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. 2023 Nov 9;11(11):2739.
doi: 10.3390/microorganisms11112739.

First Report on Leptospira Species Isolated from Patients in Slovenia

Eva Ružić-Sabljić  1 , Daša Podgoršek  1   2 , Katja Strašek Smrdel  1 , Andraž Celar Šturm  1 , Mateja Logar  3   4 , Andrea Pavlović  5 , Tatjana Remec  6 , Zvonko Baklan  7 , Emil Pal  8 , Tjaša Cerar Kišek  1   9
Affiliations

First Report on Leptospira Species Isolated from Patients in Slovenia

Eva Ružić-Sabljić et al. Microorganisms. .

Abstract

Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human Leptospira strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified lfb1 gene, determination of Leptospira serogroups using rabbit immune sera, NotI-RFLP of the whole Leptospira genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic Leptospira species (L. kirschneri, L. interrogans, L. borgpetersenii, and L. santarosai) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using NotI-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of Leptospira strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.

Keywords: Leptospira sp.; MALDI-TOF; MLST; NotI-RFLP; Slovenia; WGS; leptospirosis; lfb gene.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Cluster analysis and NotI-RFLP banding patterns of Leptospira strains isolated from patients (cyan) in Slovenia and the reference Leptospira strains (grey). A UPGMA dendrogram using a dice similarity coefficient with a 1.5% band position tolerance was constructed for the 10 clinical and 15 reference Leptospira strains using BioNumerics 7.1 software.
Figure 2
Figure 2
Cluster analysis of Leptospira strains isolated from patients in Slovenia (cyan) and the reference Leptospira strains (black) using MALDI-TOF mass spectrometry.
Figure 3
Figure 3
Neighbor-joining tree based on 500 columns, no missing values. Distance based on columns form Leptospira MLST 1 (6), MLST 2 (5), Leptospira cgMLST (489). Orange: L. interrogans, green: L. borgpetersenii, blue: L. santarosai, yellow: L. kirschnerii, magenta: L. noguchi.
Figure 4
Figure 4
Phylogenetic trees based on nucleotide differences between the secY (upper) and lfb1 (lower) genes were constructed using the unweighted pair group method with arithmetic mean (UPGMA). The size of a fragment of secY was 1383 bp; the outgroup is L. biflexa Patoc1 (acc. No. GCF_000017685.1). The size of a fragment of lfb1 ranges from 363 bp to 372 bp; L. biflexa has no lfb1 gene.
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