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Identification of bacterial and yeast isolates from clinical specimens has traditionally been performed by examination of both macroscopic and microscopic colony morphology, in addition to characterization by classic tube biochemical sets. While these methods remain the gold standard for identification, they can be laborious to perform, requiring both long incubation periods and a significant amount of technologist hands-on time, and the resulting reactions may ultimately be subjective to interpret. These drawbacks were largely alleviated with the advent of automated biochemical testing platforms and, later, with the development of molecular-based DNA sequencing and polymerase chain reaction (PCR) techniques. Though reliable and with an improved turnaround time compared to conventional identification methods, molecular testing continues to be associated with a significantly higher cost, and requires advanced user expertise for assay development, performance, and analysis. These factors altogether limit routine implementation of molecular diagnostic methods to large hospitals and reference laboratories. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is an automated, molecular platform recently adopted by many clinical laboratories worldwide, which effectively circumvents many of these drawbacks and offers a rapid, straightforward, and inexpensive method for identifying bacterial and fungal organisms.
The Basic Principles of Mass Spectrometry
At its core, mass spectrometry is a semiquantitative, analytic method traditionally used to elucidate the composition or molecular structure of an unknown sample. This characterization is performed entirely by the mass spectrometry instrument and is based on the acquisition and analysis of mass and charge values from individual, ionized sample molecules. Mass spectrometry instruments are composed of 3 basic modules: an ionization chamber, a mass analyzer, and an ion detector (Figure 1). Once placed in the ionization chamber, the unknown sample, which may be in a gas, liquid, or solid phase, is pulsed with an energy source. This energy serves dual functions: ionization of individual molecules and desorption of solid or liquid phase samples into the gas phase. The vaporized sample is next directed into and accelerated through the mass analyzer, which separates ions based on their mass-to-charge ratio. Upon emerging from the mass analyzer, ionized particles collide with the ion detector, which measures both the mass and charge of each molecule as derived from their individual force and time to impact. These signals are converted to an electrical output and ultimately depicted to the user in a mass spectrum, which graphs the relative abundance of each detected ion on the y-axis versus its mass-to-charge ratio on the x-axis (Figure 1). The composition or structure of the unknown sample is subsequently derived from careful interpretation and analysis of the ion peaks.
Figure 1. Mass Spectrometry Instrument Design
Many different types of mass spectrometry instruments are available, primarily differentiated by their method of sample ionization and the type of mass analyzer used. Selection among the various instruments is largely dependent on the phase of the input sample, in addition to the physical and chemical properties of the unknown molecules: their molecular weight, thermal stability, side-chain modifications, etc. Each of these properties strongly influence the choice of ionization method and the type of mass analyzer best suited to separate the ionized molecules. Prior to the 1980s, mass spectrometry technology was largely restricted to the analysis of small, thermostable compounds able to withstand the harsh electric ionization techniques available at the time. Larger polypeptides and other biomolecules were found to rapidly degrade under these conditions, which significantly impedes their characterization. With the onset of the proteomics era, however, research into alternative mass spectrometry methods was accelerated and resulted in the development of low energy or “soft ionization” techniques — the principle behind MALDI-TOF MS. The major advantage of this method is its ability to ionize and desorb high molecular weight biomolecules into the gas phase while preserving their intact state. Based on these general considerations, MALDI-TOF MS has emerged as the premier method of choice for the analysis and identification of large polypeptides and even whole microorganisms.
Basic Principles of MALDI-TOF Mass Spectrometry
Sample characterization by MALDI-TOF MS begins by spotting the sample (either solid or liquid) into a defined indentation on a solid target support plate (Figure 2). The composition of the input sample can vary greatly from purified protein to whole-cell microorganisms. Following application onto the target plate, the sample may be further treated, depending on composition, but is ultimately overlaid with a chemical matrix which must dry completely prior to analysis. The matrix is essential for the “soft ionization” process and is chosen for both its efficient desorption into the gas phase and for its ability to effectively absorb the majority of pulsed ionizing energy, thereby protecting sample molecules from fragmentation. A number of matrix compounds have been developed and are each composed of small (<1000 Dalton), acidic molecules dissolved in an organic solvent. An approximately 10 to 1 ratio of matrix to sample is used for MALDI-TOF MS preparation to ensure efficient dilution and protection of sample molecules from fragmentation. Once dried, the prepared target plate is placed into the ionization chamber where each sample is irradiated with 240 brief pulses of energy from an ultraviolet nitrogen laser (337 nm). This process desorbs individual sample and matrix molecules from the target plate into the gas phase, with the majority of energy absorbed by the matrix, which becomes ionized with a single positive charge. This positive charge is subsequently transferred from the matrix to native sample proteins through their random collision in the gas phase.
Figure 2. Matrix-Assisted Laser Desorption Ionization - Time of Flight Process
The cloud of ionized proteins is next funneled through a positively charged, electrostatic field which accelerates the molecules into the time of flight (TOF) mass analyzer. The TOF chamber is an empty, pressurized tube that allows ions to travel down a field-free region toward the ion detector. The velocity at which individual ions fly through the TOF chamber is dependent on their mass-to-charge ratio. Because each sample analyte has an identical, single positive charge, ions are ultimately separated based on their difference in mass — heavier ions will travel through the mass analyzer at a slower velocity, compared to lighter ions. As the ions emerge from the TOF mass analyzer, they collide with the ion detector, which measures their charge and time to impact (Figure 2). Based on standards of known mass, the time to impact for each unknown analyte is converted into a mass-to-charge ratio, which is depicted on a mass spectrum.
Each generated mass spectrum can be thought of as a unique protein “fingerprint” or a protein profile of the unknown sample. Specifically for analysis of microorganisms, MALDI-TOF MS will detect the most abundant proteins over a predefined mass range (typically 2 to 20 kDa). These are mostly intracellular, hydrophilic proteins and are primarily ribosomal components or other noncatalytic, structural complexes. Based on this protein profile, identification of the unknown microorganism is performed by computerized comparison of the acquired spectra to a database of reference spectra composed of previously well-characterized isolates.
MALDI-TOF Mass Spectrometry Workflow in the Clinical Microbiology Laboratory
Following extensive validation studies, our laboratory chose to implement the Bruker Biotyper MALDI-TOF MS system for routine identification of bacterial and yeast isolates from culture.1-5 The Bruker Biotyper system includes the Microflex LT/SH MS instrument and 2 software programs: FlexControl for acquisition of protein spectra and Biotyper real-time classification (RTC) for automated spectral analysis.
MALDI-TOF MS has dramatically altered the traditional workflow and isolate processing in both our bacteriology and mycology laboratories (Figure 3). Currently, we only perform MALDI-TOF MS analysis from isolated bacterial and yeast colonies, therefore clinical specimens continue to be plated to solid media and are observed for growth. As soon as an isolated colony is large enough for visualization, it can be picked for MALDI-TOF MS processing. The amount of colony material required for analysis is minimal, with a single colony being sufficient for complete identification. With a small inoculating loop, the colony is removed and smeared into a well on the target plate, which depending on the plate design, can accommodate at least 24 samples. To release the intracellular proteins necessary for analysis, formic acid is added directly to each sample on the target plate to perforate intact cells, and this crude preparation is allowed to dry. The chemical matrix is subsequently overlaid and dried, at which point the target plate is ready for analysis. This process is referred to as the formic acid-based, direct on-plate preparation method and depending on room humidity, has a total preparation time of 10 to 20 minutes for 24 samples. Notably, potentially hazardous organisms (eg, Brucella and Francisella species) are not processed using this method to avoid aerosolization and minimize the risk for laboratory acquired infections; such agents are prepared using a tube extraction process. Following placement of the target plate into the mass spectrometer, the remaining processing and analysis steps are entirely automated.
Figure 3. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Workflow
Result Interpretation and Reporting
As each spectral profile is acquired, it is analyzed against a database of reference spectra, referred to as the MALDI Biotyper Library (MBL), in a real-time manner via the Biotyper RTC software program. This database is a compilation of spectra generated from previously identified and well-characterized bacterial and fungal isolates. Through proprietary algorithms, the level of similarity between the acquired, unknown spectra and MBL entries is determined and reported to the user as a score, ranging from 0.0, indicating no similarity, to 3.0, indicative of a perfect match. The scores and identities of the top 10 closest matching reference spectra are provided for each tested sample. Within the 0-3 score range, the manufacturer has established cutoff intervals to provide the user with a confident identification to either the genus level (scores of ≥1.700 – 1.999) or to the species level (scores ≥2.000) (Table 1). Any isolate scoring <1.700 is considered as “no identification.” In our laboratory, these manufacturer-determined cutoff values are currently applied towards the identification of gram-negative bacilli and gram-positive cocci. For non-diphtheriae corynebacterium species and yeast, our laboratory has independently validated decreased cutoff values for the genus and species level identification of these organisms: ≥1.500 – 1.699 and ≥1.700, respectively.
Currently, the top-scoring MBL database entry is used for final identification of unknown yeast isolates to either the genus or species level, depending on the acquired score value in relation to our established cutoff criteria.5Identification of gram-negative bacilli, gram-positive cocci and non-diphtheriae Corynebacterium species is likewise based on the top-scoring MBL entry. However, to further improve the identification specificity of these organisms, we also require at least a 10% difference in scores between the top MBL reference spectra and the next different genus or species entry (internal Mayo Clinic validation studies). Isolates scoring below the cutoff for genus-level confidence are reextracted and reanalyzed by MALDI-TOF MS on the same day. If repeat analysis fails to provide at least a genus-level score, isolates are identified by an alternative method, such as 16S ribosomal RNA gene sequence analysis.
Performance of the Bruker Biotyper MALDI-TOF Mass Spectrometry System
Our evaluation of the Biotyper system was performed with single colony isolates from both clinical specimens and archived organisms representing less commonly recovered species. Each isolate was tested in parallel using the Bruker MALDI-TOF MS system and traditional identification techniques, which included identification of gram-negative bacilli by the BD Phoenix automated microbiology system (Becton Dickinson, Franklin Lakes, NJ) and biochemical characterization of other bacteria and yeast using conventional tube-biochemical sets or API strips (BioMérieux, Marcy l’Etoile, France). For our initial MALDI-TOF MS analyses, gram-negative bacilli were prepared by the direct, on-plate method without the use of formic acid, while all other evaluated groups of organisms (ie, cocci, non-diphtheriae Corynebacterium species, and yeast) were prepared using the manufacturer-recommended tube extraction protocol.2-4 This extraction process involves submerging the isolate in ethanol for inactivation, followed by decanting the ethanol and incubating the sample in a solution of formic acid and acetonitrile. This method is thought to more efficiently release intracellular proteins from both gram-positive bacteria and yeast, which typically have a more tenacious external cell wall. The mixture is next pelleted and an aliquot of the supernatant containing the released intracellular proteins is spotted onto the target plate, allowed to dry and overlaid with matrix. Notably, the processing time for this method can range from 45 minutes to 1 hour for 24 samples, which is significantly longer than the direct, on-plate preparation method for the same number of isolates. Regardless of the extraction method used, discrepant results between MALDI-TOF MS and our standard identification methods, were resolved by sequencing the 16S rRNA gene, gyrB or the rpoB for bacteria and the D2 region of the 28S rRNA gene for yeast.
Using the aforementioned genus and species level cutoff criteria, we evaluated 900 bacterial and yeast isolates, with an overall correct identification rate of 94.2% to the genus and 79.2% to the species levels (Table 2). While genus level identification was above 90% for most of the evaluated organism groups, the lower species level identification percentage was primarily driven by the gram-positive cocci (69.5%). This decreased rate of species level identification may be explained by the absence of representative reference spectra in the MBL for certain uncommon isolates. Alternatively, despite the presence of 1 or 2 MBL reference spectra for a particular species, these may not be sufficient to capture the protein variability that may exist within different isolates of the same species. These limitations can, however, be overcome by continued manufacturer improvement of the database, alongside user-based supplementation of the MBL with reference spectra from laboratory characterized isolates.
In an effort to further streamline isolate identification and decrease turnaround time for MALDI-TOF preanalytical processing, we have validated and implemented the formic acid-based, direct on-plate preparation method for all of the aforementioned groups of organisms. Through our published data and internal validation studies, we have shown that this process leads to equivalent identification percentages for gram-negative bacilli, gram-positive cocci, Corynebacterium species, and yeast, compared to processing of these isolates using either the tube or the on plate without formic acid extraction methods (Table 3).2 Additionally, the on-plate preparation minimizes the risk for sample misidentification during processing and is a significantly more environmentally friendly extraction method, reducing the use of additional tubes and chemical agents (eg, ethanol, acetonitrile, formic acid).
MALDI-TOF Mass Spectrometry Misidentifications
Our validation studies indicate that certain closely related bacterial species cannot be differentiated by MALDI-TOF MS, regardless of the system manufacturer. Most notable is the inability of this technology to reliably distinguish Escherichia coli from Shigella species or Streptococcus pneumoniae from Streptococcus mitis group species.2,4 These pairs of organisms are closely related in genetic and protein composition, leading to an inability to collect unique reference spectra for either species (at least as currently configured). To avoid potential misidentification of these organisms, we routinely perform quick, confirmatory biochemical assays (eg, quick indole for Echerichia coli and bile solubility for Streptococcus pneumoniae) prior to finalizing isolate identifications.
Despite the ability of MALDI-TOF MS to identify other closely related organisms to the species level, many names of the identified species would be unfamiliar to providers, since the individual organisms have conventionally been considered to belong to and are reported as a specific complex or group (eg, Burkholderia cepacia complex, Klebsiella pneumoniae complex, Pseudomonas fluorescens group, etc). To minimize confusion, we have decided to maintain our established nomenclature rules and report isolate identification to the complex level, as appropriate (eg, Enterobacter cloacae or Enterobacter ludwigii are reported as Enterobacter cloacae complex). Finally, we have not encountered similar clinically significant misidentifications among yeast isolates, which are all reported without additional confirmatory testing.
Advantages and Limitations of Microbial Identification by MALDI-TOF Mass Spectrometry Analysis
Perhaps the greatest advantage of MALDI-TOF MS technology over traditional diagnostic methods is the ability of this system to identify a large number of unknown isolates in a significantly reduced time span, using fewer laboratory resources (eg, media, biochemicals) and at a lower cost to the patient (Table 4).3,5 Additionally, the manufacturer-supplied spectral library is currently an open database and can be supplemented with reference spectra from laboratory-characterized clinical isolates, allowing the user to expand the database alongside routine library updates provided by the manufacturer. Among the main limitations of this system are the need for isolated colonies prior to analysis and the current inability of our laboratory to perform direct-from-specimen testing. Finally, this technology does not provide antimicrobial susceptibility information alongside organism identification. While research is ongoing in this area, to our knowledge the developed methods have not yet been applied to a clinical laboratory setting. Other advantages and limitations of MALDI-TOF MS technology are outlined in Table 4.
The field of diagnostic microbiology is rapidly evolving into a more streamlined and automated process for organism identification. The most recent driving force of this transition has been the introduction of MALDI-TOF MS into the clinical microbiology laboratory. Identification of unknown isolates by this technology is based on the acquisition of unique protein profiles from isolated colonies and comparison of this data to a library of reference spectra derived from well-characterized isolates. Our laboratory has shown that this is a rapid and inexpensive method able to accurately identify isolates within minutes, compared to the more time-consuming methods associated with other automated microbial identification systems and the potentially subjective nature of classic biochemical analyses. The substantially decreased turnaround time for isolate identification may ultimately benefit patient care by enabling health care providers to make clinical decisions in a much more timely fashion.
Authored by Elitza S. Theel, PhD