Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases.

A database of MALDI-TOF mass spectrometry (MS) profiles has been developed with the aim of establishing a high throughput system for the characterisation of microbes. Several parameters likely to affect the reproducibility of the mass spectrum of a taxon were exhaustively studied. These included such criteria as sample preparation, growth phase, culture conditions, sample storage, mass range of ions, reproducibility between instruments and the methodology prior to database entry. Replicates of 12 spectra per sample were analysed using a 96-well target plate containing central wells for peptide standards to correct against mass drift during analysis. The quality of the data was assessed statistically prior to database addition using root mean squared values of <3.0 as the criterion for rejection. Cluster analysis using a nearest neighbour algorithm also enabled subsets of data to be compared. This was achieved using the bespoke MicrobeLynx trade mark software. Columbia blood agar was used to standardise all procedures for the database, since it permitted the culture of most human pathogens and also produced spectra with a broad range of mass ions. In some instances, alternative media such as CLED were used in specific studies with greater success. Following standardisation of the procedure, a database was developed comprising ca. 3500 spectra with multiple strain entries for most species. The results to date show unequivocally that as the number of strains per species increased, so too did the success of species matching. The technique demonstrated unique mass spectral profiles for each genus/species, with the variation in mass ions among strains/species being dependent on the intra-specific diversity. The success of identification against the database for wild-type strains ranged between 33 and 100%; the lower percentage results being generally associated with poor representation of some species within the database. These findings provide a new dimension for the rapid and high throughput characterisation of human pathogens with potentially broad applications across the field of microbiology.

[1]  K. Voorhees,et al.  Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. , 1996, Rapid communications in mass spectrometry : RCM.

[2]  Adam Baldwin,et al.  Burkholderia cepacia complex infection in patients with cystic fibrosis. , 2002, Journal of medical microbiology.

[3]  Ellen Jo Baron,et al.  Manual of clinical microbiology , 1975 .

[4]  S. Gharbia,et al.  Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology. , 2002, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[5]  C. Fenselau.  Peer Reviewed: MALDI MS and Strategies for Protein Analysis , 1997 .

[6]  E Bergogne-Bérézin,et al.  Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features , 1996, Clinical microbiology reviews.

[7]  M. Collins,et al.  EVIDENCE OF PHYLOGENETIC HETEROGENEITY WITHIN THE GENUS RHODOCOCCUS: REVIVAL OF THE GENUS GORDONA (TSUKAMURA) , 1988 .

[8]  K. Kersters,et al.  Classification and identification of bacteria by electrophoresis of their proteins. , 1980, Society for Applied Bacteriology symposium series.

[9]  C. Fenselau,et al.  Use of bioactive glass slides for matrix-assisted laser desorption/ionization analysis: application to microorganisms. , 2003, Analytical Chemistry.

[10]  S. N. Davey,et al.  The rapid identification of intact microorganisms using mass spectrometry , 1996, Nature Biotechnology.

[11]  Patrick A. D. Grimont,et al.  Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii , 1986 .

[12]  S. Gharbia,et al.  The Application of Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry to Profile the Surface of Intact Bacterial Cells , 2000 .

[13]  R. Gaynes,et al.  Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. , 1999, Critical care medicine.

[14]  B. Szponar,et al.  Determination of microbial colonisation in water-damaged buildings using chemical marker analysis by gas chromatography-mass spectrometry. , 2000, Indoor air.

[15]  J. Rothgänger,et al.  Diagnostics of Neisseriaceae andMoraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms , 2001, Journal of Clinical Microbiology.

[16]  K. Welham,et al.  The characterization of micro-organisms by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1998, Rapid communications in mass spectrometry : RCM.

[17]  K. Schleifer,et al.  Taxonomic study of anaerobic, gram-negative, rod-shaped bacteria from breweries: emended description of Pectinatus cerevisiiphilus and description of Pectinatus frisingensis sp. nov., Selenomonas lacticifex sp. nov., Zymophilus raffinosivorans gen. nov., sp. nov., and Zymophilus paucivorans sp. nov. , 1990, International journal of systematic bacteriology.

[18]  M. Soufian,et al.  Rapid typing of bacteria using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and pattern recognition software. , 2002, Journal of microbiological methods.

[19]  M. Goodfellow,et al.  Microbiological classification and identification. , 1980, Society for Applied Bacteriology symposium series.

[20]  R. Cole,et al.  Rapid identification and speciation of Haemophilus bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1998, Journal of mass spectrometry : JMS.

[21]  G. Gilardi.  Nonfermentative gram-negative rods : laboratory identification and clinical aspects , 1985 .

[22]  R. Cotter,et al.  Time-of-flight mass spectrometry for the structural analysis of biological molecules. , 1992, Analytical chemistry.

[23]  C. Fenselau,et al.  Lectin-based affinity capture for MALDI-MS analysis of bacteria. , 1999, Analytical chemistry.

[24]  C. Nilsson.  Fingerprinting of Helicobacter pylori strains by matrix-assisted laser desorption/ionization mass spectrometric analysis. , 1999, Rapid communications in mass spectrometry : RCM.

[25]  W. Tsai,et al.  Identification of Enterobacteriaceae bacteria by direct matrix-assisted laser desorptiom/ionization mass spectrometric analysis of whole cells. , 1999, Rapid communications in mass spectrometry : RCM.

[26]  S. J. Flynn,et al.  Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1997, Rapid communications in mass spectrometry : RCM.

[27]  C. Fenselau,et al.  Matrix-assisted laser desorption/ionization time-of-flight analysis of Bacillus spores using a 2.94 µm infrared laser , 2000 .

[28]  J. Chun,et al.  Rhodococcal systematics: problems and developments , 1998, Antonie van Leeuwenhoek.

[29]  B. Szponar,et al.  Use of mass spectrometry for characterising microbial communities in bioaerosols. , 2001, Annals of agricultural and environmental medicine : AAEM.

[30]  J. Ley,et al.  The quantitative measurement of DNA hybridization from renaturation rates. , 1970, European journal of biochemistry.

[31]  Yuqin Dai,et al.  Detection and identification of low-mass peptides and proteins from solvent suspensions of Escherichia coli by high performance liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometry. , 1999, Rapid communications in mass spectrometry : RCM.

[32]  Liang Li,et al.  Investigation of spectral reproducibility in direct analysis of bacteria proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1998, Rapid communications in mass spectrometry : RCM.

[33]  P. Ross,et al.  Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells. , 1996, Rapid communications in mass spectrometry : RCM.

[34]  P. Demirev,et al.  Microorganism identification by mass spectrometry and protein database searches. , 1999, Analytical chemistry.

[35]  K. Welham,et al.  The effect of solvent and matrix combinations on the analysis of bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. , 1999, Rapid communications in mass spectrometry : RCM.

[36]  W. Ra,et al.  Enzyme patterns in bacterial classification and identification. , 1980 .

[37]  A. Ozbek,et al.  Identification of Three Strains of Mycobacterium Species Isolated from Clinical Samples Using Fatty Acid Methyl Ester Profiling , 2003, The Journal of international medical research.

[38]  B. V. Baar.  Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry. , 2000 .

[39]  K. Tomer,et al.  Discrimination effects and sensitivity variations in matrix‐assisted laser desorption/ionization , 1997 .

[40]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[41]  K. Schleifer,et al.  Chemical studies as a guide to the classification of Corynebacterium pyogenes and "Corynebacterium haemolyticum". , 1982, Journal of general microbiology.

[42]  M. Qian,et al.  Determination of bacterial protein profiles by matrix-assisted laser desorption/ionization mass spectrometry with high-performance liquid chromatography. , 1996, Rapid communications in mass spectrometry : RCM.

[43]  M. Erhard,et al.  Rapid typing and elucidation of new secondary metabolites of intact cyanobacteria using MALDI-TOF mass spectrometry , 1997, Nature Biotechnology.

[44]  M. Collins,et al.  Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. , 1991, International journal of systematic bacteriology.

[45]  P. Vandamme,et al.  Burkholderia anthina sp. nov. and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools. , 2002, FEMS immunology and medical microbiology.

[46]  D. A. Wolf,et al.  Differentiation of microorganisms based on pyrolysis-ion trap mass spectrometry using chemical ionization. , 1999, Analytical chemistry.

[47]  Catherine Fenselau,et al.  Rapid Characterization of Spores of Bacillus cereusGroup Bacteria by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry , 2000, Applied and Environmental Microbiology.

[48]  R. Pukall,et al.  Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus. , 2001, International journal of systematic and evolutionary microbiology.

[49]  J P Reilly,et al.  Fingerprint matching of E. coli strains with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of whole cells using a modified correlation approach. , 1998, Rapid communications in mass spectrometry : RCM.

[50]  A. Fox,et al.  Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility. , 2002, Journal of microbiological methods.