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Logo of bmcmicrBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Microbiology
 
BMC Microbiol. 2012; 12: 185.
Published online Aug 27, 2012. doi:  10.1186/1471-2180-12-185
PMCID: PMC3460781
Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)
Anna Rettinger,1 Inke Krupka,1 Karola Grünwald,2 Viktor Dyachenko,1 Volker Fingerle,2 Regina Konrad,2 Heribert Raschel,2 Ulrich Busch,2 Andreas Sing,2 Reinhard K Straubinger,corresponding author1 and Ingrid Huber2
1Bacteriology and Mycology, Institute for Infectious Diseases and Zoonoses, Department of Veterinary Sciences, Faculty of Veterinary Medicine, LMU Munich, Veterinaerstr.13, 80539, Munich, Germany
2Bavarian Health and Food Safety Authority, Veterinaerstr. 2, D-85764, Oberschleißheim, Germany
corresponding authorCorresponding author.
Anna Rettinger: anna.rettinger/at/micro.vetmed.uni-muenchen.de; Inke Krupka: i.krupka/at/lmu.de; Karola Grünwald: karola.gruenwald/at/lgl.bayern.de; Viktor Dyachenko: v.dyachenko/at/lmu.de; Volker Fingerle: volker.fingerle/at/lgl.bayern.de; Regina Konrad: regina.konrad/at/lgl.bayern.de; Heribert Raschel: heribert.raschel/at/lgl.bayern.de; Ulrich Busch: ulrich.busch/at/lgl.bayern.de; Andreas Sing: andreas.sing/at/lgl.bayern.de; Reinhard K Straubinger: r.straubinger/at/lmu.de; Ingrid Huber: ingrid.huber/at/lgl.bayern.de
Received May 10, 2012; Accepted July 30, 2012.
Abstract
Background
In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis.
Results
Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering.
Conclusions
MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.
Keywords: MALDI-TOF MS, Leptospira interrogans, Leptospira kirschneri, Leptospira borgpetersenii, multi locus sequence typing, LipL32, LipL41, rrs2, 16S rRNA, ClinProTools
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