Evaluation of the Andromas MALDI-TOF MS system for identification of aerobically 1 growing Gram ...

20 Pages·2012·1.43 MB·English

JCM Accepts, published online ahead of print on 12 June 2012
J. Clin. Microbiol. doi:10.1128/JCM.00368-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Evaluation of the Andromas MALDI-TOF MS system for identification of aerobically
growing Gram-positive Bacilli
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E. Farfour1,2, J. Leto6, M. Barritault1,2, C. Barberis7, J. Meyer1, B. Dauphin6, A-S Le Guern8,
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A. Leflèche9, E. Badell10, N. Guiso10, A. Leclercq11, A. Le Monnier11, M. Lecuit1,11, V.
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Rodriguez-Nava12, E. Bergeron12, J. Raymond1,3, S. Vimont4, E. Bille,1,2 E. Carbonnelle1,5, H.
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Guet-Revillet1,2, H. Lécuyer1,2, J-L Beretti1,2, C. Vay7, P. Berche1,2, A. Ferroni1,2, X. Nassif 1,2
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and O. Join-Lambert1,2,#
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9 1 Université Paris Descartes, Paris, France lo
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2 Laboratoire de Microbiologie, Hôpital Necker-Enfants malades, 3 Laboratoire de d
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Bactériologie, Hôpital Cochin, 4 Laboratoire de Bactériologie, Hôpital Tenon, 5 Laboratoire de
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Microbiologie, Hôpital Européen Georges Pompidou, Assistance Publique - Hôpitaux de m
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13 Paris, Paris, France tt
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6 Andromas SAS, Pépinière Paris Santé Cochin, 29 rue du faubourg Saint Jacques, 75014 :/
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Paris, France m
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7 Laboratorio de Bacteriologia. Hospital de Clínicas. Facultad de Farmacia y Bioquímica. s
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17 Universidad de Buenos Aires, Argentina. .o
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18 8 Laboratoire du Centre Médical de l’Institut Pasteur, 9 Cellule d'Intervention Biologique g/
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d'Urgence, 10 Molecular Prevention and Therapy of Human Diseases, National Centre of n
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Reference of corynebacteria of the diphtheriae complex, and 11 Centre National de Référence p
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des Listeria, Institut Pasteur, Paris, France 1
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12 Research group on Bacterial Opportunistic Pathogens and Environment, UMR5557- 2
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Observatoire Français des Nocardioses, Université Claude Bernard Lyon 1, Lyon, France 1
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Running title: identification of Gram-positive rods by MALDI-TOF MS u
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Corresponding author: Olivier Join-Lambert, M.D., Ph.D.
Mailing address: Laboratoire de Microbiologie, Hôpital Necker-Enfants malades,
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149 rue de Sèvres, 75015 Paris-France
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Phone: [phone]
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Fax: [phone]
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E-mail: [email protected]
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Key words: Gram-positive rods, MALDI-TOF mass spectrometry, microbial identification
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Abstract
Matrix-associated laser desorption time of flight mass spectrometry (MALDI-TOF MS) is a
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rapid and simple microbial identification method. Previous reports using the Biotyper system
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suggested that this technique requires a preliminary extraction step to identify Gram Positive
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rods (GPRs), a technical issue that may limit the routine use of this technique to identify
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pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS
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Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and
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73 species by the direct colony method. This bacterial collection included 40 C. diphtheriae,
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8 13 C. pseudotuberculosis, 19 C. ulcerans and 270 other Corynebacterium isolates, 32 w
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9 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 lo
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Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 d
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Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other
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GPR isolates, all identified by reference techniques. m
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13 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level tt
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respectively. Except for L. grayi isolates that were identified to the species level, all other :/
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Listeria isolates were identified to the genus level because of highly similar spectra. These m
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data demonstrate that rapid identification of pathogenic GPRs can be obtained without s
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17 extraction step by MALDI-TOF mass spectrometry. .o
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Introduction
Aerobically growing Gram-positive rods (GPRs) form a very heterogeneous and extending
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group of bacterial species (23). Some of them, such as Corynebacterium diphtheriae,
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Listeria monocytogenes and Bacillus anthracis are highly pathogenic. These bacterial species
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are associated with severe community acquired infections and may be associated with
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outbreaks, thus requiring rapid identification both for therapeutic and infection control
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measures. Other clinically important pathogenic GPR species include Nocardia spp and
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Rhodococcus equi that can cause severe opportunistic infections. Corynebacterium
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8 urealyticum and Corynebacterium jeikeium are respectively associated with urinary tract w
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9 infections and vascular catheters infections, prosthetic endocarditis and septicemia and can be lo
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multidrug resistant (31, 49). Finally, during the two last decades, a number of species of low d
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pathogenicity such as Propionibacterium acnes and new actinomycetes (Actinobaculum
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schaalii, Actinomyces neuii, Actinomyces turicensis, Actinomyces radingae), have been m
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13 associated with various infectious conditions (5, 25, 37). Rapid identification of GPRs is tt
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therefore increasingly required both for therapeutic and/or epidemiological concerns. :/
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Systematic identification of GPRs is a challenge. Phenotypic identification methods are m
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accurate for identification of pathogenic GPRs. However, most commensal GPR isolates that s
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17 are frequently cultured in microbiology laboratories are uneasy to identify using these .o
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18 techniques, due to weak or variable reactivity among single species or because additional tests /
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are required in up to 50% of cases to obtain a 90% correct identification rate (22). In addition, n
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recently described species have been characterized using molecular techniques such as 16S p
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ribosomal RNA gene sequencing. These species cannot be identified using phenotypic 1
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methods in the absence of appropriate identification systems or updated databases (1, 10, 21, 2
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22, 45, 54). In some cases, 16S ribosomal gene sequencing poorly discriminates closely
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24 related species and alternative molecular methods have been developed to solve these issues y

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(28, 36, 38). u
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26 Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF t
MS) is a rapid bacterial identification technique that is increasingly used in microbiology
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laboratories (13, 30, 48). MALDI-TOF MS identification systems are based on the
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comparison of the tested isolate mass spectrum with reference databases. Several databases
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and identification strategies have been developed, including the Biotyper (Bruker Daltonics,
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Bremen, Germany), the Saramis (bioMé́rieux, Marcy l’Etoile, France) and the Andromas
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(Paris, France) systems. Previous studies reported that the Bruker MALDI-TOF mass
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spectrometry system accurately identified GPR species such as Listeria spp, Nocardia spp,
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and Corynebacterium species (3, 6, 29, 51). In these studies, a preliminary extraction step was
mandatory to obtain satisfactory results, a procedure that is more cumbersome and time
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consuming that the direct colony method that can be used to identify Gram negative bacilli
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(43). Available MALDI-TOF MS databases have also been evaluated in the routine use of
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clinical microbiology laboratories, but few GPRs were included and in some cases, poor
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identification results were reported (7, 47).
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The Andromas identification strategy is based on a limited number of species-specific profiles
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for each entry (11, 16). A particularity of the Andromas database is that it was built without
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8 any extraction step (7, 12), and previous studies have shown that this identification strategy w
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9 provides good identification of bacteria, mycobacteria, yeasts and Aspergillus spp by the lo
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direct colony method (2, 7, 11, 16, 18, 19, 32). We evaluated the accuracy of this method to d
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identify a large collection of GPR isolates that included both pathogenic and commensal
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isolates. m
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Materials and Methods
1 Bacterial isolates. A collection of 659 GPR isolates was set up for this study, originating
from French clinical microbiology laboratories and reference centers, and from the clinical
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microbiology laboratory of the Buenos-Aires University (Argentina). Altogether, this
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collection comprised 16 bacterial genera and 73 bacterial species that are listed in table 1.
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Except for Listeria, Nocardia, C. diphtheriae, C. pseudotuberculosis and C. ulcerans isolates,
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we used 16S ribosomal RNA gene sequencing as reference technique to identify clinical
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isolates. 16S rRNA gene sequences were compared with those of bacterial strains obtained
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from the GenBank or Bioinformatic Bacterial Identification database (17). A ≥ 99.0% D
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similarity cutoff was required for definite identification. w
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Listeria isolates were identified by the French National Reference Centre for Listeria with the lo
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API Listeria identification system (bioMérieux, Marcy l’Etoile, France) (8, 20, 34) and d
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additional phenotypic confirmation tests (41, 46). All Nocardia isolates were identified by the d
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French reference centre for Nocardia using a polyphasic approach including morphological o
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examination, biochemical profiling, antimicrobial susceptibility testing and 16S rRNA gene
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sequencing using specific primers designed to identify Nocardia spp (42). Partial sequencing tp
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16 of the hsp65 gene was done only for isolates identified as N. abscessus, N. arthritidis and N. /jc
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beijingensis to confirm species identification (42). C. diphtheriae, C. ulcerans and
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C. pseudotuberculosis isolates were identified by the French National Reference Centre of s
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Corynebacteria of the diphtheriae complex using the API Coryne system (bioMérieux, Marcy .
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l’Etoile, France), additional phenotypical assays and specific molecular methods (a PCR g
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targeting the dtxR gene for C. diphtheriae and a multiplex PCR targeting the 16S rRNA, rpoB o
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and pld genes for C. pseudotuberculosis and C. ulcerans) (36, 38). A
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23 Instrumentation and data analysis. Bacterial isolates were grown on 5% horse blood ril
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Columbia agar plates and incubated 24h (48h for slow growing bacteria) at 37°C in a 10% 0
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CO2 atmosphere. Whole cell bacteria were deposited without extraction step using a cotton 2
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swab on a MALDI-TOF MS target plate and allowed to dry at room temperature. Samples 1
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were then fixed by adding 1 µl of absolute ethanol to inactivate bacteria and allowed to dry at b
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room temperature. Samples were then overlaid with 1 µl of saturated alpha-cyano-4- g
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hydroxycinnamic acid and allowed to crystallize at room temperature. Samples were e
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processed on the Microflex MALDI-TOF MS spectrometer with the FlexControl software
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(Bruker Daltonics, Bremen, Germany). Positive ions were extruded at an accelerating voltage
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of 20 kV in linear mode. Each spectrum was the sum of the ions obtained from 350 laser shots
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performed automatically on different regions of the same well. The spectra were analyzed in a
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m/z range of 3,500 - 20,000.
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Evaluation of Andromas MALDI-TOF MS system for identification of GPRs
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Bacterial isolates were identified using the Andromas software, being unaware of the
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identification result of the reference method. This software compares the mass spectrum of
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the tested isolate with the spectra included in the Andromas database, taking into account a
possible MS peak variation of ± 10 m/z (7). The Andromas database was created using both
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reference and clinical isolates routinely used in our laboratory. To date, this database
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encompasses more than 700 bacterial species, including 33 genus and 156 species of
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aerobically growing Gram-positive rods. Using this software, species identification is
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considered to be valid if the percentage of common peaks is ≥ 68% of those of a species-
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specific profile of the database. A 10% difference between the first two species diagnostics
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having the best match in the database is also required to give species identification. If the
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8 latter condition is not fullfilled, the identification is considered to be correct at the genus or w
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9 group level for closely related species if the first two matches belonged to the same genus or lo
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group of bacteria. In all other cases, results are considered to be non conclusive. d
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In this study, a misidentification was defined as a discrepancy between the identification
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result given by MALDI-TOF MS and the result obtained by 16S rRNA sequencing or other m
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13 reference identification methods described above. Identifications to the genus level or absence tt
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of identification were not considered as misidentifications. :/
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Listeria species discrimination m
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Similarity study of Listeria isolates mass spectra was performed using the BioNumerics 5.1 s
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17 software (Applied Maths, Belgium) for in gel view representation and calculation of .o
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18 dendrograms. Similarities between mass spectra were calculated using the Euclidean distance. /
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Hierarchical clustering was performed using the UPGMA (unweighted paired-group method n
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with arithmetic mean) aggregation method. p
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Results
Except for Listeria isolates, 98.5% (594/603) of strains were identified to the species level
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and only 1.2% (7/603) of them could not be identified beyond the genus level (table 1).
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Correct identifications were obtained for the closely related pathogenic species
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C. diphtheriae, C. ulcerans and C. pseudotuberculosis as well as for frequently encountered
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non lipophilic commensal Corynebacterium species that are difficult to identify using
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biochemical tests: C. striatum or C. amycolatum that have been misidentified as C. xerosis
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(21), C. simulans that fits the biochemical profile of C. striatum or C. minutissimum (53), and
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8 C. aurimucosum that is difficult to differentiate from C. minutissimum (55). MALDI-TOF MS w
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methods for definitive identification as Nocardia spp, Rhodococcus equi, Erysipelothrix d
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rhusiopathiae, Actinomyces spp, Bacillus and Lactobacillus spp (9). In most cases, MALDI-
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TOF MS performed thus as well as extended phenotypic and specific molecular methods to m
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Conversely, except for Listeria grayi isolates that were identified to the species level, all other :/
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Listeria species isolates could not be identified beyond the genus level. Indeed, the mass m
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spectrum of these isolates always matched with more than one Listeria species-specific s
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17 profile of the database with a high similarity value (≥ 68% of common peaks). Moreover, the .o
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18 10% difference between the two best matchs that is required by the Andromas algorithm to /
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provide species identification was not reached. Therefore, identification was only given at the n
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genus level. The similarity analysis of the mass spectra of Listeria isolates accounts for these p
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results, showing that Listeria grayi and L. non-grayi isolates form two distinct clusters with 1
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high intrinsic similarity value (≥ 70%), but that these two clusters only share a 40% similarity 2
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value (figure 1). These findings corroborate molecular 16s and 23s rRNA phylogenetic
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24 studies, that showed that two clusters can be identified within this genus, one for L. grayi, and y

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a second for other Listeria species (14, 44). u
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26 Only 7 other GPRs were identified to the genus level, including 4 Nocardia, 2 C. t
aurimucosum and 1 A. viscosus isolates. The 4 Nocardia isolates, that belonged to 4 different
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species, were not identified to the species level because of poor mass spectra, a technical issue
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that has been previously reported for Nocardia spp without extraction step (51). The two
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C. aurimucosum isolates and the A. viscosus isolate had satisfactory mass spectra but matched
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with another species specific profile of the same genus with a less than 10% difference.
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Finally, 1 C. aurimucosum isolate was misidentified as C. striatum and 1 C. diphtheriae
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isolate could not be identified. Analysis of the mass spectrum of the C. aurimucosum isolate
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showed that it corresponded to a new specific C. aurimucosum profile. The C. diphtheriae
isolate was not identified because its mass spectrum comprised only 5 peaks, whereas species-
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specific profiles of the closely related species C. diphtheriae, C. pseudotuberculosis and C.
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ulcerans comprise a minimum of 23 peaks. In that case, a formic acid protein extraction step
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slightly improved the mass spectrum profile (13 peaks) but species identification could not be
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obtained because the two best matches in the database were too close (C. diphtheriae: 63%
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and C. pseudotuberculosis: 60%). This non-toxigenic isolate was recovered from a patient
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with bacteremia. It grew well on agar plates and was correctly identified by phenotypic and
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8 molecular assays. w
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two species display similar species-specific spectra in the Andromas database. Two additional d
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bacillus isolates were identified as B. pumulus/safensis for the same reason and because they
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cannot be identified to the species level using 16S rRNA gene sequencing. m
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Discussion
This work is the first MALDI-TOF MS study that addressed the issue of the accuracy of this
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new bacterial identification method against a fairly extensive collection of Gram-positive
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rods, including both pathogenic and commensal species. The most important result of our
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study is that the vast majority of pathogenic GPRs could be identified to the species level. In
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addition, we also accurately identified non- or low pathogenic species. As most of these
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strains were obtained from clinical microbiology laboratories and represent the GPRs that are
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most frequently recovered in this setting, our data suggest that this identification strategy can
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8 also be used to rule out pathogenic pathogens and to describe new GPRs-associated infectious w
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In this study, MALDI-TOF MS identification of bacterial isolates were obtained by the direct d
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colony method, i.e. in workflow conditions that can be routinely used in all microbiology
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laboratories. Similar results were only obtained after a preliminary extraction step using the m
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13 Bruker Biotyper system (3, 6, 29, 51). Each MALDI-TOF MS identification system has its tt
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own specificities, including the hardware itself (the mass spectrometer), the way the database :/
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was built and the algorithms used to compare the mass spectrum of a sample with those of the m
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database. Therefore, it is an uneasy task to identify the reason why better results were s
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17 obtained with the Andromas system by the direct colony method. It is well established that the .o
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18 quality of mass spectra varies according to bacterial genera. For instance, better MALDI-TOF /
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MS acquisitions (i.e. higher number of peaks and/or higher relative intensity of peaks) are n
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usually obtained for Gram-negative bacteria (except for mucoid colonies) as compared to p
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enterococci and Gram-positive rods (4). Consequently, using a MALDI-TOF database that 1
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was built with an extraction step, it is likely that identification errors will occur more 2
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frequently for Gram-negative bacteria than for Gram-positive bacteria. The Andromas
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24 database was built without any extraction step, a technical issue that probably had an y

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important impact on the quality of results we obtained. To the best of our knowledge, the way u
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26 the bioMérieux and Bruker databases were engineered has not been reported in a manuscript. t
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Our data suggest that species identification of Listeria spp by the direct colony method can
only be achieved at the genus level except for L. grayi. It has been reported that species
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identification and typing of Listeria spp can be obtained with the Biotyper system (6).
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Nevertheless, a complex extraction step and several centrifugations that may not be routinely
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implemented in routine laboratories were required to obtain these results. As L.
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monocytogenes causes almost all cases of human listeriosis, rapid identification of a GPR
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