Mass spectrometry provides fast and reliable microbe identification

Mass spectrometry (MS), which measures the masses of molecules in a sample, is a useful technique for identifying microorganisms because of its specificity, sensitivity, speed, and robustness. In the past three years, MS has transitioned from an experimental tool to a successful commercial platform with the installation of 1000 dedicated instruments. MS has been used for food control, biodefense, and clinical chemistry applications. Now, diagnosing infectious diseases and testing microorganism properties, such as virulence and resistance to antibiotics, are becoming a reality with this technology. MS has the potential to provide an improved alternative to traditional laboratory methods for identifying microorganisms.

Basic electrospray ionization setup
Figure 1. The basic setup of electrospray ionization, a technique used in mass spectrometry to produce ions. A sample is sprayed through a charged capillary with sheath gas that assists in the formation of charged droplets. Upon release from the nozzle, the solvent within the droplets gradually evaporates with the assistance of the drying gas until the droplets have divided into single, charged molecules. (Source: V. Havlicek and K. Schug)

The rapid identification of microorganisms in clinical microbiology laboratories can help guide patient treatment and improve clinical outcomes. Waiting several days for a definitive identification of a pathogen provides an obstacle to the clinician seeking to target therapy with the most effective treatment. An array of biochemical and other tests have been developed to advance the field, which began more than a century ago with the work of Hans Christian Gram. In 1884, Gram published his staining protocol to visualize bacteria in stained lung tissue sections while working in the morgue of a city hospital in Berlin.

Today, mass spectrometry (MS) represents the most significant emerging player in clinical microbiology for microorganism identification.[1] MS is the science of displaying the masses of the molecules comprising a sample of material. The technique has evolved over the years to provide a rapid and detailed analysis of chemicals, proteins, lipids, and DNA. Although the clinical community still declares it has discovered a ‘new technology,’ the first study using mass spectrometry to investigate whole microorganisms was published in 1970,[2] and the first commercial protein mass-fingerprinting package was introduced in 2000. Using this fingerprinting approach, microorganisms are identified by comparing MS measurements of specific, mostly ribosomal, protein molecules with a database of mass spectra associated with various microbial strains.

In a typical MS procedure, a sample is ionized (see Figure 1), and the ions are separated according to their mass-to-charge ratio and detected. Next, the signal is processed into the spectra of the masses of the sample’s particles. An advantage of mass spectrometers over other analytical instruments is that they provide a high degree of accuracy and sensitivity when determining the molecular weight of biological compounds.

MALDI-TOF mass spectrometer
Figure 2. Schematic of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (Source: V. Havlicek and K. Schug)

A technique called matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry now provides a standardized, routine, fast, and reliable method of bacterial typing with reproducible and automated sample preparation protocols. In a typical procedure, a scientist transfers a sample onto a stainless steel plate, covers the sample with a matrix (i.e., a layer of a small light-absorbing organic molecule), and inserts the sample into the ion source of a mass spectrometer (see Figure 2). The role of the matrix is twofold: it absorbs the energy of the irradiating ultraviolet laser beam via momentum transfer and it serves as a proton donor to convert sample molecules into ions that can be separated and detected by the mass spectrometer. Current MALDI-TOF protocols allow for microorganism identification in less than 30 minutes after a blood culture provides a positive result. The mass spectral approach also significantly reduces the cost per test.

Two commercially available in vitro diagnostic medical devices supported by large microbial databases have received the CE mark, which signifies European regulatory approval of the product. The first is the Vitek MS, which was developed by bioMérieux and received the CE mark in 2012. The Vitek MS database is based on the Spectral ARchive And Microbial Identifications System (SARAMIS), which contained more than 35,000 single fingerprint spectra of more than 2000 bacteria, yeast, and fungal species and 500 genera in September 2010. The second device is the MALDI Biotyper MSP, which is based on the Bruker Daltonics microflex bench-top MALDI-TOF mass spectrometer. The Biotyper 3.1 database contained more than 4600 unique reference strains, 350 genera, and 2140 species as of September 2012. Two recent studies found that the Vitek MS and Biotyper systems performed similarly on a specified set of clinical isolates,[3][4] while another study indicated better performance by the Biotyper system.[5]

Recently, MS expanded from microbial subtyping to antibiotic susceptibility testing (AST)[6] and detection of virulence factors[7] (molecules secreted by pathogenic microbes that enable them to proliferate and cause disease). AST is the endpoint of the major work a microbiology laboratory performs on a daily basis. After bacteria are cultured and isolated from patient specimens, these organisms need to be identified and tested to determine which drugs will inhibit their growth. The Vitek MS integrates mass-spectrometry-based bacterial identification with Vitek 2 AST. The Bruker Daltonics system has achieved automated mass-spectral AST for several penicillin-, cephalosporin-, and carbapenem-related antibiotics. In a recent study, researchers used MS to analyze the susceptibility of yeast isolates to major antifungal substances.[8] The detection, full characterization, and inhibition of microbial virulence factors may lead to new AST approaches and the identification of new antimicrobial drugs.

Mass spectrometry image of murine brain tissue
Figure 3. Mass spectrometry image of a murine brain tissue section. The intensity of green corresponds to where certain lipids are concentrated in the sample. (Source: V. Havlicek and K. Schug)

When species cannot be identified, distinguished, or even characterized through their protein profiles, microbial metabolites (the small molecules involved in an organism’s metabolism) can be used instead. Metabolites describe the most dynamic level of biological regulation and, as such, provide the most direct reflection of an organism’s physiological status. Although the microbial metabolome is very sensitive to cultivation conditions of the microorganism of interest, it can provide very specific markers. Some metabolomics markers are of non-ribosomal origin and represent virulence factors.[9]

Another increasingly important technique is mass spectrometry imaging (MSI), which can determine the distribution of molecules directly on the surface of biological tissues without requiring labeling such as dyes (see Figure 3). MSI can be used to visualize an entire microbial colony and evaluate the response of the microorganisms to antibiotics, making it a potential tool for identifying molecular markers, analyzing pathology, and investigating disease mechanisms.[10] The technique can provide direct access to biomarkers and provide information on the interactions with the host immune system, which can affect disease progression and treatment options. A recent study showed successful uptake of 68Ga-enriched siderophores by the fungus Aspergillus fumigatus disseminated in lungs, enabling high-definition positron emission tomography imaging.[11] High-sensitivity tomographic images provide morphologic information on the location of aspergilloma within an organ, which can be supported by mass-spectral detection of fungal metabolites found in bodily fluids.

The progressive role of MS in microbiology and associated clinical areas has brought new users into the field.[1] In turn, the field has continuously expanded due to the technique’s robustness, sensitivity, selectivity, and speed, and thanks to vendor support and investments. In the future, we expect the broad expansion of MS into biological and biochemical testing areas currently occupied by conventional approaches. We also believe the market for typing microorganisms by their proteins will grow and spread, especially in the United States. Routine food quality control and detection of infectious diseases, including viral infections, will soon become a reality. MS device companies recognize the importance of the microbiological market and are developing new approaches that are not based on protein mass fingerprinting. We expect new commercial products based on MS to reach the market within five years.

References

  1. V. Havlicek, K. Lemr, and K. A. Schug, Current trends in microbial diagnostics based on mass spectrometry, Anal. Chem. 85, pp. 790–797, 2013.
  2. P. G. Simmonds, Whole microorganisms studied by pyrolysis-gas chromatography-mass spectrometry. Significance for extraterrestrial life detection experiments, Appl. Microbiol. 20, pp. 567–572, 1970.
  3. D. Martiny, L. Busson, I. Wybo, R. A. El Haj, A. Dediste, and O. Vanderberg, Comparison of the Microflex LT and Vitek MS systems for routine identification of bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry, J. Clin. Microbiol. 50, pp. 1313–1325, 2012.
  4. D. C. Marko, R. T. Saffert, S. A. Cunningham, J. Hyman, J. Walsh, S. Arbefeville, W. Howard, et al., Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients, J. Clin. Microbiol. 50, pp. 2034–2039, 2012.
  5. E. Carbonnelle, P. Grohs, H. Jacquier, N. Day, S. Tenza, A. Dewailly, O. Vissouarn, et al., Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification, J. Microbiol. Methods 89, pp. 133–136, 2012.
  6. A. Grundt, P. Findeisen, T. Miethke, E. Jager, P. Ahmad-Nejad, and M. Neumaier, Rapid detection of ampicillin resistance in Escherichia coli by quantitative mass spectrometry, J. Clin. Microbiol. 50, pp. 1727–1729, 2012.
  7. X. Didelot, R. Bowden, D. J. Wilson, T. E. A. Peto, and D. W. Crook, Transforming clinical microbiology with bacterial genome sequencing, Nat. Rev. Genet. 13, pp. 601–612, 2012.
  8. A. F. Schmalreck, B. Willinger, G. Haase, G. Blum, C. Lass-Florl, W. Fegeler, K. Becker, and the Antifungal Susceptibility Testing (AFST) Study Group, Species and susceptibility distribution of 1062 clinical yeast isolates to azoles, echinocandins, flucytosine and amphotericin B from a multi-centre study, Mycoses 55, pp. e124–e137, 2012.
  9. V. Havlicek and K. Lemr, Fungal metabolites for microorganism classification by mass spectrometry, ACS Symposium Series 1065, pp. 51–60, 2011.
  10. J. Pol, M. Strohalm, V. Havlicek, and M. Volny, Molecular mass spectrometry imaging in biomedical and life science research, Histochem. Cell Biol. 134, pp. 423–443, 2010.
  11. M. Petrik, G. M. Franssen, H. Haas, P. Laverman, C. Hortnagl, M. Schrettl, A. Helbok, C. Lass-Florl, and C. Decristoforo, Preclinical evaluation of two Ga-68-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging, Eur. J. Nucl. Med. Mol. Imaging 39, pp. 1175–1183, 2012.

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