Brachyspira hyodysenteriae strains, cultivation and purity control
The shBh strain B204 [30] and the whBh strain (G423) were used in this study. Strain B204 was isolated from a pig with SD in the United States of America in the 1970s and strain G423 was isolated during routine microbiology diagnostics from growing pigs suffering from mild diarrhea in Germany in 2014. By using a species-specific PCR that targets the nox gene region (amplicon 435 bp, [40]) both strains were confirmed as B. hyodysenteriae. Furthermore, examination of hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and putative hemolysin genes (hemolysin III gene, hemolysin activation protein gene, hemolysin channel protein gene, and hemolysin gene) showed that these eight genes were present in both strains (data not shown).
Bacteria were cultured on trypticase soy agar (TSA) plates containing 5% (v/v) sheep blood under anaerobic conditions (adapted and modified from Kunkle and Kinyon, [41]). Brain-heart-infusion broth (BHI; Oxoid, Wesel, Germany) containing 20% fetal calf serum (FCS; Biochrom, Berlin, Germany) was used as liquid culture (BHIF). The purity of both strains was confirmed phenotypically based on their typical morphology and by use of dark-field and phase-contrast microscopy (Leica DMR HC microscope, Leitz, Wetzlar, Germany). For the transcription studies (see below), 100 μl-samples were taken every day and were additionally cultured on blood agar under aerobic and anaerobic conditions to exclude bacterial contaminations.
Experimental setup, determination of hemolytic activity and calculation of growth forming units (GFU50/mL) for the hemolysin gene transcription study
BHIF (BHI containing 20% FCS) broth (50 mL) was inoculated with 1 mL B. hyodysenteriae B204 and G423, respectively, each containing 3 × 105 live cells, and incubated anaerobically at 37 °C on a shaker. Each experiment was performed in triplicate. At four different growth points (Figs. 1 and 2) samples were taken to determine the hemolytic activity of the strains, the live cell count and hemolysin gene transcription rates. Hemolytic activity in different cell-free supernatants was determined by using an agar diffusion assay. TSA agar was poured into petri dishes and wells of 8 mm in diameter were prepared in the agar and filled with the culture filtrates. After incubation of the plates for 48 h at 37 °C, hemolysis was identified by the presence of a transparent zone surrounding the wells. For semi-quantitative estimation of the hemolytic activity the hemolysis zone sizes were assessed. To calculate GFU50/mL series dilutions (on a logarithmic scale) the B. hyodysenteriae suspensions were prepared in BHIF at different growth points. Four 10 μL spots from each dilution were given on TSA agar plates. After five days of anaerobic incubation (37 °C) the number of hemolytic spots was used to calculate the titer in GFU50/mL according to the formula of Spearman [42] and Kaerber [43]. The four time points chosen for hemolysin gene transcription activity were based on the results from the hemolysin diffusion assay and growth curves (GFU50/mL).
RNA isolation and purification
Total RNA was prepared from samples taken at hemolytic and non-hemolytic stages of growth by use of the RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). Briefly, 500 μL of cultured bacteria were added to 1000 μL RNAprotect, immediately vortexed, incubated for five minutes at 20 °C, and centrifuged (10 min, 5000 x g, 20 °C). After carefully removing the supernatant, the pellet containing bacterial cells with stabilized RNA was frozen at − 70 °C until further processing. RNA from bacterial cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). For this purpose, bacterial cells were suspended in 700 μL lysis buffer, mixed with 500 μL 99% ethanol (Merck KGaA, Darmstadt, Germany) and transferred to a spin column. After centrifugation at 7000 x g for 15 s, wash buffer was added before digestion of DNA with 2 μl DNAseI (1500 Kunitz units; Qiagen, Hilden, Germany) for 15 min at 20 °C. After three washing steps mRNA was eluted with RNase-free water and finally treated a second time with 5 μl DNase (1 U/μL; Thermo Fisher Scientific, St. Leon-Rot, Germany) for 20 min at 37 °C. To protect the RNA from degradation, a total of 3.5 μL RNase inhibitor (20 U/μL, Thermo Fisher Scientific, St. Leon-Rot, Germany) was added to all solutions containing RNA. The concentration of RNA was measured with the Nano Drop 2000C photometer (Thermo Fisher Scientific, St. Leon-Rot, Germany) and the purity was evaluated through the A260/A280 ratio. RNA was stored at − 70 °C until further usage.
Reverse transcription
Complementary DNA (cDNA) was generated from purified mRNA by use of forward and reverse primers (Additional file 1). All primers used in this study were checked for their in silico binding activity to the nucleotide sequences of B. hyodysenteriae strains B204 (NCBI Reference Sequence no. NZ_JXND00000000) and G423. Nucleotide sequence alignments with sequences deposited in GenBank were performed with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Software Geneious version 8.1.9 (Biomatters, Auckland, New Zealand).
Each reaction mixture (26 μL total volume) contained 10 μL purified mRNA, 1 μL of each primer (20 μM), 2 μL DTT (0.1 mM Dithiothreitol, Roth, Karlsruhe, Germany), 1 μL MgCl2 (50 mM, PAN Systems, Aidenbach, Germany), 4 μL nucleotides (each nucleotide 4 mM, Rapidozym, Berlin, Germany), 5 μL of 5 x buffer (750 mM Tris, 500 mM KCl and RNase-free water), 0.2 μL RNase Inhibitor (20 U/μL, Thermo Fisher Scientific, St. Leon-Rot, Germany), and 0.3 μL MultiScribe Reverse Transcriptase (50 U/μL, Thermo Fisher Scientific, St. Leon-Rot, Germany). After incubation at 40 °C for 42 min samples were stored at − 20 °C until further usage.
Reference genes for real-time quantitative PCR
Three genes (adh, pgm, gdh), that are also used in the 7-MLST scheme of B. hyodysenteriae, and the gyrase gene gyrB gene, which was already used in a transcription study by Witchell et al. [32], were compared by using the Excel-based software BestKeeper version 1 (https://www.gene-quantification.de/bestkeeper.html) to identify the two most appropriate reference genes for this study [44]. This software computed a descriptive analysis for each reference gene, including geometric and arithmetic mean, minimal and maximal value, standard deviation (StDv) as well as the coefficient of variance (CV). To estimate the inter-gene relations of reference genes numerous pair-wise correlation analyses were performed.
Verification of absence of genomic DNA
Absence of genomic DNA was verified by conventional PCR with primers targeting the hemolysin and reference genes. Each reaction mixture (30 μL total volume) contained 1 U PanScript DNA Polymerase (PAN Systems, Aidenbach, Germany), 0.5 μM of the specific primers (Additional file 1), 133 μM of each nucleotide (Rapidozym GmbH, Berlin, Germany), 1 x NH4 buffer, 2 mM MgCl2, and 3 μL of the template.
The PCR conditions for the detection of hemolysin genes included an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 40 s, annealing at a primer-specific temperature (Additional file 1) for 40 s, elongation at 72 °C for 60 s, and a final elongation at 72 °C for 7 min. The PCR conditions for the detection of gyrB were an initial denaturation (5 min, 94 °C), followed by 35 cycles at 94 °C for 30 s, annealing at 60 °C for 30s, elongation at 72 °C for 30 s, and a final elongation at72 °C for 7 min. The PCR conditions for the detection of adh, pgm and gdh followed the protocol of Råsbäck et al. [45]. Amplicons were separated by horizontal electrophoresis using 2% Tris-acetic acid-EDTA (TAE) agarose gels supplemented with 0.5 μg/mL ethidium bromide (Serva Electrophoresis, Heidelberg, Germany) and visualized by UV light. In case of positive signals, the process of RNA purification was repeated.
Real-time quantitative PCR
Real-Time quantitative (q) PCR was performed on an ABI 7300 Real-time qPCR System (Thermo Fisher Scientific, St. Leon-Rot, Germany) using a qPCR MasterMix for SYBR Green I (Eurogentec, Cologne, Germany). Primers for Real-Time quantification (Additional file 1) were designed with the software Primer Express version 3.0 (Thermo Fisher Scientific, St. Leon-Rot, Germany) to amplify fragments of 62 to 131 bp of the hemolysin and reference genes. Forward and reverse primers were each mixed and stored at − 20 °C in a concentration of 1.0 μM. One reaction mix (25 μL total volume) contained 5 μL of primer mix, 0.75 μL SYBR Green I, 4.25 μL RNase-free water, 12.5 μL 2 x reaction buffer, and 2.5 μL cDNA. Cycling conditions were taken from the manufacturer of the qPCR MasterMix (UNG step 2 min 50 °C, HotGoldStar activation 10 min 95 °C and 40 cycles consisting of 15 s 95 °C and 1 min 60 °C) with an additional melting curve analysis to detect non-specific products like primer-dimers.
Efficiencies of the different qPCR assays were calculated from the slopes of standard curves obtained from 10-fold dilution series of B. hyodysenteriae B204 and G423 genomic DNA, respectively. To increase the accuracy, five repetitions of each dilution were performed. Real-time qPCR of cDNA samples (hemolysin genes and reference genes) was performed in triplicate. Outliers were detected with the Grubbs’ test [46] and omitted from further analysis.
Sequence analysis of gene fragments of whBh strain G423
To verify affiliation of the weakly hemolytic strain G423 to the species B. hyodysenteriae we compared the nucleic acid identity of the NADH oxidase (nox) gene and the 16S rRNA gene of strain G423 with other B. hyodysenteriae strains. Fragments of these genes (nox gene, 939 bp; 16S rRNA gene 870 bp) were amplified by PCR and sequenced (LGC Genomics, Berlin, Germany). In a recently published article a disruption of the promoter site of hlyA of whBh strains was described [11]. Therefore, the intergenic region of our whBh strain G423 between the fabG (ACP reductase) and the hemolysin gene hlyA with a length of 1084 bp was also amplified and sequenced. Primers and PCR conditions are listed in Additional file 1.
Data evaluation and statistical analysis
For each of the three experiments with the shBh and whBh strains, transcription activities of the eight hemolysin genes were determined at different time points at the hemolytic and non-hemolytic stages of growth.
The comparative Ct method (ddCt) was used to analyze differences in gene expression between strongly and weakly hemolytic strains. Statistical analysis was performed with the REST program (Qiagen, Hilden, Germany) which normalizes Ct values of the hemolysin genes to the reference genes and takes different PCR efficiencies into account.