Highly Efficient Classification and Identification of Human Pathogenic Bacteria by MALDI-TOF MS

Abstract

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10 4 cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future. Molecular & Cellular Proteomics 7: 448 -456, 2008.Currently the most popular methods for bacterial identification are based on microbiologic procedures, antibody recognition, and PCR amplification. Traditionally microbiologic methods are culture-based assays that examine the presence of bacterial species. These methods provide high sensitivity and specificity, but their efficiency is limited by the complexity of the procedures, including culture, selection, isolation, and morphologic and biochemical characterization, which usually take 48 h or longer. Serologic methods are presumptive and confined to the availability of antibodies and to bacteria that are included ahead in the assays. Molecular biology techniques, particularly PCR, have been regarded as non-culturebased methods with high efficiency and specificity (1). However, they are completely dependent on the known genetic sequences of the target bacteria.MS with its capability of de novo protein/peptide sequencing (such as electrospray ionization or MALDI-TOF MS for tandem MS/MS) or its high efficiency for proteome profiling (particularly MALDI-TOF MS) has been suggested as an alternative for microbial identification (2-7...

Rapid Identification of Bacteria in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

“…Analysis of bacterial or fungal colonies by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry produces reproducible, species-specific spectral patterns that can be used to identify microorganisms at the species level. A broad spectrum of organisms has been identified, including nocardia, mycobacteria, yeasts,5,[7][8][9][10]12,13). Compared to standard biochemical identification methods, this technique is rapid and reproducible with minimal consumable costs and has an accuracy comparable to that of genome sequencing (14).…”

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Clinically Important Yeast Species

“…Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is emerging as a rapid and accurate tool for identifying pathogens, including Gram-positive and Gram-negative bacteria, mycobacteria, molds, and yeast species (3, 6, 11-13, 16, 18, 22, 23, 27). The technique can be performed rapidly, with minimal consumable expenses, and produces reproducible, species-specific spectral patterns that are not dependent upon the age of culture, growth conditions, or medium selection (7,13,20,26).…”

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Direct Bacterial Identification from Positive Blood Culture Pellets

“…Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) allows identification of both gram-positive (2, 7) and gram-negative bacteria (1,4) to the species level in a few minutes by measuring the molecular masses of proteins and other bacterial components obtained from whole bacterial extracts. A relatively low crude bacterial load of about 5 ϫ 10 3 CFU is necessary for reliable MALDI-TOF analysis (3), suggesting that bacterial identification might be done directly with blood culture pellets. We applied a simple procedure for lysing erythrocytes from positive blood cultures and prepared a bacterial pellet for MALDI-TOF MS analysis.…”