Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding

Abstract

Leptospirosis is a zoonotic disease of global importance. The breadth of Leptospira diversity associated with both human and animal disease poses major logistical challenges to the use of classical diagnostic techniques, and increasingly molecular diagnostic tools are used for their detection. In New Zealand, this has resulted in an increase in positive cases reported nationally that have not been attributed to the infecting serovar or genomospecies. In this study, we used data from all pathogenic Leptospira genomes to identify a partial region of the glmU gene as a suitable locus for the discrimination of the infecting species and serovars of New Zealand-endemic Leptospira. This method can be used in culture and culture-independent scenarios making it flexible for diagnostics in humans, animals, and environmental samples. We explored the use of this locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION. Sequences obtained by this method allowed specific identification of Leptospira species in mixed and enriched environmental cultures, however read error inherent in the MinION sequencing system reduced the accuracy of strain/variant identification. Using this approach to characterise Leptospira in enriched environmental cultures, we detected the likely presence of Leptospira genomospecies that have not been reported in New Zealand to date. This included a strain of L. borgpetersenii that has recently been identified in dairy cattle and sequences similar to those of L. mayottensis. L. tipperaryensis, L. dzianensis and L. alstonii.

Application of Multilocus Sequence Typing for the Characterization of Leptospira Strains in Malaysia

“…Such information provides a vital contribution to the planning and implementation of effective prevention strategies [ 4 , 23 , 24 ]. As 16S rRNA gene sequencing has poor discriminatory strength and whole genome sequencing is currently impractical for routine strain typing, additional gene targets, such as secY and glmU genes, have been utilized to type Leptospira species [ 6 , 11 , 25 , 26 ]. LipL32 gene PCR sequencing has also been used extensively because it is a robust target for leptospirosis diagnosis and only detects pathogenic leptospires [ 27 ].…”

Understanding leptospirosis: application of state-of-the-art molecular typing tools with a One Health lens

“…Given that whole genome sequencing is still not practical for routine strain typing and 16S ribosomal RNA (rRNA) gene sequencing provides limited discriminatory power, sequence information from other gene targets, such as the ppk, secY, flaB, lfb1, and glmU genes, have been used to type Leptospira species, 15,[18][19][20] as well as for PCR-based diagnostic testing. 21 The use of lipL32 gene PCR sequencing has been particularly widespread, because lipL32 gene PCR is a sensitive target for diagnosis of leptospirosis and only detects pathogenic leptospires.…”

“…20 A simplified 3-locus (adk, lipL41, mreA) scheme for direct application to clinical specimens was recently described, 26 although this still requires gel electrophoresis followed by sequencing, which is not practical in a routine clinical laboratory setting. After examination of 598 Leptospira published genomes from 20 P1 species, investigators 19 in New Zealand identified the glmU locus as having sufficient discriminatory power to differentiate among serovars circulating in New Zealand. Direct application of glmU PCR sequencing to the urine of infected cattle 25 was used to deduce infecting serovars.…”

“…from the environment has been facilitated by filtration and adding multiple antimicrobials or 5-fluorouracil to the culture medium to inhibit growth of contaminating bacteria. 19,[67][68][69][70][71][72] MALDI-TOF MS has also been used to rapidly identify isolates from soil. 70 With the use of PCR assays that target genes such as the lipL32, secY, flab, or glmU genes, a diverse array of pathogenic Leptospira species have been detected widely and in abundance in soil and water samples from regions where leptospirosis is endemic.…”

“…70 With the use of PCR assays that target genes such as the lipL32, secY, flab, or glmU genes, a diverse array of pathogenic Leptospira species have been detected widely and in abundance in soil and water samples from regions where leptospirosis is endemic. 2,19,70,72 Because some pathogenic Leptospira strains are not readily cultured from environmental sources, direct PCR sequencing of pathogenic leptospiral DNA from these sources has been performed using lipL32 or secY gene PCR 54,73,74 or high-throughput sequencing following environmental DNA metabarcoding. 20,43,75,76 With the latter approach, environmental DNA is concentrated and subjected to multiplex or singleplex PCR; indexing PCR is used to create dual index tags for high throughput sequencing, and the resulting sequences are compared to those in the NCBI database.…”