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Background    Pseudomonas aeruginosa  is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments.    Methods  In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of  P. aeruginosa  strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed.    Results  Of all  P. aeruginosa  PA14 mutant profiles 99.7% were correctly identified as  P. aeruginosa  with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual  P. aeruginosa  Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions.    Conclusions  The PA14 mutant profile collection presented here constitutes the largest coherent  P. aeruginosa  MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different  P. aeruginosa  genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.

Comprehensive MALDI-TOF Biotyping of the Non-Redundant Harvard Pseudomonas aeruginosa PA14 Transposon Insertion Mutant Library