Leptospira broomii sp. nov., isolated from humans with leptospirosis

Footnotes

The genus Leptospira presently consists of 12 named species and four unnamed genomospecies (Brenner et al., 1999; Levett et al., 2005; Pérolat et al., 1998). Phylogenetic analysis shows that leptospires cluster in three clades, representing species that contain pathogenic serovars, non-pathogenic serovars and an intermediate group (Pérolat et al., 1998). Within the latter clade are two species, Leptospira inadai and Leptospira fainei (Pérolat et al., 1998; Yasuda et al., 1987). L. fainei was first isolated from the uteri and kidneys of pigs in south-eastern Australia (Pérolat et al., 1998). Serological reactivity to L. fainei has been found in pigs and cattle in Australia (Pérolat et al., 1998) and in humans in Australia (Chappel et al., 1998) and in the Seychelles (Yersin et al., 1998). Three cases of human infection were recently reported in European countries (Arzouni et al., 2002; Petersen et al., 2001), from which leptospires identified as L. fainei were isolated. In this paper, we report the characterization of these isolates using DNADNA relatedness, G+C content, 16S rRNA gene sequence data and pulsed-field gel electrophoresis (PFGE) and the definition of a novel species to accommodate them.

Strains 5399^T and L065 were isolated in polysorbate medium (Arzouni et al., 2002; Petersen et al., 2001) from the blood of patients with acute leptospirosis. These and other strains were maintained at room temperature in semi-solid PLM-5 medium (Serologicals Corp.) containing 1·5 % agar (Difco). Subcultures in liquid PLM-5 medium were incubated at 30 °C for 7 days. DNA was extracted and purified from strains 5399^T and L065, L. fainei BUT 6^T and L. inadai 10^T as described previously (Brenner et al., 1982). DNA from all strains except L065 was labelled with [³²P]dCTP (Brenner et al., 1982) and DNA relatedness and percentage divergence between the strains were determined by the hydroxyapatite method, with 55 °C used for optimal reassociation (Table 1). The G+C content (mol%) was determined for strains 5399^T and L065 by the thermal denaturation method (Mandel et al., 1970). All samples were run at least three times, using DNA from Escherichia coli K-12 as a control.

Table 1. DNA relatedness of strains 5399T and L065 with related Leptospira strains RBR, Relative binding ratio; D, percentage divergence. Reactions were performed at 55 °C.

For PFGE analysis, late-exponential-phase cultures in liquid PLM-5 medium at 30 °C were centrifuged to concentrate the bacteria used to prepare the agarose plugs. Slices cut from each agarose plug were digested with 30 U NotI restriction enzyme at 37 °C for 2 h. Salmonella enterica serovar Braenderup strain H9812 was digested with 50 U XbaI for use as a DNA size standard.

Plug slices containing digested DNA were placed in the wells of a 1 % agarose gel and electrophoresed in a Bio-Rad CHEF Mapper XA or CHEF-DRIII for 18 h at 14 °C with recirculating TBE buffer under the following conditions: 2·16 s initial switch time, 35·07 s final switch time, 120° angle, 6·0 V cm¹ gradient, linear ramping factor. The gel was stained with ethidium bromide following PFGE and photographed under UV transillumination. Gel analysis was performed and Pearson correlation coefficients were calculated using BioNumerics version 3.0 software.

Strains 5399^T and L065 showed morphology and motility typical of Leptospira strains under dark-field microscopy. DNA relatedness data are shown in Table 1. Strains 5399^T and L065 showed no significant relatedness to L. fainei strain BUT 6^T or L. inadai 10^T. However, there was strong relatedness between the two strains 5399^T and L065. These two strains meet the criteria for the molecular definition of a species (Brenner et al., 1999). The G+C contents of strains 5399^T and L065 were 42 mol%, within the range of other Leptospira species (Yasuda et al., 1987). Phylogenetic analysis of 16S rRNA gene sequences of strains 5399^T and L065, showed that these two strains formed a cluster distinct from L. fainei and were more closely related to, but still distinct from, L. inadai (Fig. 1). The DNA relatedness and phylogenetic analyses support the classification of strains 5399^T and L065 in a novel species, part of the intermediate clade that also contains L. inadai and L. fainei.

Isolated from the blood, cerebrospinal fluid and urine of human patients with leptospirosis in Denmark and France. Morphology is as described previously for the genus (Brenner et al., 1999; Johnson & Faine, 1984). Cells are 1015 µm in length and 0·1 µm in diameter, with wavelength of 0·5 µm and amplitude of approximately 0·3 µm (Petersen et al., 2001). The DNA G+C content is 42 mol%. The type strain is 5399^T (=ATCC BAA-1107^T=KIT 5399^T).