Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight Mass Spectrometry in Comparison to Rpob Gene Sequencing

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ORIGINAL ARTICLE BACTERIOLOGY

Evaluation of matrix-assisted laser desorption ionization-time-of-ﬂight mass spectrometry in comparison to rpoB gene sequencing for species identiﬁcation of bloodstream infection staphylococcal isolates

T. Spanu*, E. De Carolis*, B. Fiori*, M. Sanguinetti, T. D’Inzeo, G. Fadda and B. Posteraro Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Rome, Italy

Abstract

As a result of variable expression of biochemical characters, misidentification by conventional phenotypic means often occurs with clinical isolates belonging to Staphylococcus species. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of- flight mass spectrometry (MALDI-TOF MS) for the identification of 450 blood isolates of the most relevant staphylococcal species, using sequence analysis of the rpoB gene as the reference method. A correct species identification by MALDI-TOF was obtained in 99.3% (447/450), with only three isolates being misidentified. In addition, MALDI-TOF correctly identified all the staphylococcal subspecies studied, including Staphylococcus capitis subsp. capitis and subsp. urealyticus, Staphylococcus cohnii subsp. urealyticus, Staphylococcus hominis subsp. novobiosepticus and subsp. hominis, Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus schleiferi subsp. schleiferi and Staphylococcus sciuri subsp. sciuri. Thus, MALDI-TOF MS-based species identification of staphylococci can be routinely achieved without any substantial costs for consumables or the time needed for labour-intensive DNA sequence analysis.

Keywords: Bacterial identification, bloodstream infection, MALDI-TOF-MS, rpoB gene, staphylococci Original Submission: 16 November 2009; Revised Submission: 29 December 2009; Accepted: 19 January 2010 Editor: D. Raoult Article published online: 2 February 2010 Clin Microbiol Infect 2011; 17: 44–49 10.1111/j.1469-0691.2010.03181.x some strains being misidentified with closely-related species, Corresponding author: M. Sanguinetti, Istituto di Microbiologia, especially when conventional phenotypic methods are Universita` Cattolica del Sacro Cuore, Largo F. Vito, 1 00168 Rome, Italy employed. DNA sequence analysis using different nucleic acid E-mail: [email protected] targets is an alternative technique for the accurate identifica- *These authors contributed equally to this work. tion of Staphylococcus species [6], although it should be emphasized that additional/supplementary methods might be necessary for those species that cannot be delineated by sequence comparison of a single gene (i.e. 16S rDNA) [7]. Introduction Matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDI-TOF MS) has emerged as a new Among the over 40 well-recognized species and subspecies technology for the rapid analysis of protein components of of staphylococci [1,2], Staphylococcus aureus is the leading bacterial cells [8]. Extensive high-quality databases of type human pathogen not only in hospitals, but also in communi- and reference strains are available as integrated parts of soft- ties, even though other staphylococcal species (e.g. Staphylo- ware such as BioTyper (Bruker Daltonik GmbH, Leipzig, coccus epidermidis, Staphylococcus hominis, Staphylococcus Germany) or Waters MicrobeLynx (Waters Corp., Milford, capitis, Staphylococcus haemolyticus, Staphylococcus warneri and MA, USA) [8], which claim to allow accurate, reproducible, Staphylococcus lugdunensis) are increasingly insolated from and cost-effective species identification for the major groups infections in immunocompromised patients, patients using of pathogenic bacteria [9]. Carbonelle et al. [10] demon- catheters [3] and native valve endocarditis [4]. Therefore, strated that sets of selected peaks from each of 23 reference failure to unequivocally distinguish between staphylococcal strains, corresponding to the clinically relevant species and species can have far-reaching clinical and epidemiological subspecies of Micrococcaceae, could be used as a database for repercussions [5]. Nevertheless, the precise identification of the rapid identification of clinical coagulase-negative staphylo- these bacteria at the species level remains difficult, with cocci (CoNS) isolates. Using this database, Carbonelle’s

ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases CMI Spanu et al. Identiﬁcation of staphylococcal isolates by MALDI-TOF 45

group achieved a correct species identification in 97.4% of PCR amplification and sequencing of the partial rpoB gene their CoNS isolates with MALDI-TOF MS analysis [11]. from the study isolates were performed using primers 2491F By contrast, 86 (22.3%) of 385 CoNS isolates, as well as iso- and 3241R [14] and, when appropriate, additional primers lates from others of >100 bacterial species analysed by Seng specifically designed to identify all staphylococcal subspecies et al. [9], were identified only at the genus level using the [15]. An isolate was correctly identified when its partial rpoB Bruker BioTyper database. Because precision in MALDI-TOF gene sequence yielded ‡97% sequence similarity with the MS identification was significantly correlated with the fact closest bacterial species sequence in GenBank. that the database for those species included ‡10 reference spectra, the authors claimed that a complete and representa- MALDI-TOF MS tive database was a critical requirement for the accurate For MALDI-TOF MS analysis, cells from a single colony of identification of isolates by MALDI-TOF MS. fresh overnight culture were used for each isolate to prepare The present study aimed to re-evaluate the identification samples according to the microorganism profiling ethanol/ performance of the Bruker BioTyper database by testing a acid formic extraction procedure, as recommended by the large number of blind-coded clinical staphylococcal isolates manufacturer. After centrifugation at maximum speed for representing the species routinely identified from blood- 2 min, 1 lL of each supernatant containing the bacterial stream infections in the diagnostic microbiology laboratory. extract was allowed to dry after overlaying it with 2 lLofa Because rpoB gene sequence has been shown to differentiate chemical matrix (saturated solution of a-cyano-4-hydroxy- bacterial species not distinguishable by 16S rRNA gene cinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid) sequence [12], partial rpoB gene sequencing was used as the on a polished steel MALDI target plate. The samples were reference method in the present study. then processed in the microflex LT (Bruker Daltonik GmbH) mass spectrometer equipped with a 20-Hz nitrogen laser. The spectra were recorded in the positive linear mode as Materials and methods described elsewhere [10].

Bacterial isolates and molecular identiﬁcation Data analysis We studied a collection of 450 nonrepetitive staphylococcal To identify microorganisms, the raw spectra obtained for isolates (Table 1) recovered from blood specimens received each isolate were imported into BioTyper software, version by our Institution from January 2007 to September 2009. 2.0 (Bruker Daltonik GmbH), and analysed without any user Most of the CoNS isolates were from cases of bacteraemia intervention, using default parameter settings, by the as previously deﬁned [13]. standard pattern matching algorithm against the spectra of

TABLE 1. Identification results Number (%) of isolates with obtained with the matrix-assisted indicated result laser desorption ionization-time-of- flight mass spectrometry system Species (number tested) Identified Misidentification for 450 Staphylococcus isolates Staphylococcus aureus subsp. aureus (115) 115 0 Staphylococcus arlettae (1) 1 0 Staphylococcus capitis subsp. capitis (8) 8 0 Staphylococcus capitis subsp. urealyticus (4) 4 0 Staphylococcus caprae (1) 1 0 Staphylococcus cohnii subsp. urealyticus (6) 6 0 Staphylococcus epidermidis (199) 198 1a Staphylococcus haemolyticus (36) 36 0 Staphylococcus hominis subsp. novobiosepticus (32) 32 0 Staphylococcus hominis subsp. hominis (17) 17 0 Staphylococcus lugdunensis (8) 8 0 Staphylococcus pettenkoferi (1) 0 1b Staphylococcus saprophyticus subsp. saprophyticus (1) 1 0 Staphylococcus schleiferi subsp. schleiferi (1) 1 0 Staphylococcus sciuri subsp. sciuri (2) 2 0 Staphylococcus simulans (10) 10 0 Staphylococcus warneri (7) 6 1c Staphylococcus xylosus (1) 1 0 Total 447 (99.3) 3 (0.7)

aMisidentified as S. hominis subsp. novobiosepticus. bMisidentified as S. haemolyticus. cMisidentified as S. simulans.

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3294 species in the Biotyper database, version 3.0 (updated (332/335) of the CoNS isolates. Among the three species October 2008). The reference database consisted of 126 that most frequently cause disease in humans or are isolated main spectra encompassing 46 Staphylococcus species and most frequently from human samples, S. epidermidis subspecies, as depicted in the dendrogram generated from (n = 199), S. hominis (n = 49) and S. haemolyticus (n = 36), the corresponding spectra (Fig. 1). The results of the pattern MALDI-TOF MS gave a correct identification in 99.6% (283/ matching process were expressed as log(score) values in the 284), with one S. epidermidis isolate being misidentified as range 0–3, using values of ‡2.0 for identification to the spe- S. hominis (subsp. novobiosepticus) [log(score) = 2.062]. Of cies level and values of between <2 and ‡1.7 for identifica- two other isolates not correctly identified, one was identi- tion to the genus level. The highest log(score) of a match fied as Staphylococcus simulans) by MALDI-TOF MS [log(- against the database was used for species identification. score) = 2.084] and as S. warneri by rpoB sequencing, and, interestingly, the other isolate was identified as S. haemolyticus by MALDI-TOF MS [log(score) = 2.252] and as Staphylo- Results coccus pettenkoferi by rpoB sequencing (Table 1).

In a typical analysis of staphylococcal isolates by MALDI-TOF Discussion MS, 10–20 prominent ion peaks were noted in the spectra from the region in the range 2 000–11 000 Da, with the highest-intensity peaks being consistently in the range 3000– As recently stated by Dupont et al. [11], misidentification 10 000 Da. On this basis, the log(score) values obtained by often results from the heterogeneous behaviour of the MALDI-TOF MS correctly identified all but three staphylo- CoNS population obtained from clinical specimens, particu- coccal isolates at the species level [log(score), ‡2.0], as sub- larly when conventional phenotypic identification means are sequently confirmed by the rpoB gene sequence-based employed. In two different studies published in 2008, the use molecular analysis. S. aureus isolates were 100% correctly of current automated biochemical tests [the Phoenix and identified. A correct identification was obtained in 99.1% Vitek 2 (bioMe´rieux, Marcy l’Etoile, France) (Becton-Dickinson,

MSP dendrogram

Staphylococcus lactis DSM 20033 Staphylococcus haemolyticus 10024 CHB Staphylococcus carnosus ssp utilis DSM 11676 Staphylococcus piscifermentans DSM 7373 Staphylococcus condimenti DSM 11674 Staphylococcus carnosus ssp carnosus DSM 20501 Staphylococcus schleiferi ssp schleiferi DSM 4807 Staphylococcus schleiferi ssp coagulans DSM 6628 DSM Staphylococcus lutrae DSM 10244 Staphylococcus felis DSM 7377 Staphylococcus intermedius 08_STAINT MVO Staphylococcus delphini DSM 20771 Staphylococcus muscae DSM 7068 Staphylococcus hyicus DSM 17421_DSM Staphylococcus chromogenes DSM 20454 Staphylococcus auricularis DSM 20609 Staphylococcus epidermidis 10547 CHB Staphylococcus caprae DSM 20608_DSM Staphylococcus capitis ssp urealyticus DSM 6717 Staphylococcus capitis ssp capitis DSM 20325 Staphylococcus simiae DSM 17636 Staphylococcus aureus ssp aureus DSM 11822 Staphylococcus aureus ssp anaerobius DSM 20714_DSM Staphylococcus hominis ssp novobiosepticus DSM 15614 Staphylococcus hominis ssp hominis DSM 20328_DSM Staphylococcus simulans CCM 2724 CCM Staphylococcus warneri CCM 2604 CCM Staphylococcus pasteuri DSM 10656 Staphylococcus succinus ssp succinus DSM 14617T Staphylococcus succinus ssp casei DSM 15096 Staphylococcus nepalensis DSM 15150_DSM Staphylococcus kloosii DSM 20676T Staphylococcus saprophyticus ssp saprophyticus CCM 235 Staphylococcus xylosus DSM 20266 Staphylococcus cohnii ssp urealyticum DSM 6718 Staphylococcus cohnii ssp cohnii DSM 20260 Staphylococcus lugdunensis 20659_1 CHB Staphylococcus equorum ssp equorum DSM 20674 Staphylococcus arlettae DSM 20672 Staphylococcus lentus DSM 20352 Staphylococcus sciuri ssp sciuri DSM 20345T Staphylococcus sciuri ssp carnaticus DSM 15613 Staphylococcus sciuri ssp rodentium DSM 16827 Staphylococcus fleurettii DSM 13212 Staphylococcus vitulinus DSM 15615 Staphylococcus pulvereri DSM 9931 1000 900 800 700 600 500 400 300 200 100 0 Distance level

FIG. 1. Score oriented dendrogram showing the spectral similarities of staphylococcal type strains (n = 46), as obtained by Microﬂex LT (Bruker Daltonik GmbH).

ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 44–49 CMI Spanu et al. Identiﬁcation of staphylococcal isolates by MALDI-TOF 47

Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) sys- identity were close to 100% (99.5–99.7%) and, in this method, tems] gave correct identification rates for CoNS of only a similarity threshold of ‡94% is said to be reliable for correct 90.5% and 87.5%, respectively [16,17]. Thus, although various species designation of clinical staphylococcal isolates [15]. degrees of accuracy can be achieved with different available It is known that the success of species matching is depend- biochemical-based commercial kits and automated systems, ing upon the number of strain entries within the database for phenotypic characterization remains equivocal, even for clini- a given species, but it is also true that overlapping signal ions cal isolates belonging to the S. aureus species. Conversely, increase with the number of the strains registered in the but in agreement with our results, Rajakaruna et al. [18] database, making it difficult to identify species- or subspecies- showed that all of 134 clinical isolates presumptively identi- specific markers in the spectra [8]. The results obtained fied as S. aureus, were identified correctly with MALDI-TOF clearly show that accurate MALDI-TOF MS identification was MS using the MicrobeLynx software. possible for isolates belonging to species with less than five Despite a moderate degree of species representativity in reference spectra in the database, such as Staphylococcus arlet- our CoNS isolate collection, the overall percentage of cor- tae, Staphylococcus caprae, S. schleiferi, S. sciuri and S. warneri. rect identification results using MALDI-TOF MS was superior As already discussed [19], marked differences in antimi- to that of previous studies [9,11]. We obtained a correct crobial resistance patterns in some staphylococcal species identification in 84.7% of S. epidermidis, S. hominis and S. hae- and subspecies make their differentiation essential in clinical molyticus isolates compared to the 42% achieved by Dupont practice. One remarkable finding of the present study was et al. [11]. We had a 100% correct identification for the good performance of MALDI-TOF in identifying all the the S. haemolyticus isolates compared to the 85.8% of CoNS subspecies tested, including S. capitis subsp. capitis and Dupont et al. subsp. urealyticus, S. cohnii subsp. urealyticus, S. hominis subsp. In the present study, MALDI-TOF correctly identified novobiosepticus and subsp. hominis, S. saprophyticus subsp. sap- 99.1% of CoNS isolates compared to the 77.7% correct identi- rophyticus, S. schleiferi subsp. schleiferi and S. sciuri subsp. sciuri fications reported by Seng et al. [9]. This may be related to (Table 1). As an example, different peak profiles among the differences in the species distributions in these two studies. In subspecies of S. hominis and those of S. capitis could easily be our study, three CoNS isolates were misidentified; for all the noted in their MS spectra (Fig. 2). S. pettenkoferi, a novel three isolates, rpoB gene sequence similarities to the reference CoNS species isolated from human clinical specimens [20],

4 4831.233 9661.884 (a) 3237.963 3 6648.745 2 8140.188 1

0 9661.303

2.5 (b) 4831.384 2.0 7574.932 1.5 6648.215 1.0 8138.245 0.5 0.0 2486.115 6 (c) intensity 9645.329 4 7528.914 4824.673 8065.507 6716.084 2

0 9645.512

4822.990 3 (d)

2 2611.358 6715.217 5100.474 1 8094.809

0 3000 4000 5000 6000 7000 8000 9000 10000 mass (m/z) FIG. 2. Matrix-assisted laser desorption ionization-time-of-ﬂight mass spectrometry of (a) Staphylococcus hominis subsp. novobiosepticum, (b) Staph- ylococcus hominis subsp. hominis, (c) Staphylococcus capitis subsp. capitis and (d) Staphylococcus capitis subsp. urealyticus, showing the differences among the subspecies. The absolute intensities of the ions are shown on the y-axis, and the masses (Da) of the ions are shown on the x-axis. The m/z value represents the mass to charge ratio.

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