymerase chain reaction assay using TaqMan® probes (real-time PCR) was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5) genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species), nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG), human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. Conclusion A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.


Background
Real-time quantitative polymerase chain reaction (qPCR) has recently emerged as promising tool for faecal microbiota monitoring in animal and human faeces [1][2][3] since culture-based methods are not suitable for quantification of certain microbial groups, species or strains in faeces [4]. Due to the role of bifidobacteria as probiotics much attention has been focused on the qPCR-based quantification of both the autochthonous bifidobacteria in faecal microbiota and on selected strains of bifidobacteria after consumption as probiotics [5][6][7][8][9]. Compared to fluorescence in situ hybridization (FISH), the most widely used method for culture independent quantification in faeces, qPCR is less developed in terms of the availability of specific probes [10]. On the other hand qPCR was shown to be about a 10 to 100 fold more sensitive than culture-and FISH-based enumeration techniques [11], as well as to be rapid, easy and more accurate for quantification of low levels of bacteria [12]. Several oligonucleotides were designed for the Bifidobacterium species found in the human intestinal tract, most of them based on the 16S rDNA sequence [11,13]. Other target sequences like the transaldolase encoding gene [5], heat-shock protein (HSP60) gene [14], intergenic spacer of the 16S-23S rRNA gene [15] are also being investigated for species-specific detection and quantification. Oligonucleotides targeting such sequences could also be used for developing qPCR primers.
Bifidobacterium thermophilum, being considered as an animal-associated commensal species, was never included in studies on the bifidobacterial composition of the human intestinal flora and to our knowledge, no oligonucleotide was designed for the development of B. thermophilum-specific PCR or qPCR assay until now. Recently, design of a pair of oligonucleotides for PCR amplification of a portion of the 16S rDNA of B. thermophilum was reported, but effective specificity of the assay was questioned [16]. Previously, we have isolated and characterized bifidobacteria with anti-Listeria activity from stool of newborns [17,18]. Strain RBL67 was identified as B. thermophilum using 16S rDNA sequence homology, comparative HSP60 sequence analysis, DNA-DNA genome hybridization and carbohydrate fermentation patterns [19]. This was the first demonstration of the presence of B. thermophilum in human faeces. In this study, we designed oligonucleotides specific for B. thermophilum that we used to develop PCR and qPCR assays to study the distribution of this species in human faecal samples. Finally, the qPCR technology let us isolate a strain of B. thermophilum from human infant faeces closely related to B. thermophilum RBL67.

Results
Design of a B. thermophilum specific PCR assay Specificity of the B. thermophilum specific primer was assessed by PCR on colonies with primers btherm and the Bifidobacterium genus-specific primer lm3 (Table 1). Of the 17 Bifidobacterium and two Lactobacillus strains tested, positive signals (amplification of a fragment of approximately 1.5 kb) were only obtained with the three faecal isolates B. thermophilum RBL67, Bifidobacterium thermacidophilum subsp. porcinum RBL68 and RBL70 (

Detection of B. thermophilum in faecal DNA samples by PCR
Classical PCR analysis with the B. thermophilum specific primers btherm and lm3 (Table 1) on total DNA isolated from faecal samples spiked with known quantities of B. thermophilum RBL67 showed that the detection limit of the method was 10 8 B. thermophilum cells per gram faeces ( Figure 3). This high detection limit did not allow DNA amplification from any of the 17 faecal samples. Efficacy of PCR amplification on faecal DNA samples was confirmed by amplification of a 1.3 kb-DNA fragment from each faecal DNA sample generated with the Bifidobacterium-genus specific primer-pair lm26/lm3 [20] (data not shown).

Development of a qPCR assay for detection of B. thermophilum in human faeces
The qPCR technology was chosen as an alterative to the classical PCR for its higher sensitivity. B. thermophilum specificity of qPCR performed with primers bthermRTF, bthermRTR and the TaqMan probe bthermTqM (Table 1) was tested by amplification of DNA isolated from six different Bifidobacterium strains belonging to four closely related species. A positive signal was obtained for B. thermophilum RBL67 (C T = 17.3 ± 0.5), B. thermophilum DSM20210 T (C T = 24.9 ± 0.3) and the closely related species B. thermacidophilum subsp. porcinum LMG21689 T (C T = 16.3 ± 0.4), but not for B. thermacidophilum subsp. thermacidophilum LMG21395 T , Bifidobacterium breve DSM20213 T , and Bifidobacterium boum DSM20432 T . Amplification of DNA from B. thermophilum RBL67 with this new assay was shown to be linear for DNA concentrations ranging from 0.02 ng/μl (C T = 28.1 ± 0.3) to 200 ng/ μl (C T = 15.3 ± 0.4) with a regression coefficient R 2 = 0.991 (data not shown).

Screening of faecal samples by qPCR
As for the classical PCR approach, the detection limit of the qPCR assay was determined by analysis of DNA isolated from spiked faecal samples and was equal to 1 × 10 5 bacterial cells per gram of faeces. Detection in faecal samples was shown to be linear between 1 × 10 9 and 1 × 10 5 cells per gram of faeces and corresponded to C T values comprised between 21.6 ± 0.6 and 33.8 ± 0.1, respectively, with a regression coefficient R 2 = 0.995 (data not shown). One of the 17 faecal samples (sample C7 from a 6 month-old breast-fed baby) gave a positive signal within this range ( Figure 4) with a C T value of 28.6 ± 0.3, corresponding to a concentration of 5 × 10 6 cells per gram faeces.

Application of the newly developed qPCR assay for faecal samples
Raffinose-Bifidobacterium (RB) medium and MRSC-NNLP were tested for isolation of new B. thermophilum strains from human faeces. MRSC-NNLP was chosen for isolation of bifidobacteria because it allowed a better growth of RBL67 (1.5 × 10 9 cfu/ml after three days of incubation at 40°C, in comparison to 2.9 × 10 7 cfu/ml in RB-medium and under the same conditions) and was easier to prepare. Approximately 1-5 × 10 8 colonies per gram faeces could be cultivated from all of the 17 samples plated on MRSC-NNLP agar. Microscopic observations of the isolates showed that the medium was not completely selective, allowing for the growth of non-rod-shaped microorganisms. 60 rod-shaped microorganisms were selected for PCR analysis on colony. Twenty-five of them were positive with the Bifidobacterium genus specific primers lm26/ lm3, but only one of them gave a positive signal with Localization of the 16S rDNA targets for oligonucleotides designed in this study btherm/lm3. This isolate, F9K9 from a 4-month old breast-fed baby, was streaked several times on MRSC agar and the absence of contaminants other than Bifidobacterium was confirmed by three PCR reactions with lm26/ lm3, btherm/lm3 and bak4/bak11w [24] (data not shown). Sequencing of the 16S rDNA fragment amplified with lm26 and lm3 yielded a 1454-bp sequence which was 99.93% identical to the 16S rDNA of B. thermophilum RBL67. Sequence identities with other Bifidobacterium strains are summarized in Table 2.

Discussion
Real-time PCR (qPCR) is known to be a more sensitive technique than classical PCR. This is reflected by our results for specific amplification of 16S rDNA from spiked faecal samples, where changing from classical PCR to qPCR for the detection of B. thermophilum in faecal samples lowered the detection limit of the assay from 10 8 to 10 5 cells per gram faeces. The high sensitivity obtained for qPCR in this study is similar to detection limits reported by different groups for other Bifidobacterium species or genus specific qPCR assays. Matsuki et al. [11], Penders et al. [6] and Gueimonde et al. [25], for example, reported detection limits of 10 6 , 5 × 10 3 and 5 × 10 4 cells of Bifidobacterium spp. per gram faeces, respectively. The application of a recent qPCR technology using rRNA as target molecule combined with reverse transcriptase could further enhance the sensitivity down to 10 3 cells/g faeces [26]. This methodology could also be developed for the detection of other subdominant faecal bacteria such as B. thermophilum.
We have developed a qPCR assay which is specific for B. thermophilum although the assay is also positive for the type strain of B. thermacidophilum subsp.porcinum, and B. thermacidophilum subsp.porcinum RBL68 and RBL70 (these subspecies were originally named "suis" which is a synonym for "porcinum"), but not with B. thermacidophilum subsp. thermacidophilum (LMG21395 T ) [27]. Based on our published data including DNA-DNA genome hybrizations [19] we underline that B. thermacidophilum subsp. porcinum [28] should belong to the B. thermophilum spe- cies and consequently, we conclude that our qPCR system is specific for B. thermophilum.

Specificity of the PCR for B. thermophilum using primers btherm and lm3
Until now, B. thermophilum was considered as an animalassociated species, mainly present in faeces of ruminants and pigs. The amplification of a specific 16S rDNA sequence with our qPCR on the children faecal sample C7 as well as the isolation of a B. thermophilum isolate from children faeces during this work support the assumption of von Ah et al. [19] that presence of B. thermophilum in food cannot be used to discriminate between animal and human bacterial contamination, as previously suggested [29].

Conclusion
This is the first report of the development of a qPCR assay for specific detection of B. thermophilum, a species that was not included in analysis of the composition of the bifidobacterial human intestinal microbiota until now. Using this assay, we detected B. thermophilum at a concentration of 5 × 10 6 cells per gram in one faeces sample, confirming the presence of this species in human faecal material.

Bacterial strains and culture conditions
Unless otherwise indicated, bifidobacteria and lactobacilli were grown in liquid cultures overnight in 10 ml MRSC medium consisting of MRS [30], obtained from Biolife (Milan, Italy) and supplemented with 0.05% L-cysteine hydrochloride, or on MRSC-agar plates (MRSC supplemented with 1.5% w/v agar). Incubation was carried out for 24 h at 37°C in anaerobic jars with an anaerobic atmosphere generation system (Oxoid AnaeroGen TM, Basel, Switzerland). B. thermophilum RBL67, as well as B. thermophilum subsp. porcinum RBL68 and RBL70 are human infant faecal isolates [17][18][19]31]. All of the other strains are commercial strains from DSMZ (German collection of microorganisms and cell cultures, Braunschweig, Germany) or LMG (Laboratories for Microbiology and Microbial Genetics, Ghent, Belgium).

Isolation of bifidobacteria from faecal samples
Seventeen faecal samples from human adults (4) and breast-fed children between 1 to 6 months (13) were collected as already described [32]. Subjects or parents of the subjects were informed orally and in writing about the aims and procedures of the study and consent was obtained from them. The study protocol was reviewed and approved by the Ethical Committees of the canton of Zurich and the SPUK-committee of the University Children's Hospital of Zurich (project StV31/05).
For efficient growth of B. thermophilum strains from faecal samples, Raffinose-Bifidobacterium (RB) [33] and MRSC-NNLP [34] media were compared. Serial 10-fold dilutions of overnight cultures of B. thermophilum RBL67 (containing approximately 10 9 cfu/ml) in saline solution (8.5 g/ Liter NaCl, 1 g/Liter peptone, 0.05% cysteine-HCl, pH 6-7) were plated on RB and on MRSC-NNLP, incubated for 3 days anaerobically at 40°C and cell counts were determined. Incubation temperature of 40°C was chosen as an additional selective condition, due to the relative heat tolerance of B. thermophilum spp. [19]. For isolation of bifidobacteria from faecal samples, 20 mg of samples were homogenized by vigorous vortexing in 0.2 ml of saline solution, 10-fold serially diluted in the same solution and spread on MRSC-NNLP agar plates. Plates were incubated for 3 days under anaerobic conditions at 40°C and single isolates were observed under light microscope. Rodshaped bacteria were selected for further analysis.

DNA sequencing, PCR and qPCR reactions
Primers and probe used in this study were synthesized by Microsynth and are listed in Table 1. The TaqMan probe bthermTqM was labeled with 5'-FAM as fluorescent reporter dye and 3'-TAMRA as quencher. Classical PCR was performed either on 2 μl DNA prepared from faecal samples as described above, or on 40 μl cell suspensions. For that, one colony was picked from an agar plate and resuspended in 210 μl of sterile, double distilled water. A 50-μl classical PCR reaction consisted of 2.5 U EuroTaq-DNA-Polymerase (Digitana, Horgen, Switzerland), 1.5 mM magnesium chloride (Digitana), 0.2 mM dNTP's (GE Healthcare) and 0.5 μM of each primer. When DNA isolated from faecal samples was used as template, 0.1 μg/ml BSA was added to the PCR reaction. Amplification conditions were as follows: 3 min at 95°C, 40 cycles of 15 sec at 95°C, 30 sec at 62°C and 2 min at 72°C, followed by 7 min at 72°C. Sequencing of the PCR product for 16S rDNA was performed by Microsynth (Balgach, Switzerland) using the primers btherm, 520F, 520R, 1100F, 1100R and lm3 (Table 1).