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REVIEW 10.1111/j.1469-0691.2010.03364.x

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: usefulness for taxonomy and epidemiology

P. R. Murray Microbiology Service, Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD, USA

Abstract

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for the species and subspecies classification of a broad spectrum of bacteria, including Gram-positive bacteria such as Staphylococcus, and Listeria, and Gram-negative bacteria such as Neisseria, Salmonella, Aeromonas, Campylobacter and Helicobacter. MALDI-TOF MS has also been used for the rapid identification and typing of potential bioterrorism agents, including Coxiella burnetii, Francisella tularensis and Bacillus anthracis.

Keywords: Epidemiology, MALDI-TOF, mass spectrometry, review, taxonomy Article published online: 3 September 2010 Clin Microbiol Infect 2010; 16: 1626–1630

Corresponding author: P. R. Murray, 10 Center Drive MSC 1508, Building 10, Room 2C-385, National Institutes of Health, Bethesda, MD 20892, USA E-mail: [email protected]

Taxonomy is the systematic classification of microbes on ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), the basis of their quantifiable properties. In the example of used to classify organisms must be compared with genomic bacterial classification, macroscopic and microscopic proper- classification. As discussed in the accompanying reviews, ties were used initially to subdivide organisms into related microbial identification with MALDI-TOF MS compares groups, followed by metabolic and antigenic properties, and favourably with biochemical and genomic identification meth- then, more recently, genomic relationships. Each approach ods. Theoretically, because MALDI-TOF MS detects a large still has value for the clinical microbiologist. For example, spectrum of proteins, the technique should be able to dis- the is a powerful tool for the initial classification criminate between closely related species and to classify of a bacterial isolate, biochemical tests can be used to iden- organisms at the subspecies level. A review of studies that tify most commonly isolated organisms, serological testing is have evaluated MALDI-TOF MS for taxonomic and epidemi- important for subtyping organisms such as b-haemolytic ological classification of bacteria is presented here. It should Streptococcus and Salmonella, and sequencing of one or more be noted that the current commercially available MALDI- bacterial genes (or the entire genome) offers definitive clas- TOF MS systems are accurate and give reproducible results sification of most bacteria, including organisms not previ- for identification of microbes at the species and subspecies ously recognized. Genomic techniques such as pulsed-field levels; however, a limited number of studies have been gel electrophoresis (PFGE), amplified fragment length poly- reported that have used MALDI-TOF MS for microbial morphism analysis and multilocus sequence typing are also typing and, in many cases, they have used markedly different used to subtype bacteria for epidemiological studies. analytical procedures from those used for microbial identifi- Although any scheme used to classify organisms is based on cation. Thus, the studies summarized in this review should rules that define arbitrary divisions and a hierarchy of rela- be viewed as defining the potential of MS for bacterial and tionships (e.g. genus, species and subtype), genome analysis fungal strain typing, and additional work needs to be per- is currently accepted as the prevailing standard. Thus, any formed to define the robustness of MALDI-TOF MS for this new technique, such as matrix-assisted laser desorption application.

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A series of studies published by Edwards-Jones et al. [1–3] individuals. Foodborne outbreaks are associated with soft demonstrated that MALDI-TOF MS could be used to identify cheeses and other dairy products, processed meats, and Staphylococcus aureus, discriminate between methicillin-resistant lettuce and uncooked vegetables. Differentiation of L. mono- S. aureus (MRSA) and methicillin-susceptible S. aureus strains, cytogenes from other Listeria species and subtyping of and subtype MRSA strains. MRSA and methicillin-susceptible L. monocytogenes are important tools for epidemiological S. aureus strains can be accurately differentiated by MALDI- investigations of food-related illnesses. Species identification TOF MS significantly faster than with conventional antimicro- is performed traditionally by phenotypic testing, and PFGE bial susceptibility test methods and less expensively than with is used to subtype isolates into three clonal lineages: lineage I genomic techniques; thus, it will be important for other (serotypes 1/2b, 3b, 4b, 4ab, 4d, 4e and 7), lineage II investigators to confirm these early studies, using the (serotypes 1/2a, 1/2c, 3a and 3c) and lineage III (sero- currently available MALDI-TOF MS instruments and analytical types 4a and 4c). Barbuddhe et al. [12] demonstrated that software. PFGE is a widely used, sensitive technique for sub- MALDI-TOF MS accurately identified 146 strains of Listeria typing MRSA strains [4], and has been used to define distinct (representing six species) at the species level, and correctly clusters of MRSA genotypes that have significant value for subtyped all strains of L. monocytogenes, with the results epidemiological studies [5]; however, PFGE is expensive and corresponding to PFGE classifications. technically demanding, and requires 2–3 days of processing. is an uncommonly recognized bacterium, and MALDI-TOF MS, when performed under carefully standard- is frequently misidentified by phenotypic tests as Streptococ- ized conditions as described by Jackson et al. [3], can subtype cus. The discriminatory power of MALDI-TOF MS was dem- strains in a fraction of the time and with much less expense, onstrated by Tanigawa et al. [13] when they accurately and allow real-time management of MRSA infections. It identified six species and three subspecies of Lactococcus.In should be noted that a systematic comparison of PFGE contrast, although species classification was obtained with and MALDI-TOF MS subtyping of MRSA stains has not 16S rRNA gene sequencing, subspecies identification been reported, so it is unknown whether the classification required multilocus sequence analysis of three housekeeping of a population of strains by each method would yield genes. comparable results. The genus Neisseria consists of two well-known human The genus Streptococcus is broadly subdivided into two pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, groups: the b-haemolytic streptococci, which are most com- and 21 other species of non-pathogenic or opportunistic monly classified by Lancefield grouping and phenotypic tests; pathogens. Although it is relatively easy to differentiate the and the , a collection of more than 30 pathogenic species, misidentification of the other species by a-haemolytic and non-haemolytic species, organized into five phenotypic or immunological tests as N. gonorrhoeae or subgroups that are not readily identified by either phenotypic N. meningitidis can occur [14]. In contrast, Illina et al. [15] tests or sequencing of the 16S rRNA gene [6]. Kumar et al. demonstrated that MALDI-TOF MS readily separated 57 [7] and Lartigue et al. [8] demonstrated that MALDI-TOF MS strains of Neisseria into three groups: N. gonorrhoeae, N. men- could easily differentiate common Lancefield groups of strep- ingitidis, and other species. Additionally, a dendogram of 15 tococci (groups A, B, C and G) and could be used to sub- strains of non-pathogenic species indicated that these would type group A and B streptococci. A more difficult challenge also probably have been separated into individual species is to identify members of the viridans streptococci correctly; profiles if additional strains had been evaluated. however, Friedrichs et al. [9] correctly classified 99 clinical The genus Salmonella was a taxonomic quagmire until isolates into ten individual species. In this study, Streptococcus 2005, when the more than 2500 serotypes (also called sero- pneumoniae was excluded from the analysis, in part because vars and previously referred to as species) were reorganized this species is readily identified by the bile solubility test, and into two species: Salmonella enterica and Salmonella bongori in part because it cannot be reliably differentiated from other [16]. Salmonella enterica was subdivided into six subspecies, members of the group by MALDI-TOF MS with most human pathogens being placed in Salmonella enterica [10]. Despite this limitation, subtyping of Streptococcus pneu- ssp. enterica (ssp. 1). Distinguishing among the Salmonella moniae strains by MALDI-TOF MS can be reliably performed, serovars is important for clinical and epidemiological pur- even for immunologically non-typeable or non-encapsulated poses; however, the fact that most are classified in the same strains [11]. species emphasizes the close genomic relationships among Listeria monocytogenes is an important foodborne pathogen these organisms. Serological testing, and not biochemical that is responsible for opportunistic infections in neonates, tests, have historically been used to identify these organisms. pregnant women, elderly persons and immunocompromised In 2003, Leuschner et al. [17] demonstrated that MALDI-TOF

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MS could be used to distinguish five serovars of Salmonella Campylobacter jejuni and Campylobacter coli, the Campylobac- enterica (22 isolates), and in 2008 Dieckmann et al. [18] ter species most commonly associated with human diarrhoeal expanded these findings, demonstrating that 126 isolates of disease, are typically differentiated by the hippurate hydroly- Salmonella could be classified correctly at the species and sis test. Although hippurase-positive isolates can be identified subspecies levels. Although relatively few biomarker peaks as C. jejuni, this phenotypic test cannot differentiate between (e.g. five to ten peaks) are needed to identify an isolate at C. jejuni ssp. jejuni and ssp. doylei or between C. coli and hip- the species level, Dieckmann et al. [18] reported that a lar- purase-negative strains of C. jejuni. Kolinska et al. [20], using ger number of reproducible peaks were required for subspe- species-specific gene markers, reclassified into two popula- cies identification. This poses a challenge for clinical tions 42 strains of C. jejuni previously identified by pheno- laboratories that attempt to use MALDI-TOF MS for strain typic tests: 26 strains of C. jejuni and 16 strains of C. coli. typing. Whereas rigorous control of the sample preparation MALDI-TOF MS analysis of these strains produced results in is generally unnecessary for organism identification, optimiza- complete agreement with the genomic tests. Fagerquist et al. tion of testing parameters is most likely critical for strain [21] also demonstrated that changes in biomarker masses, typing. For example, technical parameters such as the type such as that caused by an amino acid substitution, could be and concentration of the matrix, the sample preparation pro- used to differentiate C. jejuni ssp. jejuni and subsp. doylei,as cedure, the matrix–solvent mixture, the concentration of well as to predict phylogenetic relationships among isolates. acid added to the matrix and the influenced Helicobacter pylori, originally classified in the genus Campylo- the mass spectral profile of biomarkers. Dieckmann et al. bacter, is an important cause of gastritis, peptic ulcers, gastric [18] evaluated three matrix mixtures, and found that sinapi- adenocarcinoma, and gastric mucosa-associated lymphoid tis- nic acid produced the most informative spectra, particularly sue B-cell lymphomas. Phenotypic testing demonstrates a in the higher molecular mass range. Through their optimiza- high degree of natural variability among strains; however, Ilina tion procedures, the number of peaks (>300), particularly et al. [22] obtained highly reproducible mass spectra for high molecular mass peaks, was significantly increased. Addi- H. pylori isolates, permitting classification of all strains within tionally, although commercial software was used to identify the species H. pylori and differentiating them from 12 species biomarker peaks that could classify Salmonella at the genus, of Campylobacter. species or subspecies level, the need for software specifically Burkholderia cepacia and eight closely related species, com- designed for typing other microbial strains is unknown. monly referred to as B. cepacia complex, are important Aeromonas infections in humans most commonly present opportunistic pathogens responsible for infections in the as diarrhoeal disease in healthy individuals, soft tissue infec- lungs, urinary tract and bloodstream in immunocompromised tion, frequently following exposure to contaminated fresh patients, particularly patients with cystic fibrosis or chronic water, or opportunistic systemic disease in immunocompro- granulomatous disease. Accurate identification of the species mised patients, particularly those with hepatobiliary disease within this complex and differentiation of these species from or an underlying malignancy. Differentiation of Aeromonas other members of Burkholderia and related genera such as species from Vibrio species and classification of species and Ralstonia, Cupriavidus and Pandoraea cannot be achieved with subspecies within the genus Aeromonas cannot be performed phenotypic tests. Whereas some species can be identified by reliably with biochemical testing, particularly when commer- sequence differences in the 16S rRNA gene, sequence analy- cial identification systems are used; likewise, classification of sis of other housekeeping genes, such as recA, must also be Aeromonas strains at the species and subspecies level cannot performed. Vanlaere et al. [23] analysed 75 strains of B. cepa- be performed by sequencing the 16S rRNA gene. Sequencing cia complex isolates and related genera by MALDI-TOF MS, of other housekeeping genes, such as gyrB and rpoD, or mul- and were able to classify each species correctly, with the tilocus sequence typing must be performed for accurate spe- exception of Burkholderia anthina (genovar VII) and Burkholde- cies identification. Thus, most laboratories are restricted to ria pyrrocinia (genomovar IX), two species that are not com- identification of isolates as members of the Aeromonas hydro- monly isolated in clinical specimens and that formed a phila complex, Aeromonas caviae complex or Aeromonas veronii homogeneous cluster. Mellmann et al. [24] also reported that complex. In contrast to the limitations of phenotypic and MALDI-TOF MS could accurately identify Burkholderia species genomic tests, Donohue et al. [19] reported that MALDI- and related non-fermentative Gram-negative rods. TOF MS accurately classified 14 species and four subspecies Legionella pneumophila and related species are important of Aeromonas, including A. hydrophila, A. caviae, Aeromonas jan- environmental pathogens responsible for well-documented daei and A. veronii ssp. sorbia and veronii, the clinically most outbreaks of pneumonia, particularly in patients with com- important species of Aeromonas. promised pulmonary function. Pennanec et al. [25] reported

ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1626–1630 No claim to original US government works CMI P. R. Murray MALDI-TOF MS: taxonomy and epidemiology 1629

that MALDI-TOF MS could reliably differentiate Legionella Gram-positive and Gram-negative cocci and rods, at the spe- species and related organisms, and Fujinami et al. [26] cies and subspecies level. Although some investigators care- extended these studies by demonstrating that MS and PFGE fully standardized the growth conditions and assay were equally reliable in identifying epidemiologically related procedures, the need for this for subtyping most organisms strains. The advantage of MALDI-TOF MS as compared with of epidemiological significance is unknown, and must be sys- PFGE was that the results of the analysis were available tematically studied. Additionally, comparison of MALDI- within a few hours rather than several days. This important TOF MS with genomic typing methods such as PFGE is study is one of the few that has directly compared MALDI- needed. Nevertheless, the current body of published studies TOF MS with PFGE. supports the thesis that MALDI-TOF MS is an important Bacteroides fragilis and related anaerobic species are com- technology for the identification and subtyping of clinical monly classified and reported as Bacteroides fragilis group, isolates. because separation by phenotypic tests is difficult. Although 16S rRNA gene sequencing is accurate, the time needed for Transparency Declaration identification is longer and the material cost is greater [27]. In contrast, Nagy et al. [28] demonstrated that MALDI- TOF MS identification of Bacteroides species is accurate and Financial support is provided by the Division of Intramural rapid, correctly identifying 98.6% of 277 clinical isolates, with Research, Clinical Center, National Institutes of Health. No isolates being identified in less than 3 h and with minimal potential conflicts of interest. expenditure on consumable reagents. 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