Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

Background Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. Results A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. Conclusion This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.


Background
Leptospirosis is the zoonotic disease caused by members of the genus, Leptospira. They are motile helical spirochaetes that metabolise long chain fatty acids as their carbon source. There are 17 species of Leptospira as determined by DNA-DNA hybridisation [1][2][3][4]. These species can be further divided into pathogenic, non-pathogenic and opportunistic/possibly pathogenic Leptospira with pathogenic species. The pathogenic Leptospira include; L. interrogans, L. kirschneri, L. santarosai, L. weilii, L. alexanderi, L. borgpetersenii, L. genomospecies 1 and L. noguchii. The non pathogenic Leptospira include: L. biflexa, L. meyeri, L. wolbachii, L. genomospecies 3, L. genomospecies 4, L. genomospecies 5 and opportunistic/intermediate pathogens Leptospira include L. broomi, L. fainei and L. inadai [3]. The grouping of the last three species as opportunistic or possible pathogens is due to the lack of information on the pathogenicity of the species, different phenotypic characteristics compared to the pathogenic Leptospira and also the limited number of reports of these species involvement in human leptospirosis.
Before molecular techniques such as DNA-DNA hybridisation or 16s rRNA gene sequencing became available, speciation of the genus Leptospira was limited to the classifications of pathogenic (L. interrogans sensu lato) or saprophytic (L. biflexa sensu lato) and was performed using phenotypic tests such as growth at 13°C/30°C or growth in the presence of a chemical such as 8-Azaguanine [5]. These tests can take up to 28 days to complete and the results can vary within a species [2]. Since the introduction of molecular techniques, the identification of Leptospira species has generally been performed using either DNA-DNA hybridisation [2,4] or 16s rRNA gene sequencing. Both of these methods have inherent disadvantages; DNA-DNA hybridisation is laborious and requires the use of radio-labelled isotopes [6] and the 16s rRNA gene has significant sequence homology between species which requires the majority of the gene to be sequenced for a definitive Leptospira identification. As an alternative target to 16s rRNA for species identification, the DNA Gyrase Subunit B gene (gyrB) has been successfully used for species identification in a wide variety of bacterial genera [7-13]. More recently the gyrB gene has been used for the identification of Leptospira borgpetersenii isolates from the Amami Islands [14] though this study used universal gyrB primers and only conducted limited phylogenetic analysis.
This paper reports the development of a molecular technique for the identification of pathogenic Leptospira species using conventional or real-time PCR amplification and sequencing of a partial fragment of the gyrB gene. The method was then used to ascertain gyrB sequences from representative reference strains of the eight pathogenic species. These sequences were used for phylogenetic and evolutionary comparisons between the species themselves and also between the gyrB gene and 16s rRNA gene. To highlight the potential value of the gyrB as an alternate identification gene, a blind trial was conducted between the two gene targets to identify previously uncharacterised clinical Leptospira isolates.

Results and Discussion gyrB Amplification
Using conventional and real-time PCR we were able to amplify a 504 bp product from the eight pathogenic Leptospira species: L. interrogans, L. borgpetersenii, L. weilii, L. santarosai, L. alexanderi, L. genomospecies 1, L. noguchii and L. kirschneri. No PCR products were amplified from the representatives of non-pathogenic species L. biflexa, L. meyeri or from the pathogenic/intermediate species such as L. inadai, L. fainei or L. broomi (Figure 1 and 2). The development of both conventional and real-time PCR methodologies for the amplification of the gyrB gene enables this method to be instituted at the majority of laboratories and is not dependant on having relatively expensive real-time PCR equipment. The advantage of using realtime PCR over conventional PCR is that it is quicker (amplification is completed in less than an hour) and there is no need to perform agarose gel electrophoresis or capture the gel image. Confirmation of the gyrB gene amplification was performed using the melting curve analysis on the LightCycler instrument. The Tm of the gyrB PCR product was found to be between 83.4°C and 84.8°C ( Figure 2). Cycle sequencing of the gyrB PCR product and comparison of the DNA sequences enabled species specific identification. The gyrB DNA sequences of the reference strains were deposited on GenBank ( Table 1).
The amplification of only the pathogenic species from the genus Leptospira has created an assay which has a wide potential in this field of research. For example, the gyrB PCR could be use to identify pathogenic Leptospira from cultures or identify Leptospira isolates that have been overgrown with bacteria or fungi. Additionally it would be possible to apply this method to clinical samples that contain high concentrations of Leptospira organisms such as kidney tissue. The non-culture identification of pathogenic Leptospira would be difficult without the use of specific gyrB primers as universal gyrB primers such as those used by Kawabata et al. [14] would amplify DNA from all bacteria present in a sample.
The potential of this assay must be balanced by three apparent limitations. Firstly the lack of sensitivity of conventional detection methodologies would not enable this test to be used in diagnosis of human infections where there are generally only low levels of Leptospira in the blood. Secondly, potentially pathogenic species such as L. fainei, L. inadai or L. broomi are not amplified, and therefore could be missed or excluded during molecular investigations. Finally, if a culture or sample contained two different Leptospira species then it would result in a mixed sequencing result, requiring use of DNA cloning and multiple sequencing reactions. The ultimate evolution of this method would be through the use of specific DNA probe detection either through Taqman/FRET probe in a real-time PCR guise or through a more conventional chemicalluminescent detection format.

Comparative phylogenetic analysis
Multiple alignments of the DNA sequences allowed phylogenetic comparisons between the species and between the gyrB gene and 16s rRNA gene to be performed. Global clustering ( Figure 3) and similarity matrix analysis ( Table  2 and 3) was performed using the aligned sequence data and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm for the 16s rRNA, gyrB and a consensus of both genes to examine the level of relatedness between the pathogenic species. 16s rRNA and gyrB genes show significant difference in total relatedness as shown by the percentage scale in Figure 3 and more accu-rately in the similarity matrix analysis (Table 2 and 3). The maximum nucleotide difference for the 16s rRNA gene ranges from 0.1% to 1.4% whilst for the gyrB gene it ranges from 3.5% to 16.1%. The differences in nucleotide divergence between the two genes is due to gyrB having a higher rate of base substitution (0.7-0.8% per 1 million years) when compared to 16s rRNA (1% per 50 million years) [15].
In addition to the cluster analysis, minimum evolution trees for 16s rRNA and gyrB were constructed using 1000 bootstrap replications ( Figure 4). The trees have nearly identical topology except the evolution of L. kirschneri and L. interrogans in the gyrB gene is quite distinctly different to that of the evolution pattern in the 16s rRNA Real-time PCR amplification of the gyrB gene from 14 Leptospira genomospecies gene. The changes in topology signifies that the gyrB gene in pathogenic Leptospira has undergone significant evolutionary divergence compared to that of the 16s rRNA gene and this result is consisted with research conducted in other bacterial genera including the preliminary work conducted by Kawabata et al. with Leptospira [7-9,11,12,14-18].

Identification of Leptospira clinical isolates using gyrB PCR
To validate the use of gyrB as an alternative target to 16s rRNA, a comparison study was performed using 50 unidentified clinical isolates all from human sources. The gyrB sequences of the unknown isolates were compared to those deposited on GenBank using a BLASTn search. Confirmation of the gyrB result was performed using 16s rRNA sequencing and BLAST analysis as described in the methods. There was 100% agreement between the 16s rRNA Melting curve analysis of the gyrB gene using Sybr green detection Figure 2 Melting curve analysis of the gyrB gene using Sybr green detection.
and gyrB gene sequencing for the identification of pathogenic Leptospira species. Within the 50 isolates tested there was found to be the following number of species; L. alexanderi (1 isolate), L. weilli (10 isolates), L. borgpetersenii (10 isolates) and L. interrogans (29 isolates). The advantage of using the gyrB gene over the 16s rRNA gene is that during the BLASTn searches on GenBank, the score values were generally higher and the E values generally lower for the predicted species when compared to the 16s rRNA gene BLASTn searches allowing for greater confidence in the final result.

Conclusion
We have developed and validated a conventional and real-time PCR method for the amplification of gyrB gene from pathogenic Leptospira. When compared to the 16s rRNA gene, the gyrB gene shows greater evolutionary divergence and an alternate evolutionary topology for L. kirschneri and L. interrogans using minimum evolution analysis. Additionally the greater divergence of the gyrB gene makes it more amendable to the identification of pathogenic Leptospira either through sequencing as shown in this study or in the future by Real-time PCR using DNA probe technology.

Leptospira strains and DNA extraction
In total, 37 reference strains from the eight pathogenic Leptospira species and 50 clinical Leptospira isolates from human sources were obtained from the WHO/FAO/OIE Collaborating Centre for Reference & Research on Leptospirosis, Brisbane, Australia. Genomic DNA was extracted by the following method: 500 µL of Ellinghausen McCullough Johnson Harris (EMJH) media containing actively growing Leptospira was centrifuged in a micro-centrifuge tube at 12,000 g for 5 min. The supernatant was removed and the pellet re-suspended in 400 µL of 1× TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0). This suspension was boiled for 10 min and then centrifuged at 12,000 g for 5 min.  (Table 4), one unit of AmpliTaq Gold, 2 µl of DNA extract and double distilled water (ddH 2 O) to make up the final volume. Thermal cycling was as following: Initial denaturation at 94°C for 10 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s with a final extension at 72°C for 10 min. 5 µL of PCR product was electrophoresis on 1.5% agarose gel at 80 V for 60 min (Figure 1).

gyrB amplification: Real-time PCR
Real-time amplification of the gyrB gene was performed in a total volume of 20 µL containing 2 µL of Fast-Start Sybr green mix (Roche), 2.4 µL of 25 mM MgCl 2 , 10 pmol of oligonucleotides; 2For and 504Rev, 2 µL of template DNA and ddH 2 O to make up the final volume. Thermal cycling was performed on a LightCycler real-time thermalcycler (Roche) using the following program: initial denaturation at 95°C for 10 min and 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. Fluorescence readings were taken at the end of each extension cycle in the F1 (FAM/ Sybr green) channel. Melting curve analysis was performed by heating the PCR product from 60°C to 95°C and monitoring the fluorescence change every 0.2°C. The melting temperature or Tm was calculated by calculated on the initial fluorescence curve (F1/I) by plotting the negative derivative of fluorescence over temperature versus temperature (-dF1/dT versus T). Amplified products were removed before sequencing from the capillaries by uncapping and inverting the capillary in a micro-centrifuge tube and centrifuging at 1,500 g for 10 s. Global cluster analysis of the 16s rRNA gene, gyrB and consensus DNA sequences performed using the UPGMA algorithm  (Table 4), one unit of AmpliTaq Gold, 2 µl of DNA extract and (ddH 2 O) to make up the final volume. Thermal cycling was as following: Initial denaturation at 94°C for 10 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s with a final extension at 72°C for 10 min. Agarose electrophoresis was performed as above. The 1382 bp product were sequenced as described below in both the forward and reverse directions using the original primers and with internal primers; 515F, 91e, 11e, 16s1RRB, 907R and 342R (Table 4).

DNA Sequencing
Excess primers and dNTP's were removed from the remaining PCR product using the following enzymatic method: 2.5 µl of 10× Antarctic phosphatase buffer (New England Biolabs, NEB), 10 units of Exonuclease I, E. coli (Fermentas), 2.5 units of Antarctic phosphatase (NEB) and 1.5 µl of ddH 2 O were added to each sample. The PCR product plus enzyme mix were incubated at 37°C for 45 min followed by 85°C for 15 min to inactivate the enzyme. DNA sequencing was performed using the Big Dye Terminator (BDT) sequencing version 3.1 (Applied Biosystems) with the following modifications: each 20 µL reaction contained 0.5 µL of BDT mix (1/16 th dilution in final volume), 3.75 µL of 5× dilution buffer, 3.2 pmol of primer, 5-10 ng of DNA and ddH 2 O to make up the final volume. Cycle sequencing was performed using 33 cycles of 95°C for 10 s, 50°C for 10 s and 60°C for 4 min. The cycle sequencing products were purified using the sodium acetate/alcohol precipitation method as per manufacturers' instructions (Applied Biosystems). The purified products were forwarded to the Griffith university DNA sequencing facility (GUDSF), Brisbane, Australia for capillary electrophoresis using the ABI 3130 × l instrument. The sequences were assembled and trimmed to a minimum of two contiguous sequences using the Vector NTI software (Invitrogen).

Data Analysis
Analysis of the DNA sequences was performed using the Bionumerics (Applied maths) and MEGA version 3.1 software packages [22]. 37 reference sequences for both gyrB and 16s rRNA were deposited on GenBank (Table 1). Unknown isolates were submitted to a nucleotide Basic Local Alignment Search Tool (BLASTn) [23] search available at the National Centre for Biotechnology Information (NCBI) website [24].