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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool in the clinical microbiology laboratory, enabling accurate identification of bacteria, fungi, and mycobacteria grown in culture. First adopted in some European microbiology laboratories, its ease of use, accuracy, rapid turnaround time, and low cost have led to it becoming standard of care in clinical microbiology laboratories around the world.
This chapter briefly discusses the history of MALDI-TOF MS leading to its commercialization and adoption in clinical microbiology laboratories. Identification of aerobic and anaerobic organisms, as well as mycobacteria and fungi, with a focus on the US Food and Drug administration (FDA)-approved/cleared platforms, is discussed. Additional applications, such as direct identification from blood cultures are reviewed as is implementation of MALDI-TOF MS into routine laboratory workflow.
Bacteria and other microorganisms have an abundance of unique proteins allowing for their taxonomic identification based on the mass “fingerprint” of their proteins. MALDI is considered a soft ionization technique with the matrix aiding in ionization of proteins without causing them to fragment when the laser is applied (see Chapter 20 ). Sample preparation is required before performing MALDI-TOF MS. Preparation entails applying a colony of bacteria onto a spot on the MALDI-TOF MS plate (with or without formic acid) or the more traditional method of using formic acid to disrupt the cells, followed by extraction of proteins with acetonitrile and then spotting onto a MALDI-TOF MS plate. The first method is used for routine identification of bacteria and yeast, with the latter typically reserved for difficult-to-lyse organisms. Direct colony processing is much simpler, requires less hands-on time, and is less expensive to perform than off-plate extraction. Other methods of preparation are used for processing liquid cultures because of the need of a purification step. Purification may be accomplished by filtering out impurities, or pelleting microbial cells via centrifugation, combined with repeated washings. An example of a common application of preparation from liquid cultures is the processing of positive blood cultures, discussed later in this chapter.
For direct colony processing, a colony is first “picked” from a culture plate and then spotted onto a position on a MALDI-TOF MS plate ( Fig. 22.1 ). Plates can be reusable (made of metals) or disposable and come in multiple configurations. The two common commercial systems worldwide are the MALDI Biotyper (Bruker Daltonics, Billerica, Massachusetts) and VITEK MS (bioMérieux, SA, Marcy-l’Etoile, France), although, as discussed later in the chapter, other systems are available. Formic acid may be added to improve the quality of the generated mass spectra, a method referred to as extended direct transfer or on plate formic acid testing (compared with direct testing where formic acid is not used). The spot (either the microbial mass alone or following drying of formic acid for the extended direct transfer method) is overlaid with matrix (typically α-cyano-4-hydroxycinnamic acid dissolved in an acidified mixture of organic solvents and water). A third approach involves a traditional extraction methodology with inactivation by ethanol followed by a pelleting step. The pellet is then suspended in formic acid followed by addition of acetonitrile. The suspension is then centrifuged and the supernatant spotted on the plate. Once dried, the spot is overlaid with matrix as with the direct or extended direct transfer methods. Once the matrix has dried, the plate is placed into the instrument.
After the plate is introduced to the ionization chamber, a pulsed laser is directed onto the matrix/analyte combination of each spot, causing desorption and ionization of microbial proteins and other molecules in a plume of ionized sample and matrix molecules. The matrix absorbs most of the energy from the laser, leading to an ionized state that transfers charge from matrix to proteins and other molecules through random molecular collisions. Ionized microbial proteins are accelerated in an electromagnetic field and enter a TOF mass analyzer maintained under vacuum in which proteins are separated based on their velocity, which is inversely proportional to their mass-to-charge (m/z) ratio. In the application discussed, the charge is most often +1. Finally, at the end of the flight tube, the ions hit an ion detector, with smaller proteins colliding with the ion detector first, followed by larger ones. The resulting time-resolved spectrum is eventually transformed to a mass spectrum by calibrating the system with peptides of known sizes, relating TOF to m/z . The resulting mass spectrum for a given sample microorganism is then compared with reference spectra of known microorganisms ( Fig. 22.2 ).
The first proposal that mass spectrometry (MS) be used for identification of bacteria was in 1975, although the process proposed used an ionization method that fragmented proteins, thereby preventing effective analysis. The technology to study intact proteins by MS was not invented until the 1980s, when two groups developed similar ideas for detection of intact macromolecules. Koichi Tanaka and coworkers described use of an ultrafine metal powder mixed with glycerin to detect macromolecules without fragmentation, for which Tanaka was awarded the Nobel Prize in chemistry (shared with John Fenn and Kurt Wüthrich). Around the same time that Tanaka published his research, Franz Hillenkamp and Michael Karas published their work on soft desorption/ionization using an organic compound matrix, nicotinic acid, also allowing the analysis of large biomolecules ; the term matrix-assisted laser desorption/ionization (MALDI) was coined from this work. Over the past few decades, computer science, information technology, and aggregation of comprehensive databases of well-characterized reference spectra have led to MALDI-TOF MS becoming a standard method used in clinical microbiology laboratories. The two commercial systems mentioned earlier have been cleared by the US Food and Drug Administration (FDA) for routine identification of bacteria and fungi in clinical microbiology laboratories. Other platforms include the recently developed MicroIDSys (ASTA, Suwon, Korea), and the Chinese platforms Clin-TOF (Yixin, Beijing), Autof ms1000 (Autobiio, Henan) and microTyper MS (Tianru, Jiangsu); these will not be mentioned further. The VITEK MS (bioMérieux) and MALDI Biotyper CA System (Bruker Daltonics) have been approved/cleared by the FDA for identification of bacteria and fungi cultured on solid media. A list of claimed organisms as of December 1, 2019 is shown in Box 22.1 . Each system consists of a mass spectrometer, software, and a database; however, differences exist in each of the three components, as well as the organisms approved/cleared by the FDA for identification. Differences also exist in the size of the instruments; Bruker’s mass spectrometer is a bench-top model, and bioMérieux’s is a floor model instrument similar in size to a vending machine.
Abiotrophia defectiva V
Aerococcus sanguinicola B
Aerococcus urinae B
Aerococcus viridans
Alloiococcus otitis B
Arthrobacter cumminsii B
Brevibacterium casei B
Corynebacterium accolens B
Corynebacterium afermentans group B
Corynerbacterium afermentans
Corynebacterium pilbarense
Corynebacterium amycolatum B
Corynebacterium aurimucosum group B
Corynebacterium aurimucosum
Corynebacterium singulare
Corynebacterium bovis B
Corynebacterium coyleae B
Corynebacterium diphtheriae B
Corynebacterium freneyi B
Corynebacterium glucuronolyticium B
Corynebacterium glutamicum B
Corynebacterium jeikeium V
Corynebacterium kroppenstedtii B
Corynebacterium macginleyi B
Corynebacterium minutissimum B
Corynebacterium mucifaciens/ureicelerivorans group B
Corynebacterium mucifaciens
Corynebacterium ureicelerivorans
Corynebacterium propinquum B
Corynebacterium pseudodiphtheriticum B
Corynebacterium pseudotuberculosis B
Corynebacterium resistens B
Corynebacterium riegelii B
Corynebacterium striatum group B
Corynebacterium tuberculostearicum B
Corynebacterium ulcerans B
Corynebacterium urealyticum B
Corynebacterium xerosis B
Corynebacterium striatum group B
Corynebacterium striatum
Corynebacterium simulans
Curtobacterium flaccumfaciens V
Dermabacter hominis B
Dermacoccus nishinomiyaensis B
Enterococcus avium
Enterococcus casseliflavus
Enterococcus durans
Enterococcus faecalis
Enterococcus faecium
Enterococcus gallinarum
Enterococcus hirae
Enterococcus mundtii B
Enterococcus raffinosus B
Gardnerella vaginalis
Gemella haemolysans
Gemella morbillorum
Gemella sanguinis B
Granulicatella adiacens
Helcococcus kunzii B
Kocuria kristinae B
Kocuria rhizophila V
Kytococcus sedentarius B
Lactobacillus gasseri B
Lactobacillus jensenii B
Lactobacillus rhamnosus B
Lactococcus garvieae
Lactococcus lactis
Leuconostoc citreum B
Leuconostoc mesenteroides
Leuconostoc pseudomesenteroides
Listeria monocytogenes
Macrococcus caseolyticus B
Micrococcus luteus
Micrococcus lylae B
Nocardia abscessus V
Nocardia africana/nova V
Nocardia asteroides V
Nocardia brasiliensis
Nocardia cyriacigeorgica
Nocardia farcinica V
Nocardia farcinica group B
Nocardia farcinica
Nocardia kroppenstedtii
Nocardia nova B
Nocardia otitidiscaviarum
Nocardia paucivorans V
Nocardia pseudobrasiliensis V
Nocardia transvalensis V
Nocardia veterana V
Nocardia wallacei V
Pediococcus acidilactici
Pediococcus pentosaceus B
Rothia aeria B
Rothia dentocariosa B
Rothia mucilaginosa
Staphylococcus aureus
Staphylococcus auricularis
Staphylococcus capitis
Staphylococcus caprae B
Staphylococcus carnosus B
Staphylococcus chromogenes V
Staphylococcus cohnii B
Staphylococcus cohnii ssp. cohnii V
Staphylococcus cohnii ssp. urealyticus V
Staphylococcus delphini
Staphylococcus epidermidis
Staphylococcus equorum B
Staphylococcus felis B
Staphylococcus haemolyticus
Staphylococcus hominis
Staphylococcus hyicus V
Staphylococcus intermedius
Staphylococcus kloosii V
Staphylococcus lentus
Staphylococcus lugdunensis
Staphylococcus pasteuri B
Staphylococcus pettenkoferi B
Staphylococcus pseudintermedius
Staphylococcus saccharolyticus B
Staphylococcus saprophyticus
Staphylococcus schleiferi
Staphylococcus sciuri
Staphylococcus simulans
Staphylococcus vitulinus B
Staphylococcus warneri
Staphylococcus xylosus
Streptococcus agalactiae
Streptococcus alactolyticus V
Streptococcus anginosus
Streptococcus canis
Streptococcus constellatus
Streptococcus cristatus V
Streptococcus dysgalactiae B
Streptococcus dysgalactiae ssp. dysgalactiae V
Streptococcus dysgalactiae ssp. equisimilis V
Streptococcus equi B
Streptococcus equi ssp. equi V
Streptococcus equi ssp. zooepidemicus V
Streptococcus equinus
Streptococcus gallolyticus B
Streptococcus gallolyticus ssp. gallolyticus V
Streptococcus gallolyticus ssp. pasteurianus V
Streptococcus gordonii
Streptococcus infantarius ssp. coli (S. lutetiensis) V
Streptococcus infantarius ssp. infantarius V
Streptococcus intermedius
Streptococcus lutetiensis B
Streptococcus mitis/Streptococcus oralis V
Streptococcus mitis oralis group B
Streptococcus mitis
Streptococcus oralis
Streptococcus australis
Streptococcus cristatus
Streptococcus dentisani
Streptococcus infantis
Streptococcus oligofermentans
Streptococcus pseudopneumoniae
Streptococcus rubneri
Streptococcus sanguinis
Streptococcus tigu rinus
Streptococcus mutans
Streptococcus parasanguinis
Streptococcus pneumoniae
Streptococcus pseudoporcinus V
Streptococcus pyogenes
Streptococcus salivarius/vestibularis group
Streptococcus salivarius
Streptococcus vestribularis
Streptococcus salivarius ssp. salivarius V
Streptococcus sanguinis
Streptococcus sobrinus
Streptococcus suis V
Streptococcus thermophilus B
Streptococcus uberis V
Streptococcus vestibularis V
Trueperella bernardiae B
Turicella otitidis B
Vagococcus fluvialis B
Cedecea davisae V
Cedecea lapagei V
Cedecea neteri V
Citrobacter amalonaticus V
Citrobacter amalonaticus complex B
Citrobacter amalonaticus
Citrobacter farmeri
Citrobacter braakii V
Citrobacter farmeri V
Citrobacter freundii V
Citrobacter freundii complex B
Citrobacter braakii
Citrobacter freundii
Citrobacter gillenii
Citrobacter murliniae
Citrobacter rodentium
Citrobacter sedlakii
Citrobacter werkmanii
Citrobacter youngae
Citrobacter koseri
Citrobacter youngae V
Cronobacter muytjensii V
Cronobacter sakazakii V
Cronobacter sakazakii group B
Cronobacter sakazakii
Cronobacter dubliniensis
Cronobacter muytjensii
Cronobacter turicensis
Cronobacter turicensis V
Edwardsiella hoshinae V
Edwardsiella tarda
Enterobacter aerogenes
Enterobacter amingenus B
Enterobacter asburiae V
Enterobacter cancerogenus V
Enterobacter cloacae V
Enterobacter cloacae complex B
Enterobacter asburiae
Enterobacter cancerogenus
Enterobacter cloacae
Enterobacter hormaechi
Enterobacter kobei
Enterobacter ludwigii
Enterobacter gergoviae V
Enterobacter hormaechei V
Enterobacter kobei V
Enterobacter ludwigii V
Escherichia coli
Escherichia fergusonii V
Escherichia hermannii
Escherichia vluneris
Ewingella americana
Hafnia alvei
Klebsiella oxytoca V
Klebsiella oxytoca/Raoultella ornithinolytica B
Klebsiella pneumoniae
Klebsiella variicola
Kluyvera ascorbata V
Kluyvera cryocrescens V
Kluyvera intermedia V
Leclercia adecarboxylata
Lelliottia amnigena V
Morganella morganii
Pantoea agglomerans
Plesiomonas shigelloides B
Pluralibacter georgoviae
Proteus mirabilis
Proteus penneri V
Proteus vulgaris V
Proteus vulgaris group B
Proteus hauseri
Proteus penneri
Proteus vulgaris
Providencia alcalifaciens V
Providencia rettgeri
Providencia rustigianii V
Providencia stuartii
Raoultella ornithinolytica V
Raoultella planticola V
Raoultella terrigena V
Salmonella enterica ssp. enterica V
Salmonella sp. B
Serratia ficaria V
Serratia fonticola
Serratia grimesii V
Serratia liquefaciens
Serratia marcescens
Serratia odorifera
Serratia plymuthica
Serratia proteamaculans V
Serratia quinivorans V
Serratia rubidaea
Yersinia aldovae V
Yersinia enterocolitica
Yersinia frederiksenii
Yersinia intermedia
Yersinia kristensenii
Yersinia pseudotuberculosis
Yersinia ruckeri V
Achromobacter denitrificans V
Achromobacter xylosoxidans
Acinetobacter baumannii V
Acinetobacter baumannii/nosocomialis group B
Acinetobacter baumannii
Acinetobacter nosocomialis
Acinetobacter calcoaceticus
Acinetobacter haemolyticus
Acinetobacter johnsonii
Acinetobacter junii
Acinetobacter lwoffi
Acinetobacter nosocomialis V
Acinetobacter pittii
Acinetobacter radioresistens B
Acinetobacter ursingii B
Actinotignum schaalii group
Actinotignum schaali
Actinotignum sanguinis
Aeromonas hydrophila V
Aeromonas hydrophila/caviae group
Aeromonas allosaccharophila
Aeromonas aquariorum
Aeromonas caviae
Aeromonas culicicola
Aeromonas enteropelogenes
Aeromonas fluvialis
Aeromonas hydrophila
Aeromonas icthiosmia
Aeromonas jandaei
Aeromonas media
Aeromonas punctate
Aeromonas rivuli
Aeromonas sanarellii
Aeromonas sobria
Aeromonas taiwanensis
Aeromonas veronii
Aeromonas jandaei V
Aeromonas punctata (caviae) V
Aeromonas salmonicida
Aeromonas sobria V
Aeromonas veronii V
Alcaligenes faecalis B
Alcaligenes faecalis ssp. faecalis V
Bordetella avium V
Bordetella bronchiseptica V
Bordetella pertussis/bronchiseptica/parapertussis B
Bordetella hinzii B
Bordetella parapertussis V
Bordetella pertussis V
Brevundimonas diminuta V
Brevundimonas diminuta group B
Brevundimonas diminuta
Brevundimonas naejangsanensis
Brevundimonas vesicularis V
Burkholderia cenocepacia V
Burkholderia cepacia V
Burkholderia cepacia complex B
Burkholderia ambifaria
Burkholderia anthina
Burkholderia cenocepacia
Burkholderia cepacia
Burkholderia diffusa
Burkholderia dolosa
Burkholderia lata
Burkholderia latens
Burkholderia metallica
Burkholderia pyrrocina
Burkholderia seminalis
Burkholderia stabilis
Burkholderia vietnamiensis
Burkholderia contaminans V
Burkholderia gladioli B
Burkholderia multivorans
Burkholderia vietnamiensis V
Capnocytophaga ochracea B
Capnocytophaga sputigena B
Chryseobacterium gleum
Chryseobacterium indologenes
Comamonas testosteroni V
Cupriavidus pauculus group B
Cupriavidus pauculus
Cupriavidus metallidurans
Delftia acidovorans V
Delftia acidovorans group B
Delftia acidovorans
Delftia lacustris
Delftia litopenaei
Delftia tsuruhatensis
Elizabethkingia anophelis V
Elizabethkingia meningoseptica V
Elizabethkingia meningoseptica group B
Elizabethkingia meningoseptica
Elizabethkingia anopheles
Elizabethkingia miricola
Elizabethkingia miricola V
Mannheimia haemolytica V
Mannheimia haemolytica group B
Mannheimia haemolytica
Mannheimia glucosida
Myroides odoratimimus B
Myroides odoratus B
Myroides sp. V
Ochrobactrum anthropi V
Pantoea dispersa V
Pasteurella aerogenes V
Pasteurella multocida
Pseudomonas aeruginosa
Pseudomonas alcaligenes V
Pseudomonas fluorescens V
Pseudomonas fluorescens group B
Pseudomonas azotoformans
Pseudomonas brenneri
Pseudomonas cedrina
Pseudomonas congelans
Pseudomonas extremorientalis
Pseudomonas fluorescens
Pseudomonas gessardii
Pseudomonas libanensis
Pseudomonas mandelii
Pseudomonas marginalis
Pseudomonas migulae
Pseudomonas mucidolens
Pseudomonas orientalis
Pseduomonas poae
Pseudomonas rhodesiae
Pseudomonas synxantha
Pseudomonas tolaasii
Pseudomonas trivialis
Pseudomonas veronii
Pseudomonas luteola V
Pseudomonas mendocina V
Pseudomonas oryzihabitans
Pseudomonas putida V
Pseudomonas putida group B
Pseudomonas fulva
Pseudomonas monteilii
Pseudomonas mosselii
Pseudomonas plecoglossicida
Pseudomonas putida
Pseudomonas stutzeri
Ralstonia pickettii
Rhizobium radiobacter
Shewanella putrefaciens V
Sphingobacterium multivorum
Sphingobacterium spiritivorum
Sphingomonas paucimobilis V
Sphingomonas paucimobilis group B
Sphingomonas paucimobilis
Sphingomonas zeae
Stenotrophomonas maltophilia
Sutterella wadsworthensis B
Vibrio alginolyticus V
Vibrio cholerae V
Vibrio fluvialis V
Vibrio metschnikovii V
Vibrio mimicus V
Vibrio parahaemolyticus
Vibrio vulnificus
Aggregatibacter actinomycetemicomitans
Aggregatibacter aphrophilus
Aggregatibacter segnis
Brucella sp. V
Campylobacter coli
Camplylobacter jejuni
Campylobacter rectus V
Campylobacter ureolyticus B
Eikenella corrodens
Fluoribacter bozemanae
Haemophilus haemolyticus B
Haemophilus influenzae
Haemophilus parahaemolyticus V
Haemophilus parahaemolyticus group B
Haemophilus parahaemolyticus
Haemophilus paraphrohaemolyticus
Haemophilus parainfluenzae
Kingella denitrificans
Kingella kingae
Legionella longbeachae B
Legionella pneumophila V
Moraxella catarrhalis
Moraxella lacunata V
Moraxella nonliquefaciens
Moraxella osloensis
Neisseria bacilliformis B
Neisseria cinerea
Neisseria elongota
Neisseria flavescens/subflava group B
Neisseria flavescens
Neisseria perflava
Neisseria subflava
Neisseria gonorrhoeae
Neisseria lactamica B
Neisseria meningitidis
Neisseria mucosa/sicca V
Neisseria sicca group B
Neisseria macacae
Neisseria mucosa
Neisseria sicca
Neisseria weaveri
Oligella ureolytica
Oligella urethralis
Weeksella virosa
Actinomyces bovis V
Actinomyces europaeus B
Actinomyces funkei B
Actinomyces graevenitzii B
Actinomyces hyovaginalis B
Actinomyces israelii V
Actinomyces meyeri
Actinomyces naeslundii V
Actinomyces neuii
Actinomyces odontolyticus
Actinomyces oris B
Actinomyces radingae B
Actinomyces turicensis B
Actinomyces urogenitalis B
Alloscardovia omnicolens B
Anaerococcus murdochii B
Anaerococcus vaginalis B
Bacteroides caccae
Bacteroides eggerthii V
Bacteroides fragilis
Bacteroides nordii
Bacteroides ovatus/xylanisolvens V
Bacteroides ovatus group B
Bacteroides ovatus
Bacteroides xylanisolvens
Bacteroides pyogenes
Bacteroides salyesiae B
Bacteroides stercoris V
Bacteroides stercoris group B
Bacteroides stercoris
Bacteroides eggerthii
Bacteroides thetaiotaomicron V
Bacteroides thetaiotaomicron group B
Bacteroides thetaiotaomicron
Bacteroides faecis
Bacteroides uniformis
Bacteroides vulgatus
Bacteroides vulgatus group B
Bacteroides vulgatus
Bacteroides dorei
Bifidobacterium breve B
Bifidobacterium sp. V
Biolphila wadsworthia V
Clostridium baratii V
Clostridium beijerinckii
Clostridium bifermentans B
Clostridium butyricum
Clostridium cadaveris V
Clostridium clostridioforme V
Clostridium clostridioforme group B
Clostridium clostridioforme
Clostridium bolteae
Clostridioides difficile
Clostridium innocuum
Clostridium novyi V
Clostridium paraputrificum B
Clostridium perfringens
Clostridium ramosum
Clostridium septicum
Clostridium sordellii B
Clostridium sporogenes V
Clostridium sporogenes/Clostridium botulinium (group I) B
Clostridium tertium
Clostridium tetani V
Cutibacterium acnes
Cutibacterium avidum V
Cutibacterium granulosum V
Facklamia hominis B
Finegoldia magna
Fusobacterium canifelinum B
Fusobacterium mortiferum V
Fusobacterium necrophorum
Fusobacterium nucleatum
Fusobacterium periodonticum V
Hathewaya histolytica V
Mobiluncus curtisii
Paenclostridium sordellii V
Parabacteroides distasonis B
Parabacteroides goldsteinii B
Parabacteroides johnsonii/merdae group B
Parabacteroides johnsonii
Parabacteroides merdae
Paraclostridium bifermentans V
Parvimonas micra V
Peptoniphilus asaccharolyticus V
Peptoniphilus harei group B
Peptoniphilus harei
Peptoniphilus inodolicus
Peptostreptococcus anaerobius
Porphyromonas asaccharolytica/uenonis V
Porphyromonas gingivalis
Porphyromonas somerae B
Prevotella bivia
Prevotella buccae
Prevotella denticola
Prevotella intermedia
Prevotella loescheii V
Prevotella melaninogenica
Prevotella oralis V
Prevotella oris V
Propionibacterium acidipropionici V
Propionibacterium propionicum V
Tannerella forsythia V
Veillonella dispar V
Veillonella parvula group B
Veillonella parvula
Veillonella dispar
Veillonella rugosae
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