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Journal of Clinical Microbiology, March 2004, p. 1270-1273, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1270-1273.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Hyo-Soon Park,¹ Jung-Hee Lee,¹ Eun-Ju Jeong,¹ Seong-Eun Koh,² Tae-Kyu Park,³ Won-Jong Jang,¹ Kyung-Hee Park,¹ Bum-Joon Kim,⁴ Yoon-Hoh Kook,⁴ and Seung-Hyun Lee^1*

Department of Microbiology,¹ Department of Rehabilitation Medicine, College of Medicine,² Department of Biotechnology, Konkuk University, Chungju, Chungchungbuk-Do 380-701,³ Department of Microbiology, Seoul National University College of Medicine, Seoul 110-799, Korea⁴

Received 22 July 2003/ Returned for modification 13 November 2003/ Accepted 21 November 2003

Top Abstract Introduction References

 
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.

Top Abstract Introduction References

 
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.

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TABLE 1. Borrelia reference strains used in this study

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TABLE 2. Sequences of primers and PCR conditions

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 with those in the flagellin genes (positions 280 to 789 in the B. burgdorferi B31^T numbering; 510 bp) and 16S rRNA genes (positions 44 to 849 in B. burgdorferi B31^T numbering; 806 bp). Moreover, intraspecies differences in the groEL genes from B. burgdorferi, B. afzelii, and B. garinii were compared with those in the flagellin and 16S rRNA genes. However, intraspecies differences in the groEL genes of other Borrelia species could not be compared with those in the flagellin and 16S rRNA genes, because the groEL gene sequence of just one strain per species was available (9).

groEL gene analysis has several characteristics different from those of analyses of other genes. Compared with the 16S rRNA genes, groEL sequences have higher divergence for strains of B. burgdorferi sensu lato. More than 91.6% similarity of the groEL gene sequences was observed among strains of B. burgdorferi sensu lato. On the other hand, more than 95.4% similarity of the 16S rRNA gene sequences was observed among strains of B. burgdorferi sensu lato (data not shown). The groEL gene sequence similarities in B. burgdorferi, B. afzelii, and B. garinii strains were 99.7 to 100%, 99.0 to 99.4%, and 96.8 to 100%, respectively (Table 3). On the other hand, the 16S rRNA gene sequence similarities in B. burgdorferi, B. afzelii, and B. garinii strains were 99.6 to 100%, 99.6 to 100%, and 99.1 to 100%, respectively (Table 3). These results showed that the groEL gene is more heterogeneous than the 16S rRNA gene and is useful in intraspecies differentiation. Compared with the flagellin gene analysis, more than 92.0% similarity of the flagellin gene sequences was observed in strains of B. burgdorferi sensu lato (data not shown). The groEL gene sequence similarities in B. burgdorferi, B. afzelii, and B. garinii strains were 99.7 to 100%, 99.0 to 99.4%, and 96.8 to 100%, respectively, whereas the flagellin gene sequence similarities in B. burgdorferi, B. afzelii, and B. garinii strains were 99.0 to 99.8%, 99.6 to 100%, and 98.0 to 100%, respectively (Table 3). These results showed that the groEL gene is more heterogeneous than the flagellin gene in B. afzelii and B. garinii, whereas the flagellin gene is more heterogeneous than the groEL gene in B. burgdorferi.

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TABLE 3. Intraspecies variation in the groEL, flagellin, and 16S rRNA gene sequences of Borrelia strains

The B. garinii IP89 strain showed the lowest similarity (96.8 to 98.4%) to other B. garinii strains (data not shown). This strain was previously classified as a different group with B. garinii using multilocus enzyme electrophoresis (2). This strain also showed a different restriction fragment length polymorphism (RFLP) pattern of 5S-23S intergenic spacer amplicons from one of the B. garinii strains (11). These results showed that groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of B. garinii strains. groEL gene sequence analysis may be useful for intraspecies differentiation of B. afzelii and B. garinii strains, whereas flagellin gene sequence analysis may be useful for intraspecies differentiation of B. burgdorferi strains.

Twenty-three Korean isolates were characterized by phylogenetic analysis based on groEL gene sequences. Eleven strains (KK1, KK2, KK5, KM4, KM10, CJ1, CJ2, CJ3, CJ21, HN9, and HN17), identified as B. afzelii through PCR-RFLP analyses of the ospC gene and the rrf-rrl intergenic spacer in a previous study (10), were also identified as B. afzelii by groEL gene analysis (Fig. 1). The nucleotide sequence of strain KW3 was identical to that of B. garinii IP89 (Fig. 1), and KW3 also showed the same restriction pattern as B. garinii IP89 in RFLP analysis of the 5S-23S intergenic spacer amplicons (data not shown). Eleven Haenam strains formed a distinctive cluster, separated from other strains of B. burgdorferi sensu lato in the phylogenetic tree (Fig. 1). The sequence similarities among 11 Haenam strains (HN6, HN7, HN8, HN11, HN12, HN13, HN14, HN15, HN16, HN18, and HN19) were 98.7 to 100%. In general, they showed 89.7 to 94.8% similarity with other strains of B. burgdorferi sensu lato. The MseI and DraI restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other strains of B. burgdorferi sensu lato. Furthermore, in the phylogenetic tree based on 16S ribosomal DNA sequences, Haenam strains also formed a distinctive cluster (8).

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FIG. 1. Phylogenetic tree based on groEL gene sequences of Borrelia strains. The phylogenetic tree was constructed by the unweighted pair group method with arithmetic averages using MEGA software. Bootstrap analysis was performed with 100 replicates. The GenBank accession numbers are shown in parentheses.

In conclusion, the groEL gene is useful for the identification and characterization of B. burgdorferi sensu lato despite the fact that it has a shorter nucleotide sequence (310 bp) than the flagellin gene (510 bp) and the 16S rRNA gene (806 bp).

 
This work was supported in part by the Ministry of Science and Technology through the Bio-Food and Drug Research Center at Konkuk University, Chungju, Korea.

 
* Corresponding author. Mailing address: Department of Microbiology, College of Medicine, Konkuk University, 322 Danwol-dong, Chungju, Chungchongbuk-Do 380-701, Korea. Phone: 82 (43) 840-3725. Fax: 82 (43) 851-9329. E-mail: shlee{at}kku.ac.kr.

Top Abstract Introduction References

 

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Journal of Clinical Microbiology, March 2004, p. 1270-1273, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1270-1273.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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• Richter, D., Postic, D., Sertour, N., Livey, I., Matuschka, F.-R., Baranton, G. (2006). Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.. Int. J. Syst. Evol. Microbiol. 56: 873-881 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Park, H.-S. Articles by Lee, S.-H. Search for Related Content PubMed PubMed Citation Articles by Park, H.-S. Articles by Lee, S.-H.