Abstract

The usual target for sequence-based identification of Leptospira species is the 16S rRNA gene. However, because the 16S rRNA gene is not polymorphic enough, it is necessary to sequence a 1500 bp segment of this gene for accurate identification. Based on the alignment of previously determined rpoB of three Leptospira strains, we designed and tested a primer pair that enabled us to amplify and sequence a 600 bp segment of Leptospira rpoB. This segment was species-specific for the 16 species tested, but was unable to separate Leptospira interrogans serovars accurately. For the 11 L. interrogans serovars tested, only seven genotypes could be determined. We thus think that analysis of partial rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira and detection or identification of Leptospira in clinical or environmental samples, but not for serovar determination.

Introduction

The genus Leptospira is an incredibly varied group of organisms, containing hundreds of serovars and genetic types, which can occupy diverse environments, habitats and life cycles. Included within the genus are highly pathogenic host-specific strains and harmless free-living waterborne strains. Historically, this genus is divided into two species, namely the pathogenic (e.g., Leptospira interrogans) and the free-living saprophytic (e.g. Leptospira biflexa) species. The latter is indigenous to fresh surface water (Johnson & Faine, 1984). Of late, DNA–DNA hybridization techniques have been used to classify the genus Leptospira into 17 genomospecies (Yasuda et al., 1987; Brenner et al., 1999; Levett, 2001). According to this scheme, pathogenic Leptospira comprise eight species. These are L. interrogans sensu stricto, Leptospira weilii, Leptospira borgpetersenii, Leptospira noguchii, Leptospira santarosai, Leptospira alexanderi, Leptospira kirschneri (formerly Leptospira alstoni) and Leptospira genospecies 1. This species assignment is consistent with the results of the phylogenetic analyses based on the rrs gene, which codes for the 16S rRNA gene (Hookey et al., 1993; Postic et al., 2000). However, the discriminatory power of even complete rrs (size: c. 1500 bp) may be limited, due to the absence of a high degree of polymorphism in this gene.

Previously, we have demonstrated the usefulness of rpoB sequence analysis to help differentiate bacterial species, including spirochetes (Renesto et al., 2000; Khamis et al., 2004). This prompted us to design a pair of universal primers to amplify and sequence a small fragment of rpoB gene. The objective was to evaluate whether an such analysis would be useful for routine identification of Leptospira species.

Materials and methods

To begin with, we aligned the 3678 bp sequences of the rpoB of Leptospira available in the GenBank (L. biflexa Patoc, AF150880; L. interrogans Lai, NC-004342; L. interrogans Copenhageni, NC-005823). For this, we used a standard software (clustalw: http://npsa-pbil.ibcp.fr). Then, we used svarap software to determine the most variable area bordering the conserved regions (Khamis et al., 2004; Colson et al., 2005). This enabled us to design a pair of primers. The pair included Lept 1900f (5′-CCT-CAT-GGG-TTC-CAA-CAT-GCA-3′) and Lept 2500r (5′-CGC-ATC-CTC-RAA-GTT-GTA-WCC-TT-3′) bordering a hypervariable region between the position 1900 and 2500. Using this pair of primers, we have amplified and sequenced this area in 19 L. interrogans strains (Table 1). In this study, we have focused our attention on each species and several serovars of L. interrogans. We did this considering the fact that L. interrogans is the most common Leptospira spp. pathogenic to humans. Strains DNA was extracted using the QIAamp DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. All PCR mixtures contained 2.5 × 10−2 U of Taq polymerase µL−1, 1 × Taq buffer, 1.8 mM MgCl2 (Gibco BRL, Life Technologies, Cergy Pontoise, France), 200 µM concentrations of dATP, dCTP, dTTP and dGTP (Boehringer Manheim GmbH, Hilden, Germany) and 0.2 µM concentrations of each primer (Eurogentec, Seraing, Belgium). PCR mixtures were subjected to 35 cycles of denaturation at 94°C for 30 s, primer annealing at 51°C for 30 s and extension at 72°C for 2 min. Every amplification program began with a denaturation step of 95°C for 2 min and ended with a final elongation step of 72°C for 10 min. Amplicons were purified for sequencing using a QIAquick spin PCR purification kit (Qiagen) following the protocol of the supplier. Sequencing reactions were carried out with the same pair of primers and the reagents of ABI Prism 3100 DNA sequencer (dRhod. Terminator RR Mix, Perkin Elmer Applied Biosystems) following the standard automated sequencer protocol. Multiple sequence alignments and percentages of similarity between the different species with rpoB and 16S rRNA gene were made using clustalw on the EMBL-EBI web server (http://www.ebi.ac.uk/clustalw/). Phylogenetic trees were obtained from DNA sequences by the neighbour-joining method using the mega 3.1 software (http://www.megasoftware.net).

1

List of strains used in this study for partial rpoB gene sequencing and phylogenetic trees construction

   Genbank accession number 
Species Serovar Strain rpoB 16S rRNA 
Leptospira genomospecies 1 Pingchang 80–142 DQ296130  
 Sichuan 79601  AY631881 
L. santarosai Shermani 1342K DQ296131  
 LT 821  AY631883 
L. kirschneri Cynopteri 3522C DQ296139 AY631895 
L. interrogans Australis Akiyami A DQ296144  
 Batavia Van Tienen DQ296146  
 Icterohaemorrhagiae RGA DQ296133 AY631894 
 Verdun   
 Copenhageni M20   
 Copenhageni Fiocruz L1-130 AE017290  
 Pyrogenes Salinem DQ296147  
 Lai Lai NC_004342  
 Autumnalis Ballico DQ296145  
 Canicola Hond Utrecht   
 Hebdomadis Hebdomadis   
 Pomona Pomona   
L. noguchii Panama CZ214K DQ296141 AY631886 
L. borgpetersenii Javanica Veldrat Batavia 46 DQ296134 AY887899 
L. wolbachii Codice CDC DQ296138 AY611879 
L. biflexa Patoc Patoc1 DQ296142 AY631876 
L. meyeri Ranarum ICF DQ296135 AY631878 
Leptospira genomospecies 3 Holland Waz Holland DQ296143 AY631897 
Leptospira genomospecies 4 Hualin ST11-33  AY631888 
Leptospira genomospecies 5 Saopaulo Saopaulo DQ296136 AY631882 
L. weilii Celledoni Celledoni DQ296132 AY631877 
L. fainei Hurstbridge BuT 6T DQ296137 AY631885 
L. inadai Lyme 10 DQ296140 AY631896 
L. alexanderi Manhao 3 L60 DQ296129 AY631880 
   Genbank accession number 
Species Serovar Strain rpoB 16S rRNA 
Leptospira genomospecies 1 Pingchang 80–142 DQ296130  
 Sichuan 79601  AY631881 
L. santarosai Shermani 1342K DQ296131  
 LT 821  AY631883 
L. kirschneri Cynopteri 3522C DQ296139 AY631895 
L. interrogans Australis Akiyami A DQ296144  
 Batavia Van Tienen DQ296146  
 Icterohaemorrhagiae RGA DQ296133 AY631894 
 Verdun   
 Copenhageni M20   
 Copenhageni Fiocruz L1-130 AE017290  
 Pyrogenes Salinem DQ296147  
 Lai Lai NC_004342  
 Autumnalis Ballico DQ296145  
 Canicola Hond Utrecht   
 Hebdomadis Hebdomadis   
 Pomona Pomona   
L. noguchii Panama CZ214K DQ296141 AY631886 
L. borgpetersenii Javanica Veldrat Batavia 46 DQ296134 AY887899 
L. wolbachii Codice CDC DQ296138 AY611879 
L. biflexa Patoc Patoc1 DQ296142 AY631876 
L. meyeri Ranarum ICF DQ296135 AY631878 
Leptospira genomospecies 3 Holland Waz Holland DQ296143 AY631897 
Leptospira genomospecies 4 Hualin ST11-33  AY631888 
Leptospira genomospecies 5 Saopaulo Saopaulo DQ296136 AY631882 
L. weilii Celledoni Celledoni DQ296132 AY631877 
L. fainei Hurstbridge BuT 6T DQ296137 AY631885 
L. inadai Lyme 10 DQ296140 AY631896 
L. alexanderi Manhao 3 L60 DQ296129 AY631880 

Only sequences in bold type were determined and deposited in Genbank in the present study.

Results and discussion

The sequence similarities in the 600 bp segment of rpoB among the species tested varied between 60.5% and 92.3% (Table 2). In case of the 1500 bp segment of rrs, such figures varied between 88% and 99.7% (Table 3). For three major groups of species, the topology of the tree constructed using partial rpoB (Fig. 1) was identical to the tree we constructed using rrs (Fig. 2). However, the intragroup topology was different among these three major groups of species. For example, the branches were more widely separated in the rpoB tree. However, the bootstrap value obtained using partial rpoB and rrs was not significantly different (considering nodes with bootstrap value ≥75%, 6/13 vs. 9/14, respectively, P=0.3 by a χ2 test). Using a partial rpoB sequence, we were able to distinguish accurately 10 serovars of L. interrogans from other species. This demonstrates the usefulness of partial rpoB sequence in the identification of Leptospira down to the species level.

2

Percent similarity observed for partial rpoB sequences among Leptospira species and some Leptospira interrogans serovars

 10 11 12 13 14 15 16 17 18 19 20 
100                    
89.6 100                   
89.6 90.4 100                  
88.4 88.0 91.1 100                 
88.5 88.2 91.3 99.4 100                
88.4 88.2 91.5 99.3 99.1 100               
88.2 88.0 91.3 99.1 98.9 99.8 100              
87.1 87.6 91.1 97.0 96.9 97.6 97.4 100             
87.8 86.7 90.2 97.2 97.4 96.9 97.0 97.6 100            
10 88.4 87.8 90.6 97.4 97.6 97.0 96.9 97.0 97.2 100           
11 89.3 92.1 91.9 88.5 88.7 88.9 88.7 88.0 87.8 88.4 100          
12 89.1 88.7 92.2 89.8 90.0 90.2 90.0 90.2 89.6 89.3 89.1 100         
13 63.2 62.1 62.7 63.2 63.2 63.1 62.9 63.2 63.2 63.8 61.9 60.3 100        
14 61.8 60.3 60.3 61.4 61.2 61.6 61.4 61.8 62.1 62.3 61.9 58.6 87.7 100       
15 62.9 62.9 63.2 64.2 64.0 64.5 64.3 64.2 63.6 64.2 64.5 61.9 87.9 87.1 100      
16 62.5 62.1 61.9 62.9 62.7 63.1 62.9 62.7 62.9 63.1 63.2 60.5 87.7 88.8 88.1 100     
17 63.2 61.2 62.1 62.1 62.1 62.3 62.1 62.3 62.7 62.7 62.3 59.7 85.8 89.0 85.8 88.2 100    
18 86.9 88.7 88.7 88.9 89.1 89.3 89.1 88.9 88.4 88.7 88.4 88.7 63.2 61.2 64.5 61.2 61.2 100   
19 74.9 74.1 72.8 73.8 73.8 73.8 73.6 72.6 72.6 73.9 73.6 73.8 60.7 59.0 59.2 59.9 58.8 73.9 100  
20 74.1 73.9 72.1 73.4 73.4 73.2 73.0 72.6 72.6 74.1 73.2 72.1 60.1 59.0 59.6 58.8 59.0 72.6 91.3 100 
21 87.1 87.4 87.6 86.1 86.3 86.9 86.7 86.3 86.1 86.0 89.5 87.8 62.5 62.1 64.2 63.1 63.2 90.2 73.9 72.3 
 10 11 12 13 14 15 16 17 18 19 20 
100                    
89.6 100                   
89.6 90.4 100                  
88.4 88.0 91.1 100                 
88.5 88.2 91.3 99.4 100                
88.4 88.2 91.5 99.3 99.1 100               
88.2 88.0 91.3 99.1 98.9 99.8 100              
87.1 87.6 91.1 97.0 96.9 97.6 97.4 100             
87.8 86.7 90.2 97.2 97.4 96.9 97.0 97.6 100            
10 88.4 87.8 90.6 97.4 97.6 97.0 96.9 97.0 97.2 100           
11 89.3 92.1 91.9 88.5 88.7 88.9 88.7 88.0 87.8 88.4 100          
12 89.1 88.7 92.2 89.8 90.0 90.2 90.0 90.2 89.6 89.3 89.1 100         
13 63.2 62.1 62.7 63.2 63.2 63.1 62.9 63.2 63.2 63.8 61.9 60.3 100        
14 61.8 60.3 60.3 61.4 61.2 61.6 61.4 61.8 62.1 62.3 61.9 58.6 87.7 100       
15 62.9 62.9 63.2 64.2 64.0 64.5 64.3 64.2 63.6 64.2 64.5 61.9 87.9 87.1 100      
16 62.5 62.1 61.9 62.9 62.7 63.1 62.9 62.7 62.9 63.1 63.2 60.5 87.7 88.8 88.1 100     
17 63.2 61.2 62.1 62.1 62.1 62.3 62.1 62.3 62.7 62.7 62.3 59.7 85.8 89.0 85.8 88.2 100    
18 86.9 88.7 88.7 88.9 89.1 89.3 89.1 88.9 88.4 88.7 88.4 88.7 63.2 61.2 64.5 61.2 61.2 100   
19 74.9 74.1 72.8 73.8 73.8 73.8 73.6 72.6 72.6 73.9 73.6 73.8 60.7 59.0 59.2 59.9 58.8 73.9 100  
20 74.1 73.9 72.1 73.4 73.4 73.2 73.0 72.6 72.6 74.1 73.2 72.1 60.1 59.0 59.6 58.8 59.0 72.6 91.3 100 
21 87.1 87.4 87.6 86.1 86.3 86.9 86.7 86.3 86.1 86.0 89.5 87.8 62.5 62.1 64.2 63.1 63.2 90.2 73.9 72.3 

1, Leptospira genomospecies 1 (Pingchang 80–142); 2, Leptospira santarosai (Shermani 1342K); 3, Leptospira kirschneri; 4, Leptospira interrogans Australis; 5, L. interrogans Batavia; 6, L. interrogans Icterohaemorrhagiae RGA (strains Verdun and Copenhageni M20 shared identical sequence); 7, L. interrogans Icterohaemorrhagiae Copenhageni Fiocruz L1-130; 8, L. interrogans Pyrogenes; 9, L. interrogans Lai; 10, L. interrogans Autumnalis (serovars Canicola, Hebdomadis and Pomona shared the same sequence); 11. Leptospira noguchii; 12. Leptospira borgpetersenii; 13, Leptospira wolbachii; 14, Leptospira biflexa; 15, Leptospira meyeri; 16, Leptospira genomospecies 3; 17, Leptospira genomospecies 5; 18, Leptospira weilii; 19, Leptospira fainei; 20, Leptospira inadai; 21, Leptospira alexanderi.

3

Percent similarity observed for partial 16S rRNA gene sequences among Leptospira species (Genbank accession number)

 10 11 12 13 14 15 
100               
99.4 100              
95.1 95.2 100             
95.2 95.2 99.3 100            
88.0 87.8 89.0 89.0 100           
95.1 95.0 99.2 98.9 89.2 100          
88.1 87.9 89.0 89.1 98.3 88.9 100         
94.8 94.8 98.9 99.5 89.0 98.4 89.1 100        
88.0 87.8 89.0 89.1 98.6 88.9 99.6 89.1 100       
10 95.0 95.2 99.2 99.4 89.0 99.0 89.1 99.0 89.1 100      
11 95.1 95.2 99.0 99.1 88.7 98.6 88.9 98.7 88.9 99.0 100     
12 95.1 95.1 99.4 99.1 88.7 98.7 88.8 98.7 88.8 98.9 98.8 100    
13 94.8 95.0 99.6 99.1 88.7 99.0 88.9 98.7 88.9 99.2 98.9 99.2 100   
14 88.0 87.8 89.0 89.0 98.5 88.8 99.5 89.0 99.9 89.0 88.8 88.7 88.8 100  
15 87.9 87.7 88.8 88.9 98.0 88.7 99.4 88.9 99.4 88.8 88.6 88.6 88.6 99.4 100 
16 88.0 87.8 88.9 89.0 98.1 88.7 99.7 89.0 99.5 88.9 88.7 88.7 88.7 99.5 99.4 
 10 11 12 13 14 15 
100               
99.4 100              
95.1 95.2 100             
95.2 95.2 99.3 100            
88.0 87.8 89.0 89.0 100           
95.1 95.0 99.2 98.9 89.2 100          
88.1 87.9 89.0 89.1 98.3 88.9 100         
94.8 94.8 98.9 99.5 89.0 98.4 89.1 100        
88.0 87.8 89.0 89.1 98.6 88.9 99.6 89.1 100       
10 95.0 95.2 99.2 99.4 89.0 99.0 89.1 99.0 89.1 100      
11 95.1 95.2 99.0 99.1 88.7 98.6 88.9 98.7 88.9 99.0 100     
12 95.1 95.1 99.4 99.1 88.7 98.7 88.8 98.7 88.8 98.9 98.8 100    
13 94.8 95.0 99.6 99.1 88.7 99.0 88.9 98.7 88.9 99.2 98.9 99.2 100   
14 88.0 87.8 89.0 89.0 98.5 88.8 99.5 89.0 99.9 89.0 88.8 88.7 88.8 100  
15 87.9 87.7 88.8 88.9 98.0 88.7 99.4 88.9 99.4 88.8 88.6 88.6 88.6 99.4 100 
16 88.0 87.8 88.9 89.0 98.1 88.7 99.7 89.0 99.5 88.9 88.7 88.7 88.7 99.5 99.4 

1, Leptospira inadai; 2, Leptospira fainei; 3, Leptospira borgpetersenii; 4, Leptospira kirschneri; 5, Leptospira genomospecies 3; 6, Leptospira santarosai (Shermani LT 821); 7, Leptospira wolbachii; 8, Leptospira interrogans Icterohaemorrhagiae RGA; 9, Leptospira biflexa; 10, Leptospira noguchii; 11, Leptospira genomospecies 1 (Sichuan 79601); 12, Leptospira alexanderi; 13, Leptospira weilii; 14, Leptospira genomospecies 4; 15, Leptospira genomospecies 5; 16, Leptospira meyeri.

1

Dendrogram representing phylogenetic relationships among Leptospira species. This dendrogram was constructed using the neighbour-joining method. The tree was derived from alignment of partial rpoB sequences. The support of each branch, as determined from 1000 bootstrap samples, is indicated by the value at each node (in %). IH means Icterohaemorrhagiae. The sequences used are those listed in Table 1.

1

Dendrogram representing phylogenetic relationships among Leptospira species. This dendrogram was constructed using the neighbour-joining method. The tree was derived from alignment of partial rpoB sequences. The support of each branch, as determined from 1000 bootstrap samples, is indicated by the value at each node (in %). IH means Icterohaemorrhagiae. The sequences used are those listed in Table 1.

2

Dendrogram representing phylogenetic relationships among Leptospira species. This dendrogram was constructed using the neighbour-joining method. The tree was derived from alignment of 16S rRNA gene sequences. The support of each branch, as determined from 1000 bootstrap samples, is indicated by the value at each node (in %). The sequences used are those listed in Table 1.

2

Dendrogram representing phylogenetic relationships among Leptospira species. This dendrogram was constructed using the neighbour-joining method. The tree was derived from alignment of 16S rRNA gene sequences. The support of each branch, as determined from 1000 bootstrap samples, is indicated by the value at each node (in %). The sequences used are those listed in Table 1.

Our data demonstrate that rpoB is a good reflection of the gene contents of Leptospira. Apart from what we observed in Leptospira genomospecies 3, there was a good correlation (Fig. 3) between the G+C content of the genome and partial rpoB [coefficient of determination (R2) > 0.7]. Our findings were similar to those obtained by others (Fournier et al., 2006). In Leptospira genomospecies 3, there was no good correlation between the G+C content of the genome and partial rpoB. The reason for this discrepancy remains unclear to us. Future studies should address this issue, as due to technical limitations the determination of G+C content may not always be reproducible.

3

Plot of the rpoB G+C content vs. complete genome for 13 Leptospira species (Leptospira genomospecies 1, Leptospira santarosai, Leptospira interrogans, Leptospira noguchii, Leptospira borgpetersenii, Leptospira wolbachii, Leptospira biflexa, Leptospira meyeri, Leptospira genomospecies 3 each, Leptospira genomospecies 5, Leptospira weilii, Leptospira inadai, Leptospira alexanderi). Apart from Leptospira genomospecies 3 (dot), all species are represented by squares. A regression line is fitted to the data. The coefficient of determination and the tendency curve equation are indicated.

3

Plot of the rpoB G+C content vs. complete genome for 13 Leptospira species (Leptospira genomospecies 1, Leptospira santarosai, Leptospira interrogans, Leptospira noguchii, Leptospira borgpetersenii, Leptospira wolbachii, Leptospira biflexa, Leptospira meyeri, Leptospira genomospecies 3 each, Leptospira genomospecies 5, Leptospira weilii, Leptospira inadai, Leptospira alexanderi). Apart from Leptospira genomospecies 3 (dot), all species are represented by squares. A regression line is fitted to the data. The coefficient of determination and the tendency curve equation are indicated.

As is the case with rrs, a single pair of primers enables us to amplify partial rpoB of any representative strain of the 15 species/genomospecies belonging to this diverse genus. However, in Leptospira species, the analysis of partial rpoB offers two additional advantages. Firstly, a 600 bp fragment of rpoB may be amplified and sequenced in two runs of sequence using the same pair of primers used for PCR amplification. This is not the case for large fragments such as rrs, which requires approximately six primers and thus six runs of sequence to be accurately sequenced. Secondly, compared with rrs, the degree of polymorphism is higher in rpoB amplicons. This means that rpoB may be more useful than rrs for the identification of Leptospira species. For examples, in L. kirschneri Cynopteri and L. interrogans Canicola, the positions of only three nucleotides are different in 1500 bp sequences of rrs. In contrast, for the same species, the positions of 51 nucleotides are different in 600 bp fragment of rpoB. Owing to the convenience of this method, we amplified genomospecies 1, 3 and 5. To our knowledge, there is no published report that has examined the phenotypic characters of these genomospecies. Previously, it has been suggested that genomospecies 3 and 5 might be free–2 living species. Our data (Fig. 1) confirm this notion, for both genomospecies 3 and 5 cluster with Leptospira wolbachii, L. biflexa and Leptospira meyeri. In contrast, genomospecies 1 clusters with pathogenic Leptospira. Genomospecies 1 comprises two serovars, both of which have been isolated from frogs in China. The other serovars isolated from frogs include Ranarum and Bim in the USA and Barbados, respectively. The former serovar belongs to L. meyeri, a free–2 living species and the latter to L. kirschneri, a pathogenic species.

Several molecular techniques have been evaluated for the identification of Leptospira species or serovars. These include random amplified polymorphic DNA, arbitrarily primed PCR, use of insertion sequences in PCR-based assays, restriction length polymorphism, specific probes, variable number tandem repeat analysis and pulsed-field gel electrophoresis (Barocchi et al., 2001; Levett, 2001; Majed et al., 2005). However, none of these techniques is based on sequence analysis. This may be problematic as far as the reproducibility among different laboratories is concerned. The Leptospira genes coding for outer membrane proteins have been sequenced for six pathogenic species. These genes transfer horizontally from one species to the other. This makes these genes unsuitable for the identification of various Leptospira species (Haake et al., 2004).

Using rpoB sequence analysis, we were able to separate 11 L. interrogans serovars tested in this study and place them under seven genotypes. As had been the case with rrs (Postic et al., 2000), this technique separated several genogroups. However, it failed to distinguish clearly the member of one serovar from that of the other. This confirms the previously held notion (Herrmann et al., 1992; Brenner et al., 1999; Levett, 2001) that the same serovar can be found in different genetic groups/species. Analyses of several strains of the most commonly encountered serovars may prove useful for defining the contribution of rpoB sequencing to the molecular screening of Leptospira isolates. Such information may, in turn, prove invaluable for the determination of various serovars of Leptospira. Alternatively, analysis of a segment of rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira using a system of similarity cut-off to define species (La Scola et al., 2003). If the partial rpoB similarity of a test isolate is lower than 92%, it should be regarded as a new species. On the other hand, if such a value goes above 97%, the isolate under scrutiny should be regarded as being representative of a known species. However, to be used for the routine identification of Leptospira, these cut-offs will have to be validated on large collections of isolates, as we did for Corynebacterium sp. (Khamis et al., 2005). Finally, this technique may be useful for the detection as well as identification of Leptospira in clinical or environmental samples, provided the specificity of the primers has been verified previously.

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Author notes

Editor: Reggie Lo