Evaluation of groEL Gene Analysis for Identification of Borrelia burgdorferi Sensu Lato

Abstract

The nucleotide sequences of the groEL genes, the flagellin genes, and the 16S rRNA genes from 22 reference strains of Borrelia were compared. groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of Borrelia afzelii and Borrelia garinii isolates.Borrelia burgdorferi sensu lato, the causative spirochete of Lyme disease, is transmitted to humans and animals through Ixodes ticks (1, 3). Lyme disease is one of the most prevalent tick-borne infectious diseases in Europe and North America and now occurs all over the world (1, 12). Various DNA-based techniques have recently been developed for the species identification of B. burgdorferi because characterization and identification by conventional methods are time-consuming and expensive (2,5,6,11). A previous study proposed that groEL gene analysis is useful for the differentiation of B. burgdorferi sensu lato (9). In the present study, groEL gene analysis was compared with the sequence analyses of the 16S rRNA and the flagellin gene to determine the role of the groEL gene in defining evolutionary relationships among strains of B. burgdorferi sensu lato.Twelve Borrelia strains were recently isolated from Ixodes granulatus, Ixodes nipponensis (tick vectors for Lyme spirochetes in rare cases), and Ixodes persulcatus, and 11 strains were isolated from Apodemus agrarius (8,10). In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10). In the present study, a comparative sequence analysis of the groEL gene from Korean isolates was performed to determine their relationships with the known species of the genus Borrelia.Twenty-two reference strains (Table 1) and 23 Korean isolates of the genus Borrelia were used in this study. The strains were cultivated at 32°C in Barbour-Stoenner-Kelly II (BSKII) medium. DNA was extracted by a modified version of a previously described method (4). The groEL genes of 22 reference strains and 23 Korean isolates and the flagellin genes and the 16S rRNA genes of 22 reference strains were amplified as presented in Table 2. The nucleotide sequences of the recombinant DNA were determined using the CEQ L DNA Analysis System and the CEQ 2000 Dye Terminator Cycle Sequencing kit (Beckman Coulter Inc., Fullerton, Calif.) with forward and reverse sequencing primers (M13) and sequencing primers ( Table 2). The multiple-alignment algorithm in the MegAlign software package (Windows version 3.12e; DNASTAR, Madison, Wis.) was used to align the sequences. All positions with alignment gaps were excluded from the pairwise sequence comparison. Phylogenetic trees were constructed by the unweighted pair group method with arithmetic averages using the MEGA program (7). A bootstrap analysis (100 replicates) was performed to evaluate the topology of the phylogenetic tree.In this study, interspecies differences in the groEL genes (positions 552 to 861 in B. burgdorferi B31 T numbering; 310 bp) of B. burgdorferi strains sensu lato were compared ...

Population genetics, taxonomy, phylogeny and evolution of Borrelia burgdorferi sensu lato

“…Many different genes and loci have been targeted in studies depending on the level of variation and the discriminatory power required and which species were being investigated. These included intergenic spacer (IGS) regions, the rrs (16S rRNA) locus, the plasmid located genes encoding the outer surface proteins A and C ( ospA , ospC ), decorin-binding protein A ( dbpA ), the chromosomally located housekeeping genes recombinase A ( recA ), groEL , hbb or flagellin B ( flaB ) (Casati et al, 2004; Dykhuizen and Baranton, 2001; Fukunaga et al, 1996c; Liveris et al, 1995; Marconi et al, 1995; Michel et al, 2004; Park et al, 2004; Postic et al, 1994; Schulte-Spechtel et al, 2006; Valsangiacomo et al, 1997; Will et al, 1995; Wilske et al, 1996). …”

Characterization Through Multilocus Sequence Analysis of Borrelia turdi Isolates from Portugal

“…Sequencing procedures were performed at the sequencing unit of the Portuguese National Institute of Health Dr. Ricardo Jorge. B. turdi-positive cultures that showed Borrelia growth and that were passaged at least three times were further tested by a nested PCR targeting the 5S-23S rRNA intergenic spacer region [20] and the 16S rRNA using the primers described by Park et al [21]. MLST was performed as described in [15], by the amplification of internal fragments of eight housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA) with elongation times of 60 s. Amplicons were sequenced as described above using the inner primers (in the case of nested PCR reactions).…”

Rapid Identification of Borrelia burgdorferi Sensu Lato by PCR‐Restriction Fragment Length Polymorphism Analysis of groEL Gene

“…The MseI and DraI restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other B. burgdorferi sensu lato strains (10). Haenam strains also formed a distinctive cluster in the phylogenetic trees constructed with the 16 S rDNA and groEL gene sequences (10,16).…”

“…B. afzelii ACA1 and HN17 were not clustered with other B. afzelii strains in the phylogenetic tree constructed based on the 5S-23S intergenic spacer sequences (data not shown) whereas those were clustered with other B. afzelii strains in the phylogenetic tree constructed based on groEL gene sequences (16).…”

“…Secondly, the groEL gene sequence analysis is useful for the differentiation and identification of B. burgdorferi sensu lato (11,16 conclusion (17). B. afzelii ACA1 and HN17 were not clustered with other B. afzelii strains in the phylogenetic tree constructed based on the 5S-23S intergenic spacer sequences (data not shown) whereas those were clustered with other B. afzelii strains in the phylogenetic tree constructed based on groEL gene sequences (16).…”