up to 6 months. This can also allow the shipment of the
inactivated Leptospira suspension to a distant laboratory.
4. The pellet must be completely dry. Any remaining ethanol will
decrease the quality of the reads.
5. The volume of formic acid to be added must be adjusted to the
size of the cell pellet, still paying attention to avoid excessive
dilution of the pellet. In case of doubt, simply add 10 μL to
small pellets and 15–20 μL to large pellets.
6. Once the spots are dry, you can check for the density of the
biological material on each spot. If needed, you can add
another 1 μL to spots with low material density and let dry
again.
7. Using biological replicates (independent preparation from an
independent culture of the same strain), additional reads can be
included in the MSP. This will allow taking into account some
peaks that may not be systematically present for a given strain.
8. Of note, the MSP is portable. A database with MSP for 35 Leptospira species was published as a supplementary material as a
btmsp file that can be imported into a Bruker Biotyper system
[7]. It is of utmost importance to include only well-identified
strains.
9. Scores below 2.0 or even below 1.5 do not preclude the identification of the genus Leptospira as observed with novel species
in a former study [7]. Scores of 2.300 or higher might be
observed for closely related species, notably species with 100%
nucleotide identity of their respective 16S rRNA genes, as also
observed between Escherichia coli and Shigella.
References
1. Johnson RC, Rogers P (1964) Differentiation
of pathogenic and saprophytic leptospires with
8-azaguanine. J Bacteriol 88:1618–1623
2. Johnson RC, Harris VG (1967) Differentiation
of
pathogenic
and
saprophytic
Leptospires. I. Growth at low temperatures. J
Bacteriol 94(1):27–31
3. Yasuda PH, Steigerwalt AG, Sulzer KR, Kaufmann AF, Rogers F, Brenner DJ (1987) Deoxyribonucleic
acid
relatedness
between
serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira
species. Int J Syst Bacteriol 37(4):407–415.
https://doi.org/10.1099/00207713-49-2839
4. Ramadass P, Jarvis BD, Corner RJ, Penny D,
Marshall RB (1992) Genetic characterization
of pathogenic Leptospira species by DNA
hybridization. Int J Syst Bacteriol 42
(2):215–219.
https://doi.org/10.1099/
00207713-42-2-215
5. Perolat P, Chappel RJ, Adler B, Baranton G,
Bulach DM, Billinghurst ML, Letocart M,
Merien F, Serrano MS (1998) Leptospira fainei
sp. nov., isolated from pigs in Australia. Int J
Syst Bacteriol 48:851–858. https://doi.org/
10.1099/00207713-48-3-851
6. Thibeaux R, Iraola G, Ferre ´s I, Bierque E,
Girault D, Soupe ´-Gilbert ME, Picardeau M,
Goarant C (2018) Deciphering the unexplored
Leptospira diversity from soils uncovers genomic evolution to virulence. Microb Genom 4
(1):000144. https://doi.org/10.1099/mgen.
0.000144
7. Thibeaux R, Girault D, Bierque E, Soupe ´-Gilbert ME, Rettinger A, Douyere A,
Meyer M, Iraola G, Picardeau M, Goarant C
(2018)
Biodiversity
of
environmental
28
Dominique Girault et al.
inactivated Leptospira suspension to a distant laboratory.
4. The pellet must be completely dry. Any remaining ethanol will
decrease the quality of the reads.
5. The volume of formic acid to be added must be adjusted to the
size of the cell pellet, still paying attention to avoid excessive
dilution of the pellet. In case of doubt, simply add 10 μL to
small pellets and 15–20 μL to large pellets.
6. Once the spots are dry, you can check for the density of the
biological material on each spot. If needed, you can add
another 1 μL to spots with low material density and let dry
again.
7. Using biological replicates (independent preparation from an
independent culture of the same strain), additional reads can be
included in the MSP. This will allow taking into account some
peaks that may not be systematically present for a given strain.
8. Of note, the MSP is portable. A database with MSP for 35 Leptospira species was published as a supplementary material as a
btmsp file that can be imported into a Bruker Biotyper system
[7]. It is of utmost importance to include only well-identified
strains.
9. Scores below 2.0 or even below 1.5 do not preclude the identification of the genus Leptospira as observed with novel species
in a former study [7]. Scores of 2.300 or higher might be
observed for closely related species, notably species with 100%
nucleotide identity of their respective 16S rRNA genes, as also
observed between Escherichia coli and Shigella.
References
1. Johnson RC, Rogers P (1964) Differentiation
of pathogenic and saprophytic leptospires with
8-azaguanine. J Bacteriol 88:1618–1623
2. Johnson RC, Harris VG (1967) Differentiation
of
pathogenic
and
saprophytic
Leptospires. I. Growth at low temperatures. J
Bacteriol 94(1):27–31
3. Yasuda PH, Steigerwalt AG, Sulzer KR, Kaufmann AF, Rogers F, Brenner DJ (1987) Deoxyribonucleic
acid
relatedness
between
serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira
species. Int J Syst Bacteriol 37(4):407–415.
https://doi.org/10.1099/00207713-49-2839
4. Ramadass P, Jarvis BD, Corner RJ, Penny D,
Marshall RB (1992) Genetic characterization
of pathogenic Leptospira species by DNA
hybridization. Int J Syst Bacteriol 42
(2):215–219.
https://doi.org/10.1099/
00207713-42-2-215
5. Perolat P, Chappel RJ, Adler B, Baranton G,
Bulach DM, Billinghurst ML, Letocart M,
Merien F, Serrano MS (1998) Leptospira fainei
sp. nov., isolated from pigs in Australia. Int J
Syst Bacteriol 48:851–858. https://doi.org/
10.1099/00207713-48-3-851
6. Thibeaux R, Iraola G, Ferre ´s I, Bierque E,
Girault D, Soupe ´-Gilbert ME, Picardeau M,
Goarant C (2018) Deciphering the unexplored
Leptospira diversity from soils uncovers genomic evolution to virulence. Microb Genom 4
(1):000144. https://doi.org/10.1099/mgen.
0.000144
7. Thibeaux R, Girault D, Bierque E, Soupe ´-Gilbert ME, Rettinger A, Douyere A,
Meyer M, Iraola G, Picardeau M, Goarant C
(2018)
Biodiversity
of
environmental
28
Dominique Girault et al.