Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

BMC Microbiology

Ingrid Huber¹  Reinhard K Straubinger²  Andreas Sing¹  Ulrich Busch¹  Heribert Raschel¹  Regina Konrad¹  Volker Fingerle¹  Viktor Dyachenko²  Karola Grünwald¹  Inke Krupka²  Anna Rettinger² 

[1]Bavarian Health and Food Safety Authority, Veterinaerstr. 2, D-85764, Oberschleißheim, Germany

[2]Bacteriology and Mycology, Institute for Infectious Diseases and Zoonoses, Department of Veterinary Sciences, Faculty of Veterinary Medicine, LMU Munich, Veterinaerstr.13, 80539, Munich, Germany

关键词: ClinProTools;    16S rRNA;    rrs2;    LipL41;    LipL32;    multi locus sequence typing;    Leptospira borgpetersenii;    Leptospira kirschneri;    Leptospira interrogans;    MALDI-TOF MS;   

Others  :  1221779
DOI  :  10.1186/1471-2180-12-185

 received in 2012-05-10, accepted in 2012-07-30,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Rettinger et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Adler B, de la Pena MA: Leptospira and leptospirosis. Vet Microbiol 2010, 140:287-296.
• [2]Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, et al.: Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003, 3:757-771.
• [3]Levett PN: Leptospirosis. Clin Microbiol Rev 2001, 14:296-326.
• [4]Andre-Fontaine G: Canine leptospirosis–do we have a problem? Vet Microbiol 2006, 117:19-24.
• [5]Geisen V, Stengel C, Brem S, Muller W, Greene C, Hartmann K: Canine leptospirosis infections - clinical signs and outcome with different suspected Leptospira serogroups (42 cases). J Small Anim Pract 2007, 48:324-328.
• [6]Goldstein RE: Canine leptospirosis. Vet Clin North Am Small Anim Pract 2010, 40:1091-1101.
• [7]Adler B, Bragger JM: Leptospiral infections in humans. Med J Aust 1976, 2:357.
• [8]Yang CW, Wu MS, Pan MJ: Leptospirosis renal disease. Nephrol Dial Transplant 2001, 16(Suppl 5):73-77.
• [9]Visith S, Kearkiat P: Nephropathy in leptospirosis. J Postgrad Med 2005, 51:184-188.
• [10]Bolin CA, Cassells JA, Hill HT, Frantz JC, Nielsen JN: Reproductive failure associated with Leptospira interrogans serovar bratislava infection of swine. J Vet Diagn Invest 1991, 3:152-154.
• [11]Ellis WA: Leptospirosis as a cause of reproductive failure. Vet Clin North Am Food Anim Pract 1994, 10:463-478.
• [12]Kingscote BF: Diagnosis of Leptospira serovar hardjo Infection in Cattle in Canada. Can Vet J 1985, 26:270-274.
• [13]Curling A: Equine recurrent uveitis: classification, etiology, and pathogenesis. Compend Contin Educ Vet 2011, 33:E1-E4.
• [14]Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RS: Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Bacteriol 1999, 49(Pt 2):839-858.
• [15]Cerqueira GM, Picardeau M: A century of Leptospira strain typing. Infect Genet Evol 2009, 9:760-768.
• [16]Toyokawa T, Ohnishi M, Koizumi N: Diagnosis of acute leptospirosis. Expert Rev Anti Infect Ther 2011, 9:111-121.
• [17]Cerqueira GM, McBride AJ, Queiroz A, Pinto LS, Silva EF, Hartskeerl RA, et al.: Monitoring Leptospira strain collections: the need for quality control. AmJTrop Med Hyg 2010, 82:83-87.
• [18]Miller MD, Annis KM, Lappin MR, Lunn KF: Variability in results of the microscopic agglutination test in dogs with clinical leptospirosis and dogs vaccinated against leptospirosis. J Vet Intern Med 2011, 25:426-432.
• [19]da SJ B, Carvalho E, Hartskeerl RA, Ho PL: Evaluation of the Use of Selective PCR Amplification of LPS Biosynthesis Genes for Molecular Typing ofLeptospiraat the Serovar Level. Curr Microbiol 2010, 62:518-524.
• [20]Branger C, Blanchard B, Fillonneau C, Suard I, Aviat F, Chevallier B, et al.: Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1. FEMS Microbiol Lett 2005, 243:437-445.
• [21]Levett PN, Morey RE, Galloway RL, Turner DE, Steigerwalt AG, Mayer LW: Detection of pathogenic leptospires by real-time quantitative PCR. J Med Microbiol 2005, 54:45-49.
• [22]Slack AT, Symonds ML, Dohnt MF, Smythe LD: Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene. BMC Microbiol 2006, 6:95. BioMed Central Full Text
• [23]Sauer S, Kliem M: Mass spectrometry tools for the classification and identification of bacteria. Nat Rev Microbiol 2010, 8:74-82.
• [24]Claydon MA, Davey SN, Edwards-Jones V, Gordon DB: The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 1996, 14:1584-1586.
• [25]Stephan R, Ziegler D, Pfluger V, Vogel G, Lehner A: Rapid genus- and species-specific identification of Cronobacter spp. by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2010, 48:2846-2851.
• [26]Wieser A, Schneider L, Jung J, Schubert S: MALDI-TOF MS in microbiological diagnostics-identification of microorganisms and beyond (mini review). Appl Microbiol Biotechnol 2011, 93:965-974.
• [27]Djelouadji Z, Roux V, Raoult D, Kodjo A, Drancourt M: Rapid MALDI-TOF mass spectrometry identification of Leptospira organisms. Vet Microbiol 2012, 158:142-146.
• [28]Zhou N, Wang N, Xu B, Wang J, Fang J, Dong F, et al.: Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of bacteria cultured in liquid media. Sci China Life Sci 2011, 54:48-53.
• [29]Czekalowski JW, Mcleod JW, Rodican J: Observations on the nutrition ofLeptospirabased on the development of Dinger's phenomenon. J Gen Microbiol 1954, 10:199-208.
• [30]Alatoom AA, Cunningham SA, Ihde SM, Mandrekar J, Patel R: Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2011, 49:2868-2873.
• [31]Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, et al.: Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clin Microbiol 2010, 48:1169-1175.
• [32]Smole SC, King LA, Leopold PE, Arbeit RD: Sample preparation of Gram-positive bacteria for identification by matrix assisted laser desorption/ionization time-of-flight. J Microbiol Methods 2002, 48:107-115.
• [33]Ahmed N, Devi SM, Valverde ML, Vijayachari P, Machang'u RS, Ellis WA, et al.: Multilocus sequence typing method for identification and genotypic classification of pathogenicLeptospiraspecies. Ann Clin Microbiol Antimicrob 2006, 5:28. BioMed Central Full Text
• [34]Murray PR: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: usefulness for taxonomy and epidemiology. Clin Microbiol Infect 2010, 16:1626-1630.
• [35]Freiwald A, Sauer S: Phylogenetic classification and identification of bacteria by mass spectrometry. Nat Protoc 2009, 4:732-742.
• [36]Ferreira L, Sanchez-Juanes F, Gonzalez-Avila M, Cembrero-Fucinos D, Herrero-Hernandez A, Gonzalez-Buitrago JM, et al.: Direct identification of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2010, 48:2110-2115.
• [37]Ferreira L, Vega CS, Sanchez-Juanes F, Gonzalez-Cabrero S, Menegotto F, Orduna-Domingo A, et al.: Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures. PLoS One 2010, 5:e14235.
• [38]Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, Beretti JL, et al.: MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem 2011, 44:104-109.
• [39]Saenz AJ, Petersen CE, Valentine NB, Gantt SL, Jarman KH, Kingsley MT, et al.: Reproducibility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for replicate bacterial culture analysis. Rapid Commun Mass Spectrom 1999, 13:1580-1585.
• [40]Welker M: Proteomics for routine identification of microorganisms. Proteomics 2011, 11:3143-3153.
• [41]Haake DA, Suchard MA, Kelley MM, Dundoo M, Alt DP, Zuerner RL: Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol 2004, 186:2818-2828.
• [42]Morey RE, Galloway RL, Bragg SL, Steigerwalt AG, Mayer LW, Levett PN: Species-specific identification of Leptospiraceae by 16 S rRNA gene sequencing. J Clin Microbiol 2006, 44:3510-3516.
• [43]Victoria B, Ahmed A, Zuerner RL, Ahmed N, Bulach DM, Quinteiro J, et al.: Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira. PLoS One 2008, 3:e2752.
• [44]Adler B, Lo M, Seemann T, Murray GL: Pathogenesis of leptospirosis: the influence of genomics. Vet Microbiol 2011, 153:73-81.
• [45]Sauer S, Freiwald A, Maier T, Kube M, Reinhardt R, Kostrzewa M, et al.: Classification and identification of bacteria by mass spectrometry and computational analysis. PLoS One 2008, 3:e2843.
• [46]Dieckmann R, Helmuth R, Erhard M, Malorny B: Rapid classification and identification of salmonellae at the species and subspecies levels by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 2008, 74:7767-7778.
• [47]Zhang X, Leung SM, Morris CR, Shigenaga MK: Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma. J Biomol Tech 2004, 15:167-175.
• [48]Ketterlinus R, Hsieh SY, Teng SH, Lee H, Pusch W: Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software. Biotechniques 2005, 38:37-40.
• [49]Friedrichs C, Rodloff AC, Chhatwal GS, Schellenberger W, Eschrich K: Rapid identification of viridans streptococci by mass spectrometric discrimination. J Clin Microbiol 2007, 45:2392-2397.
• [50]Jackson KA, Edwards-Jones V, Sutton CW, Fox AJ: Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus. J Microbiol Methods 2005, 62:273-284.
• [51]Tanigawa K, Kawabata H, Watanabe K: Identification and typing of Lactococcus lactis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Appl Environ Microbiol 2010, 76:4055-4062.
• [52]Leuschner RG, Beresford-Jones N, Robinson C: Difference and consensus of whole cell Salmonella enterica subsp. enterica serovars matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra. Lett Appl Microbiol 2004, 38:24-31.
• [53]Picardeau M, Bulach DM, Bouchier C, Zuerner RL, Zidane N, Wilson PJ, et al.: Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS One 2008, 3:e1607.
• [54]Ahmed A, Thaipadungpanit J, Boonsilp S, Wuthiekanun V, Nalam K, Spratt BG, et al.: Comparison of two multilocus sequence based genotyping schemes for Leptospira species. PLoS Negl Trop Dis 2011, 5:e1374.
• [55]Nalam K, Ahmed A, Devi SM, Francalacci P, Baig M, Sechi LA, et al.: Genetic affinities within a large global collection of pathogenic Leptospira: implications for strain identification and molecular epidemiology. PLoS One 2010, 5:e12637.