Leptospiral 16S rRNA gene sequencing has long been used as a typing method for molecular characterization of isolates, certification of bacterial panels, and taxonomic applications.10–12,17,18
DNA sequencing has several advantages over other typing methods, because it is relatively cheap and available, does not require complex reagents such as type-specific sera or purified DNA, and is not a time-demanding or laborious technique. Among some sequences, only partial coverage of the query sequence with the existing GenBank databases was observed. We also found several undefined bases in some sequences (both query and subject) that precluded the finding of 100% identity and consequently, impacted negatively on the identification of the reference strains. Although efforts have been made by several groups to deposit full-length (~1,500 bp), high-quality 16S rRNA sequences, there is still more work with respect to creating a complete set of sequences available in databases, such as GenBank, so that this method can be easily applied and interpreted. Indeed, as previously noted by Victoria and others,19 Leptospira
speciation errors can occur in reference collections, raising the possibility that some of the 16S rRNA sequences available in public databases may not be valid.
In this study, the sequencing of the 16S rRNA gene was used for quality control of reference strains from RC-99 and RC-04 (). The results were mainly concordant, although some discrepancies were observed in RC-04. Four strains of the 89 evaluated presented as contaminated cultures, and three were identified as existing strains from RC-04, probably caused by switching or mislabeling of strains during subculturing. Although 16S sequencing is a useful technique, it can only discriminate Leptospira
strains to the species level because of the highly conserved nature of the 16S rRNA genes. Before departure from KIT, the RC-04 panel was fully characterized using monoclonal antibodies, and it did not reveal the existence of any problems. Immediately on arrival, the RC-04 collection was quality controlled by the 16S sequencing, which showed the existence of possible switching or mislabeling of strains. Although the 16S rRNA gene sequencing presents limitations for the identification beyond the species level, we believe that a retyping of the strains confirmed as problems would provide similar results. The approach used for quality control seemed to be satisfactory for monitoring Leptospira
strain collections and highlighted the importance of serological verifications. The ramifications of switching strains in Leptospira
reference collections can be serious. During outbreak investigations, the serogroup is identified by the MAT and the panel of strains maintained at the local reference laboratory. Furthermore, few reference laboratories have the necessary facilities to maintain frozen stocks of their strain collections. Rather, the panels are maintained for years by repeated subculturing, significantly increasing the chances of strain switching or mislabeling. In a proficiency trial of the MAT, it was found that diagnostic laboratories often reported erroneous results.7
The serogroup was often incorrectly identified, resulting in false-negative and positive results. They concluded that contamination, mislabeling, and deterioration of the live cultures because of repeated subculturing likely contributed to these errors. Therefore, there is a need to establish an inexpensive quality-control method to identify these problems.
Polymorphisms within the 16S rRNA gene sequences of pathogenic Leptospira
spp. were reported to range from 1 to 19 nt among the pathogenic species.12
In this study, we observed a similar number of mismatches (1–13 nt, data not shown). As with previous studies, although it was possible to differentiate the species, we found that it was not possible to discriminate between the serovars because of the high homology of the 16S rRNA genes.12,20
Several groups have reported using shorter sequences (≤ 500 bp) in other genes to improve the discriminatory power over the 16S rRNA gene, including secY
Alternative techniques such as variable-number tandem-repeat22,23
and multi-locus sequence typing24,25
also offer potential improvements over 16S rRNA gene sequencing. Further work is needed to evaluate these alternative strategies as applied to quality-control testing of Leptospira
reference strains. This study has shown the need for periodical verifications and quality control in the maintenance of Leptospira
culture collections. In addition, this study has highlighted the importance of the availability of high-quality 16S rRNA gene sequences in public databases.