Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

ABSTRACT Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.

[1]  L. Cazares,et al.  Characterization of a Staphylococcus aureus USA300 protein signature using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 2012, Journal of medical microbiology.

[2]  Martin Aepfelbacher,et al.  MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages. , 2011, International journal of medical microbiology : IJMM.

[3]  A. Fox,et al.  Rapid discrimination between methicillin-sensitive and methicillin-resistant Staphylococcus aureus by intact cell mass spectrometry. , 2000, Journal of medical microbiology.

[4]  C. Fenselau,et al.  Characterization of the protein subset desorbed by MALDI from whole bacterial cells. , 2001, Analytical Chemistry.

[5]  K. Myambo,et al.  Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure , 1987, Journal of bacteriology.

[6]  M. Bischoff,et al.  Microarray-based analysis of the Staphylococcus aureus sigmaB regulon. , 2004, Journal of bacteriology.

[7]  Daphne Macapagal,et al.  Microarray-Based Analysis of the Staphylococcus aureus σB Regulon , 2004 .

[8]  D. Sturdevant,et al.  RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. , 2008, Molecular cell.

[9]  Rainer Paape,et al.  Top‐down label‐free LC‐MALDI analysis of the peptidome during neural progenitor cell differentiation reveals complexity in cytoskeletal protein dynamics and identifies progenitor cell markers , 2011, Proteomics.

[10]  Graham Ball,et al.  High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells. , 2009, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[11]  F. Götz,et al.  Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. , 1990, FEMS microbiology letters.

[12]  F. Tsai,et al.  Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF. , 2012, Analytical chemistry.

[13]  J. Rothgänger,et al.  Typing of Methicillin-Resistant Staphylococcus aureus in a University Hospital Setting by Using Novel Software for spa Repeat Determination and Database Management , 2003, Journal of Clinical Microbiology.

[14]  Hendrik Neubert,et al.  Label-free detection of differential protein expression by LC/MALDI mass spectrometry. , 2008, Journal of proteome research.

[15]  S. Engelmann,et al.  ςB Activity Depends on RsbU inStaphylococcus aureus , 2001, Journal of bacteriology.

[16]  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.

[17]  Ruifu Yang,et al.  Identification of Staphylococcus aureus and determination of its methicillin resistance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 2002, Analytical chemistry.

[18]  P. Dorrestein,et al.  Novel Phenol-soluble Modulin Derivatives in Community-associated Methicillin-resistant Staphylococcus aureus Identified through Imaging Mass Spectrometry* , 2012, The Journal of Biological Chemistry.

[19]  S. Morandi,et al.  Characterization of Staphylococcus aureus strains isolated from Italian dairy products by MALDI‐TOF mass fingerprinting , 2012, Electrophoresis.

[20]  F. Vandenesch,et al.  Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry , 2012, PloS one.

[21]  K. Voorhees,et al.  Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells. , 1999, Analytical chemistry.

[22]  A. Edwards,et al.  Methicillin Resistance Reduces the Virulence of Healthcare-Associated Methicillin-Resistant Staphylococcus aureus by Interfering With the agr Quorum Sensing System , 2012, The Journal of infectious diseases.

[23]  H. Seifert,et al.  Identification and discrimination of Staphylococcus aureus strains using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry , 2002, Proteomics.

[24]  S. Löfdahl,et al.  Identification and nucleotide sequence of the delta-lysin gene, hld, adjacent to the accessory gene regulator (agr) of Staphylococcus aureus , 1989, Molecular and General Genetics MGG.

[25]  Julian Parkhill,et al.  Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study , 2011, The Lancet. Infectious diseases.

[26]  S. Gatermann,et al.  The matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based protein peaks of 4448 and 5302 Da are not associated with the presence of Panton-Valentine leukocidin. , 2011, International journal of medical microbiology : IJMM.

[27]  D. Robinson,et al.  Evolutionary Models of the Emergence of Methicillin-Resistant Staphylococcus aureus , 2003, Antimicrobial Agents and Chemotherapy.

[28]  A. Simon,et al.  Successful management of an MRSA outbreak in a neonatal intensive care unit , 2011, European Journal of Clinical Microbiology & Infectious Diseases.

[29]  S. Foster,et al.  σB Modulates Virulence Determinant Expression and Stress Resistance: Characterization of a Functional rsbU Strain Derived from Staphylococcus aureus 8325-4 , 2002, Journal of bacteriology.

[30]  S. Foster,et al.  Investigations into σB-Modulated Regulatory Pathways Governing Extracellular Virulence Determinant Production in Staphylococcus aureus , 2006, Journal of bacteriology.

[31]  D. Raoult,et al.  MALDI-TOF-MS for rapid detection of staphylococcal Panton-Valentine leukocidin. , 2009, International journal of antimicrobial agents.

[32]  T. McKenna,et al.  The discriminatory power of MALDI-TOF mass spectrometry to differentiate between isogenic teicoplanin-susceptible and teicoplanin-resistant strains of methicillin-resistant Staphylococcus aureus. , 2006, FEMS microbiology letters.

[33]  Howard Xu,et al.  A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus , 2002, Molecular microbiology.

[34]  Alex van Belkum,et al.  Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection , 2007, Journal of Clinical Microbiology.