evaluation of species-specific score cutoff values of routinely isolated clinically relevant...

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European Journal of ClinicalMicrobiology & Infectious Diseases ISSN 0934-9723Volume 31Number 6 Eur J Clin Microbiol Infect Dis (2012)31:1109-1119DOI 10.1007/s10096-011-1415-7

Evaluation of species-specific score cutoffvalues of routinely isolated clinicallyrelevant bacteria using a direct smearpreparation for matrix-assisted laserdesorption/ionization time-of-flight massspectrometry-based bacterial identificationF. Szabados, H. Tix, A. Anders,M. Kaase, S. G. Gatermann & G. Geis

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ARTICLE

Evaluation of species-specific score cutoff values of routinelyisolated clinically relevant bacteria using a direct smearpreparation for matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry-based bacterialidentification

F. Szabados & H. Tix & A. Anders & M. Kaase &

S. G. Gatermann & G. Geis

Received: 29 July 2011 /Accepted: 31 August 2011 /Published online: 27 September 2011# Springer-Verlag 2011

Abstract Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was intro-duced a few years ago as a new method for bacterialidentification. A variety of studies have been publishedconcerning MALDI-TOF MS-based identification, most ofthem using culture collections for the validation of therespective databases in a retrospective manner in favor of aparallel investigation. The score cutoff value is of specialimportance for reliable species identification in the Biotyperdatabase. The score cutoff values suggested by themanufacturer have been validated using a previouslypublished formic acid extraction protocol. In most of thepreviously published studies investigating the Biotyperdatabase, only little information was given concerning

species-specific score values. In addition, the mass spec-trometer instruments, the number of replicates, the numberof spectra used to calculate a sum-spectrum by the suppliedsoftware, and the score cutoff values which have beenapplied varied within these studies. In this study, wecompared a straightforward direct smear preparation andmeasurement without replicate testing to defined biochem-ical identifications in a parallel manner. In addition, wedescribed new species-specific score cutoff values for theidentification of certain bacteria.

Introduction

Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS) was introduced afew years ago as a new method for bacterial identification[1]. The presence of many proteomic peaks, especiallybelow 2,000 Da, was variable and dependent on a variety offactors, such as agar media, temperature of incubation, ageof the bacteria, and other environmental factors. From thesefirst “proof-of-principle” experiments to the entering of thismethod into the routine work flow of clinical laboratories,reliable databases had to be established. For selectedbacteria, several studies have shown that bacterial speciesidentification is possible using distinct protein peaks [2].Nevertheless, these experiments were different with regardto the matrix used for MALDI-TOF MS, the protein-extraction protocols, the mass spectrometer instruments, thebacterial culturing conditions, and other environmentalconditions. Moreover, the use of different proteomicfingerprint ranges make it difficult to compare the published

Electronic supplementary material The online version of this article(doi:10.1007/s10096-011-1415-7) contains supplementary material,which is available to authorized users.

F. Szabados (*) :H. Tix :A. Anders :M. Kaase :S. G. Gatermann :G. GeisInstitute for Hygiene and Microbiology,Department for Medical Microbiology, University Bochum,National Reference Laboratory for Multidrug-ResistantGram-Negative Bacteria for Germany,Bochum, Germanye-mail: [email protected]

G. GeisInstitut für Medizinische Laboratoriumsmedizin (IML),Universitätsaninstitut, Ruhr-University Bochum,Bochum, Germany

H. TixHygiene Institut Gelsenkirchen,Gelsenkirchen, Germany

Eur J Clin Microbiol Infect Dis (2012) 31:1109–1119DOI 10.1007/s10096-011-1415-7

Author's personal copy

http://dx.doi.org/10.1007/s10096-011-1415-7

data. To date, several commercial databases for the identi-fication of bacteria have been described [3–5]. In addition,non-commercial approaches have been published [2].For the Biotyper 2.0 database (Bruker), protein peaks inthe mass-to-charge ratio of 3,000 to 15,000 Da are used,and most of them are thought to represent ribosomalprotein peaks.

A variety of studies which investigate the accuracy of theBiotyper 2.0 database have been described [6–9]. Never-theless, the mass spectrometer instruments, the number ofreplicates tested, the number of spectra used for a sum-spectrum, and the score cutoff values (which were appliedto define probable and secure species identification) variedwithin these studies. The identification score value has beenused by the manufacturer in terms of a surrogate for theidentification quality. Species misidentification in cases ofmixed cultures, irrespective of the score value, has beendescribed [10]. Nevertheless, the same score has usuallybeen applied to all identifications, independent of putativespecies-specific variations. The measured protein peaks of acertain bacterium were compared to a database. A scorevalue of up to 3 (log 1,000) could be obtained. Differencesin the peak profile will reduce an optimal identificationvalue of 3. Due to the log dependence, even smalldifferences in the score cutoff value represent largerdifferences in the peak profile. Moreover, none of thepreviously published studies gave information regardingspecies-specific score cutoff values. The same scorecutoff values have been applied to all identifications,irrespective of putative species-specific variations. Theusually applied Biotyper score values and score cutoffvalues should have been investigated in further detail.We, therefore, used a fast and straightforward directsmear preparation protocol, which has often been used inclinical laboratories, instead of the formic acid extractionprotocol suggested by the manufacturer and investigatedthe accuracy of a MALDI-TOF MS-based identificationin a parallel manner. In addition, species-specific scorecutoff values were defined using a 20th percentile ofconcordantly identified bacteria, such as for many strictanaerobic bacteria.

Materials and methods

Identification of clinically relevant bacteria

Routinely isolated, clinically relevant bacteria (n=878)were collected from February 2009 to March 2009. Thiscollection represents 13 hospitals, including both urban andrural settings from the Bochum area, as well as a variety ofcommunity and institutional facilities. Clinically relevantbacteria were preliminarily identified by typical colony

morphology, selective agar plating, Gram-staining, andcatalase or oxidase activity, if appropriate (Table 1). Thecollected bacteria represent clinically relevant bacteriafrequently isolated from human sources from variousorigins. This collection comprises blood culture samples(n=191), bronchial swabs (n=85), urine sample (n=123),and wound swabs/aspirated sample materials of differentorigins (n=479). Some samples sources, such as feces,were not included in this collection. Bacteria were culturedon typically used sets of agar plates, such as Columbia bloodagar, Chocolate agar, and MacConkey agar (bioMérieux,Marcy l’Etoile, France) and were grown for up to 48 h ofincubation at 37°C with the addition of 5% CO2. Dependingon the sample source, not all cultivated bacteria wereidentified to the species level, and, therefore, were notincluded in this collection, since these bacteria wereidentified by preliminarily methods (e.g., Staphylococcusaureus isolates of different origins were identified byspecific latex agglutination tests, and most coagulase-negative staphylococci and corynebacteria from skin-derived samples were not identified to the species level).Suspected isolates of Streptococcus agalactiae (n=10) orStreptococcus pyogenes (n=8) that induce typical beta-hemolysis on Columbia sheep blood agar plates (bioMérieux)were agglutinated by the Streptococcal Grouping Latex kitDML 1001 (Diamondial, Sees, France), according to themanufacturer’s instructions. They were defined as group Aand group B beta-hemolytic streptococci. Suspected Gram-positive bacteria (n=210) were identified using the Gram-positive (GP) identification card by the Vitek 2 automatedidentification system (bioMérieux). Supposed isolates ofpneumococci were diagnosed by testing them for theirresistance to optochin and their bile solubility. Optochin-susceptible and bile-soluble isolates were defined as Strep-tococcus pneumoniae. Suspected non-lactose fermentingProteus species were identified by the Gram-negative (GN)identification card. Isolates identified as the Proteus vulgarisgroup were tested for the presence of ornithine decarboxylase(ODC) and reactions determined by sulfide–indole(Kovacs reagent)–motility (SIM) medium. Indole-positive,ODC-negative, and hydrogen sulfide-positive strains weredefined as Proteus vulgaris. Suspected lactose non-fermenting Pseudomonas species preliminarily identified bytypical aspect, positive reaction with oxidase and by theproduction of a typical pigment were identified by the GNidentification card and defined as Pseudomonas aeruginosa.Suspected Haemophilus spp. preliminarily identified bycolony morphology and resistance to oleandomycin werecollected from chocolate agar. Strains that strictly need bothfactors X and V for growth were defined as Haemophilusinfluenzae, strains that were variably dependent on factors Xand V for growth were defined to be Haemophilus spp.(Haemophilus non-influenzae).

1110 Eur J Clin Microbiol Infect Dis (2012) 31:1109–1119


Other suspected Gram-negative bacteria were identifiedby using the GN identification card by the Vitek 2automated identification system (bioMérieux). Suspectedyeasts were identified by growth on Sabouraud selectiveagar (bioMérieux) and were uniquely identified by usingthe YST identification card by the Vitek 2 automatedidentification system (bioMérieux). Suspected obligateanaerobes were preliminarily identified by growth onlyunder strict anaerobic conditions on Columbia sheep bloodagar plates (bioMérieux), Gram staining, and catalasetesting. Identification was narrowed by the ANC card bythe Vitek 2 automated identification system (bioMérieux)and interpreted according to the manufacturer’s instruc-tions. An identification approach was repeated once if noresult was gained.

MALDI-TOF MS

All strains were examined by MALDI-TOF-MS using theMicroflex LT instrument (Bruker Daltonics), Flexcontrol3.0 software, and the Biotyper 2.0 database Build 2.0.10.0(Bruker Daltonics). The instrument was used to calculateand process the analytical data as previously described [6,9]. For sample preparation, one colony was transferred ontoan MSP 96 polished steel target (Bruker Daltonics), asdescribed previously [7]. The samples were covered with1 μl of α-cyano-4-hydroxycinnamic acid (HCCA) matrixsolution (saturated solution of α-cyano-4-hydroxycinnamicacid in 50% acetonitrile and 2.5% trifluoroacetic acidsupplied by the manufacturer). The analyzed mass rangeof spectra was from 3,000 to 15,000 m/z. Each sample wastested on a single spot and was not tested in replicates.Moreover, each sum-spectra was obtained after 240 shots inan automatic acquisition mode. The quality of the gainedprotein fingerprint increases with the number of shots andthe number of replicates (data not shown). To eliminate aputative preparation error, the identification was repeated

once if the initially obtained score was low. Retesting wasnecessary for an estimated 60 samples due to low scorevalues (those situations giving a “no-peak” result or a scorevalue of below 1.5, an estimated 2% of all samples). Toimplement new species-specific score cutoff values, allinitial scores ≥1.5 were used within this study. In theretesting of such certain isolates, all measured scores weredocumented. A “no-peak” result was interpreted as insuf-ficient identification by MALDI-TOF MS. The rounded20th percentile score values (estimated to be between the14.3 to 33.3 percentiles and also depending on the samplenumber) were used to define a new species-specific scorecutoff value for a secure species identification.

Molecular species confirmation

Staphylococci and streptococci were identified by theamplification and sequencing of the sodA gene [11], yeastsby the amplification and sequencing of the 26S rDNA [12],and other bacteria by the amplification and sequencing ofthe 16S rDNA [13], where indicated. Isolates identified asAcinetobacter baumanii complex by the GN identificationcard were confirmed by species-specific polymerase chainreaction (PCR) assays as previously described [14].

Results and discussion

MALDI-TOF MS-based identification is a novel, recentlydescribed method for the identification of bacteria. Specificprotein fingerprints were compared with databases ofvarious bacteria. In our study, the Biotyper 2.0 databasewas used. During this comparison, the software calculated ascore value depending on the number of corresponding anddiscrepant proteomic peaks up to a maximum score of 3(log 1,000). According to the manufacturer, a score ≥2 isrecommended for a probable species and a score >2.3 is

Table 1 Clinically relevantgroups of bacteria and theircorresponding Biotyper2.0 score values

Organisms Number ofspecies, n

Number ofisolates, n

Arithmetic meanscore value

Minimumscore

Maximumscore

Gram-positive facultative aerobic

Staphylococci 10 105 2.16 1.77 2.81

Streptococci 14 68 2.15 1.68 2.47

Enterococci 7 45 2.28 1.86 2.53

Other 7 10 1.79 1.54 2.23

Gram-negative facultative aerobic

Enterobacteriaceae 39 406 2.26 1.6 2.57

Non-fermenter 13 52 2.24 1.7 2.54

Other 2 28 2.17 1.8 2.35

Strict anaerobic 19 117 2.03 1.42 2.4

Yeasts 7 47 2.2 1.91 2.43

Eur J Clin Microbiol Infect Dis (2012) 31:1109–1119 1111


recommended for a highly probable species identification.In our collection, only 34.2% of the isolates had acorresponding score value ≥2.3, 52.2% of the isolates hada score value between 2.0 and 2.29, and 13.8% of theisolates had a score value below 2.0. According to the scorecutoff values suggested by the manufacturer, only 34% ofthe isolates were interpreted as a “highly probable” speciesidentification, and an additional 52.2% were interpreted as“probable” species identification. The score values havebeen used by the manufacturer as a surrogate for theaccuracy of the MALDI-TOF MS-based identification. Inaddition, this score system has been described for the use ofa formic acid preparation protocol. The mean score valuesof certain bacteria, such as Staphylococci and Enterobac-teriaceae, is higher when using the formic acid preparationprotocol (data not shown) compared to the direct extractionprotocol used in our study. Recently, a variety of studieshave evaluated the Biotyper database. Nevertheless, thesestudies varied in their extraction protocols, replicate testing,and in the number of spectra used to calculate a sum-spectrum. They also occasionally differ in the MALDI-TOFMS instrument used [6, 7, 9, 15–20]. Moreover, putativespecies-specific differences in the gained score values wereobserved in a previously investigated strain collection (datanot shown). In order to confirm these supposed species-specific differences and to evaluate species-specific scorecutoff values for clinically important bacteria, a commonstraightforward direct smear extraction protocol was usedand the bacteria were identified in parallel, in a prospectivemanner under routine conditions. In addition, samples werenot tested in replicates and the measurement was performedon a simple MALDI-TOF MS Microflex LT instrument(e.g., non-deflector detection of the proteomic mass), onewhich is usually supplied for this diagnostic purpose.Included in this study were 228 isolates of ten generaand 40 species of Gram-positive organisms, 486 isolatesof 26 genera and 54 species of Gram-negative organisms,47 isolates of seven Candida species, and 117 isolatesstrict anaerobic bacteria of 19 genera and 28 species(Table 1 and the supplementary material). As seen in manyprevious studies, the accuracy of a MALDI-TOF MS-basedidentification is high, even if a single direct smearpreparation was performed (Table 2).

Gram-positive facultative anaerobe bacteria

Isolates of S. agalactiae (Lancefield group B) or S.pyogenes (Lancefield group A), defined by GPI and latexagglutination, were compared to MALDI-TOF MS-basedidentification. The mean score values of these streptococciwere high and reliable compared to the reference method(Table 3). Similarly, the identification of beta-hemolyticstreptococci by MALDI-TOF-MS has been described as

accurate independently of the underlying database [21–23].Nevertheless, the use of species-specific scores increasedspecies identification, amongst others, Streptococcusdysgalactiae spp. equisimilis and Streptococcus anginosus.Both species had suggested score cutoff values of 1.9(Table 3).

The separation of pneumococci from other alpha-hemolytic streptococci seems to be difficult, since S.pneumoniae has been falsely identified independent of thedatabases [3, 6]. Our study indicates that most isolates of S.pneumoniae were correctly identified. Nevertheless, oneisolate was falsely identified as S. pneumoniae with a highscore value of 2.24. In addition to this, the lower number ofalpha-hemolytic streptococci in our study cautions againstan interpretation lacking reflection, but argues for theadditional testing of the bile solubility of suspected S.pneumoniae, irrespective of their score values, to clearlyrule out falsely positive MALDI-TOF MS results.

We have shown that the concordance of clinicallyrelevant streptococci other than pneumococci compared tothe biochemical reference method (GP card) was onlymoderate (Table 3). A similar result has previously beendescribed [3]. In addition, the species-specific differencesof the mean score values of tested isolates argue for the useof species-specific cutoff values. Most isolates of staphy-lococci were concordantly identified with a high meanscore value (Table 3). Our study indicates that, using directsmear extraction and non-replicate testing, the MALDI-TOF MS-based identification of staphylococci is highlyreliable and comparable to the identification yielded by theGP card. Only in selected species, such as Staphylococcusepidermidis, Staphylococcus haemolyticus, and Staphylo-coccus lugdunensis, did we find that the mean score valuesand the estimated 20th percentile species-specific scorevalues were lower and argued for the use of species-specificscore cutoff values. The accuracy of the identification to thespecies level is high (Table 3), similar to previously

Table 2 Biotyper 2.0-based identification to the species levelcompared to their reference identification

Organisms Referenceidentification

Discrepantresults, n

Concordantidentificationin %

Isolates, n

Gram-positive facultative anaerobic

GP card 15 92.6 210

Other 0 100.0 18

Gram-negative facultative anaerobic

GN card 13 96.8 460

Other 0 100.0 28

Strict anaerobic ANC card 12 89.7 117

Yeasts YST card 2 95.7 47



published studies [2, 9, 24], independent of the databasesapplied in these studies. All isolates of Enterococcusfaecalis and Enterococcus faecium were correctly identifiedwith moderate mean score values (Table 3). Our studysuggests the use of new species-specific score cutoff valuesfor some isolates such as Enterococcus faecalis andEnterococcus faecium. Three isolates of Dermabacterhominis and Micrococcus luteus were identified correctly,gaining repeatedly low score values. Our study argues forthe use of a species-specific score value for at least thesetwo species, with suggested new score cutoff values of 1.85and 1.9 for a secure species identification. Our studyindicates that some Gram-positive organisms, such asLeuconostoc spp. and Granulicatella spp., were correctlyidentified, but with low corresponding score values. Due tothe low sample numbers, an interpretation is difficult. Theresults indicate that the use of the new species-specificscore cutoff values enhances the identification for a varietyof Gram-positive organisms.

Gram-negative facultative aerobic bacteria

Isolates of H. influenzae (n=25), defined by the previouslydescribed algorithm, were concordantly identified withan arithmetic mean score value of 2.18 (min.=1.8,

max.=2.35). Haemophilus spp. other than H. influenzae,n=3), defined by the previously described algorithm,were identified as H. parainfluenzae by MALDI-TOF MSand were all distinguished from H. influenzae isolateswith score values of 1.93, 2.16, and 2.20. We suggest theuse of a species-specific score value of 2.0 for theidentification of H. influenzae.

Many species of the Enterobacteriaceae, such asEscherichia coli, Morganella morganii, Klebsiella pneumo-niae, and Serratia marcescens, were concordantly identi-fied with high mean score values (Table 4). Not allCitrobacter spp. were concordantly identified to the specieslevel by MALDI-TOF MS compared to the GN identifica-tion card. Some species of Citrobacter were incorrectlyidentified to the species level by MALDI-TOF MS, due to ahigh similarity within related species members. Neverthe-less, the chromosomal ampC gene-harboring strains (e.g. C.freundii and C. braakii) [25] could be clearly distinguishedfrom other species where this resistance mechanism was notpresent, such as C. koseri and C. diversus (Table 4). Manyisolates of Enterobacter spp. were concordantly identifiedby the GN-card. Some isolates were identified by MALDI-TOF as synonymous species, such as Enterobacterhormaechei, Enterobacter kobei, Enterobacter ludwigiiand Enterobacter asburiae as compared to the identification

Table 3 Suggested species-specific score values for selected clinically relevant bacteria identified by the Gram-positive (GP) identification card

Species concordant to thereference method

Referencemethod

Samplenumber,n

Biotyper scorecutoff value:secure species

Biotyper scorecutoff value:probable species

Biotyper scorecutoff value:probable genus

Min.score

Meanscore

Max.score

Suggested scorecutoff valuesfor speciesidentificationScore >2.3 Score 2.0 to 2.29 Score 1.79 to 1.99

Streptococcus pyogenes Aggl. 8 3 5 0 2.17 2.3 2.45 2.20

Streptococcus agalactiae Aggl. 10 6 4 0 2.17 2.29 2.35 2.20

Streptococcus pneumoniae GP 12 0 6 2 1.9 2.05 2.24 2.00

Streptococcus dysgalactiaespp. equisimilis

GP 13 0 8 5 1.77 2.05 2.21 1.90

Streptococcus anginosus GP 4 0 1 3 1.83 1.94 2.22 1.90

Staphylococcus aureus GP 4 1 3 0 2.25 2.32 2.37 2.25

Staphylococcus epidermidis GP 49 1 43 6 1.77 2.1 2.57 2.0

Staphylococcus haemolyticus GP 9 2 6 1 1.97 2.21 2 2.05

Staphylococcus hominis GP 21 12 9 0 2.09 2.29 2.49 2.15

Staphylococcus lugdunensis GP 6 3 1 2 1.86 2.22 2.35 1.90

Staphylococcus capitis GP 6 0 6 0 2.07 2.26 2.36 2.15

Staphylococcus warneri GP 6 0 6 0 2.04 2.21 2.33 2.10

Enterococcus faecalis GP 15 11 3 1 1.86 2.32 2.53 2.2

Enterococcus faecium GP 20 11 8 1 1.96 2.28 2.44 2.15

Enterococcus hirae 16S 3 1 2 0 2.2 2.26 2.38 2.20

Enterococcus avium 16S 3 0 3 0 2.13 2.19 2.24 2.15

Dermabacter hominis 16S 2 0 0 2 1.81 1.91 1.99 *

Micrococcus luteus GP 2 1 1 1.81 2.05 2.29 *

*Sample number too low



of E. cloacae by GP card. The latter species have beenpreviously described to be members of the Enterobactercloacae complex [26]. Nevertheless, some isolates ofEnterobacter spp. were misidentified at the species level,but were concordant at the genus level. This misidentifica-tion at species level is, from the presence of thechromosomal AmpC resistance mechanism point of view,of minor importance, since most Enterobacter spp. (withthe exception of Enterobacter gergoviae) harbor a chromo-somal AmpC resistance mechanism [25]. Two isolatesidentified as Raoultella planticola by the GN card were

identified as Raoultella ornitholytica by MALDI-TOF MS.Raoultella has previously been separated from Klebsiellaand described as a new genus [27]. A misidentification ofRaoultella ornitholytica as Klebsiella oxytoca has recentlybeen described [7]. Whether the proteomic peaks of thedescribed Raoultella species were related among each otherand also related to Klebsiella oxytoca has to be elucidatedin further studies. Four isolates were not identified by theGN card but were correctly identified by MALDI-TOF MSas E. coli, K. pneumoniae, C. freundii, and E. cloacae.These identities were confirmed by the amplification and

Table 4 Suggested species-specific score values for selected clinically relevant bacteria identified by the Gram-negative (GN) identification card


Referencemethod

Samplenumber,n




Min.score

Meanscore

Max.score


Escherichia coli GN 125 86 35 4 1.77 2.24 2.43 2.15

Citrobacter braakii GN 4 2 2 0 2.2 2.3 2.35 2.20

Citrobacter freundii GN 6 3 3 0 2 2.26 2.45 2.25

Citrobacter koseri/diversus GN 8 8 0 0 2.36 2.43 2.48 2.30

Campylobacter jejuni GN 2 0 1 1 1.86 2.01 2.21 *

Enterobacter aerogenes GN 6 5 1 0 2.1 2.35 2.49 2.20

Enterobacter cloacae GN 44 10 33 1 1.98 2.23 2.4 2.15

Hafnia alvei GN 4 1 2 1 1.96 2.16 2.54 2.05

Klebsiella pneumoniae GN 24 5 19 0 2.2 2.3 2.35 2.15

Klebsiella oxytoca GN 18 7 11 0 2.11 2.26 2.56 2.20

Klebsiella pneumoniaespp. pneumoniae

GN 35 19 15 1 1.88 2.24 2.47 2.20

Klebsiella pneumoniaespp. ozaenae

GN 2 1 0 1 1.96 2.14 2.38 *

Morganella morganii GN 4 1 3 0 2.23 2.44 2.57 2.30

Morganella morganiispp. morganii

GN 17 15 2 0 2.27 2.44 2.57 2.30

Morganella morganiispp. siboni

GN 4 4 0 0 2.32 2.35 2.48 *

Proteus mirabilis GN 46 33 13 0 2.06 2.35 2.53 2.30

Proteus vulgaris GN 17 9 7 1 1.86 2.28 2.53 2.05

Providencia stuartii GN 2 0 2 0 2.2 2.24 2.26 *

Providencia rettgeri GN 2 2 0 0 2.24 2.29 2.34 *

Serratia marcescens GN 13 7 6 0 2.05 2.26 2.36 2.15

Serratia liquefaciens GN 2 0 2 0 2.25 2.25 2.26 *

Salmonella enterica GN 5 3 2 0 2.16 2.33 2.43 2.25

Acinetobacter baumanii Species-specific PCR

6 6 0 0 2.33 2.38 2.44 2.30

Acinetobactergenomospecies 3

Species-specific PCR

5 1 3 1 1.94 2.13 2.35 2.00

Pseudomonas aeruginosa GN 24 15 9 0 2.09 2.36 2.54 2.20

Pseudomonas fluorescens GN 2 0 1 1 1.78 1.9 2.02 *

Pseudomonas stutzeri GN 2 0 0 2 1.88 1.93 1.98 *

Pseudomonas putida GN 4 0 4 0 2.02 2.05 2.06 2.00

Stenotrophomonasmaltophilia

GN 9 3 3 3 1.97 2.15 2.36 2.05




sequencing of the 16S rDNA. Isolates of Salmonellaenterica (n=4) were identified as Salmonella group by theGN card and as Salmonella enterica by MALDI-TOF MS.The MALDI-TOF MS-based identification results wereconcordant but partly misleading in the used databaseversion, since serovars of S. enterica spp. enterica weregiven as species results. Meanwhile, the Salmonelladatabase entries have been corrected by the manufacturer.

Subspecies identification of Klebsiella pneumoniaeand Morganella morganii

A subspecies identification of K. pneumoniae was mainlyconcordant to the GP identification: the identification tothe subspecies level was concordant in 33 isolates, twoisolates were only identified to the species level, and oneisolate was discrepant in both methods in terms ofsubspecies identification. Two isolates of K. pneumoniaespp. ozaenae were identified as K. pneumoniae spp.pneumoniae by MALDI-TOF MS. One isolate of K.pneumoniae spp. ozaenae was correctly identified as K.oxytoca by MALDI-TOF, as confirmed by 16S amplifica-tion and sequencing. Subspecies of M. morganii, identifiedas M. morganii spp. morganii by the GN card, were alsoconcordantly identified to the subspecies level with a highscore value of 2.44. Subspecies of M. morganii identified asM. morganii spp. sibonii (n=4) by the GN card were onlyconcordantly identified to the subspecies level in two out offour isolates. The other two isolates were concordantlyidentified by MALDI-TOF MS as M. morganii spp.morganii, even to the subspecies level. Further studiesmust evaluate whether the, until now, clinically minorsubspecies information gained by MALDI-TOF MS isreliable.

Pseudomonas aeruginosa, Pseudomonas fluorescens,and Pseudomonas putida identified by the GN card wereconcordantly identified by MALDI-TOF MS with highscores. Pseudomonas aeruginosa group species memberswere clearly discriminated from other Pseudomonasspecies. Our study suggests the use of species-specificcutoff values in some of these species (Table 4).

Isolates of Stenotrophomonas maltophilia (n=9) wereidentified as S. maltophilia (n=5) and Pseudomonashibiscola (n=4). P. hibiscola is an out-of-date synonymousstrain nomenclature of S. maltophilia.

Acinetobacter baumanii and Acinetobacter genomospecies3 could be discriminated by MALDI-TOF MS

Eleven isolates were identified by the GN card as A.baumannii/calcoaceticus group. This A. baumanii groupcontains the Acinetobacter baumanii, Acinetobacter cal-coaceticus, Acinetobacter genomospecies 3, and the

Acinetobacter genomospecies 13TU. Only limited datahas been published as to whether the Biotyper 2.0database could identify human pathogenic A. baumaniiisolates with reliable accuracy [28]. Notably, among these,six isolates of A. baumanii were correctly identified withhigh mean score values and three isolates of Acinetobactergenomospecies 3 were correctly identified but with scoreslower than that of A. baumanii. The Acinetobacter speciesidentification was confirmed using a species-specific PCR,as previously described [14]. In order to confirm thisresult, previously collected and molecularly defined A.baumanii (n=30) and Acinetobacter genomospecies 3 (n=30) were tested and were all correctly identified byMALDI-TOF MS. In contrast to a previous report [28],our study demonstrated that A. baumanii and Acineto-bacter genomospecies 3 were correctly identified byMALDI-TOF MS and suggests the use of a score cutoffof 2.0 for a secure species identification of Acinetobactergenomospecies 3.

We have shown that most clinically relevant enter-obacteriaceae and non-fermenting bacteria were not onlyconcordantly identified (compared to automated identifica-tion methods using a single direct smear preparation,similar to previously described studies [6, 8, 19]), but,also, that, in some cases, species-specific score cutoffvalues could enhance the rate of correct identifications.

Obligate anaerobic bacteria identified by the ANC card

Many strict anaerobic bacteria, such as Bacteroides spp.,were all concordantly identified, as seen in previous studies[15, 29], but with variable mean score values (Table 5).Nevertheless, our study argues for the use of species-specific score values in many species to improve identifi-cation, even in the genus of Bacteroides spp., where theidentification has been described as reliable [30]. We couldshow that the mean score values of common and clinicallyrelevant, strict, anaerobic bacteria significantly variedbetween species. Our study indicates that certain strictanaerobic organisms, such as members of the genusPeptostreptococcus spp. and Prevotella spp., and speciessuch as Porphyromonas asaccharolytica and Finegoldiamagna, were concordantly identified but with lowcorresponding score values. This result also indicates thatour suggested species-specific score cutoff values should beused for the identification instead of the given score cutoffvalues in these bacteria. Further studies with increasedsample numbers of the above mentioned species should becarried out in order to validate reasonable cutoff values fora probable species identification. A comparison of theaccuracy of the identification of different databases alsorevealed that some strict anaerobic species were difficult toidentify [29, 31].



Yeasts

Candia spp. were all correctly identified with high scorevalues (Table 6).

We could show that the use of a direct smear preparation,even for molds, in addition to the use of species-specificscore cutoff values, could enhance the identification ofcommon and clinically relevant Candida species. Theaccuracy of the MALDI-TOF MS-based identification washigh and similar to previous results [32, 33].

Interpreting results obtained by the Biotyper 2.0 database

During this study, several cases of major discordant results(genus or discordant family) were observed. In most of

these cases, two different bacteria were detected on the agarplate. If mixed bacterial cultures are not observed duringthe preliminarily identification, a major discordant resultcan be obtained. In addition, a control of the cleaningprocedure is very important. If the targets were notcorrectly cleaned from bacterial proteins (data not shown),according to protocols given by the manufacturer, thendiscordant (falsely positive) results were seen. In addition,if other proteins are prevalent in applications, such as adirect identification from positive blood culture bottles, thecomparison of the detected peaks to the reference databasecan produce false-positive identification results [10]. Inaddition, if mixed cultures are tested, only one species wasidentified by the identification algorithm. This identifica-tion score can be up to a score which is usually thought to

Table 5 Suggested species-specific score values for selected clinically relevant bacteria identified by the ANC identification card


Referencemethod

Samplenumber,n




Min.score

Meanscore

Max.score


Bacteroides fragilis ANC 27 10 17 0 2.03 2.25 2.4 2.00

Bacteroides ovatus ANC 10 0 1 9 1.54 1.85 2.1 1.70

Bacteroides vulgatus ANC 11 0 10 1 1.92 2.15 2.33 1.90

Bacteroides thetaiotaomicron ANC 7 0 5 2 1.91 2.08 2.24 1.90

Bacteroides uniformis ANC 2 0 2 0 2.05 2.13 2.21 *

Parabacteroides distasonis ANC 3 0 0 3 1.72 1.81 1.92 1.70

Propionibacterium acnes ANC 7 0 0 7 1.56 1.76 1.96 1.60

Eubacterium brachii 16 S 3 0 0 3 1.83 1.92 1.99 1.80

Clostridium perfringens ANC 15 1 14 0 2.08 2.22 2.35 2.10

Prevotella bivia ANC 4 0 2 2 1.81 1.9 2.05 1.80

Prevotella buccae ANC 2 0 1 1 1.76 2 2.24 *

Peptostreptococcus anaerobius ANC 2 0 0 2 1.93 1.94 1.96

Finegoldia magna ANC 2 0 0 2 1.56 1.57 1.6 *

Fusobacterium nucleatum ANC 2 0 0 2 1.59 1.64 1.68 *

Fusobacterium necrophorum ANC 2 0 1 1 1.95 2.09 2.13 *


Table 6 Suggested species-specific score values for selected clinically relevant bacteria identified by the YST identification card

Species concordant tothe reference method

Referencemethod

Samplenumber,n




Min.score

Meanscore

Max.score


Candida albicans YST 14 2 11 1 1.94 2.13 2.43 2.05

Candida glabrata YST 18 7 11 1 1.91 2.27 2.39 2.1

Candida tropicalis YST 6 1 4 1 1.96 2.12 2.3 2.05

Candida krusei YST 4 0 4 0 2.12 2.1 2.23 2.1

Candida parapsilosis YST 2 0 2 0 2.2 2.22 2.25 *




be excellent [10]. For more than 95% of the measurements,a single spot testing was accurate compared to anautomated identification by the Vitek 2 system (Table 2).If the best score out of several replicates was used for theidentification and the samples were prepared by a formicacid preparation protocol, slightly higher score values couldbe obtained in many species (data not shown). Recently, astraightforward modified sample preparation procedure hasbeen published in order to improve the gained score values[34]. Similar to others [7], our study can show that the useof a direct smear preparation is sufficient in accuracy. Inaddition, our study is the first to indicate that even areplicate testing is not necessary. Nevertheless, our studyand others’ indicate that a MALDI-TOF MS-based speciesidentification should be compared to previously gainedpreliminarily identification results, such as typical colonymorphology, selective agar plates, Gram staining, andcatalase or oxidase activity. Since some of these importantpathogens, such as Francisella spp., Brucella spp., andBurkholderia (Pseudomonas) pseudomallei, have been notincluded in the Biotyper 2.0 database, but are included in aspecial independent database of organisms relevant forsecurity purposes, the sole use of the Biotyper 2.0 databasecannot rule out such pathogens. Our study indicates theretesting of unexpected results. If suspected organismsrelevant for security purposes have to be ruled out, otheridentification tools or databases should be used for theidentification. Moreover, organisms relevant for securitypurposes should not be tested using our direct protocol infavor of an extraction protocol which uses non-viablebacteria [9], since aerosols of viable bacteria couldcontaminate the instruments and potentially infect labora-tory employees. On the other hand, in most cases, MALDI-TOF MS-based identification will give an accurate speciesidentification. In many species, this identification issuperior to the classical biochemical identificationapproaches [7, 9, 19]. A high amount of latex beadagglutination-negative S. aureus isolates have recently beendescribed [35]. The use of such fast agglutination-basedtests for the identification of suspected S. aureus isolatesshould, thus, be reconsidered and a MALDI-TOF MS-based identification should be favored instead. This shouldespecially be done when local agglutination negative strainsare prevalent. Whether the whole-bacteria MALDI-TOFMS is also suitable for the detection of certain pathogenicityfactors, such as the PVL toxin [36], or not [37, 38] should beinvestigated in further detail. Sometimes, the accuratespecies identification by MALDI-TOF MS can influenceand even confuse laboratory work flow, since, in manyclinical diagnostic laboratories, often, groups of severalspecies were included in one “simplified” species groups,such as P. aeruginosa group and E. cloacae complex, and arare group member could imply a misidentification of the

species level. Sometimes, the same species has been treatedas “something” different (as happened for E. coli andShigella dysenteriae [39]). This study indicates that aMALDI-TOF MS-based identification is a suitable surro-gate for biochemical identification in clinically routinelaboratory services. Moreover, our study suggests a varietyof species-specific score cutoff values in order to improvespecies identification for a variety of routinely isolated,clinically relevant bacteria.

Conclusion

Our data indicates that a whole-cell bacterial identificationusing the Biotyper 2.0 database, a direct smear preparation,and a single spot testing is a fast and accurate identificationalternative for the bacterial identification of many bacterialspecies encountered in medical microbiological laboratoryservices. In addition, our study clearly indicates that alaborious sample preparation using a formic acid extractionprotocol and replicate testing is not necessary in order togain results equivalent to an automated, biochemicalidentification system. Nevertheless, our study also arguesagainst the use of this technology, where there is notaccompanying microbiological expertise. The results of ourstudy suggest using species-specific score cutoff valuesinstead of general score cutoff values of 2.0 or 2.3 given bythe manufacturer. The use of these cutoff values couldenhance identification accuracy and will help to avoidunnecessary retesting for a variety of clinically relevantisolates with a lower score value.

Acknowledgments We thank Gurpreet Khaira (Vancouver, Canada)for the linguistic advice.

Conflict of interests The authors certify that there is no actual orpotential conflict in relation to this article.

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