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A matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for discrimination between molecular types of Cryptococcus neoformans and Cryptococcus gattii

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... BMC Infect. Dis. 6:4.[CrossRef][Medline]; 17. Lay, JO, Jr. 2001. MALDI-TOF mass spectrometry of bacteria. Mass Spectrom. Rev. 20:172-194.[CrossRef][Medline]; 18. Lipuma, JJ 2005. Update on the Burkholderia cepacia complex. Curr. Opin. Pulm. Med.
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   Identification of non fermenting Gram negative bacilli isolated in cystic fibrosis by Matrix assisted laser desorption ionization time-of-flight mass spectrometry 1 2 3 4 5 6 7 8 9 10 11 12 13  Nicolas Degand 1,2  , Etienne Carbonnelle 1, 2 , Brunhilde Dauphin 2 , Jean-Luc Beretti 1 , Muriel Le Bourgeois 3 , Isabelle Sermet-Gaudelus 4 , Christine Segonds 5 , Patrick Berche 1,2 , Xavier  Nassif 1,2 , Agnès Ferroni 1* . 1 Assistance Publique-Hôpitaux de Paris, Laboratoire de Microbiologie, Hôpital Necker-Enfants Malades, Paris, France, 2 Université Paris Descartes, Faculté de médecine, Paris, France , 3 Service de Pneumologie Pédiatrique , 4 Service de Pédiatrie Générale, Hôpital  Necker-Enfants Malades, Paris, France, 5 Observatoire Cepacia, Hôpital Purpan, Toulouse, France. Short title : Cystic fibrosis bacterial identification Key words : MALDI-TOF-MS, cystic fibrosis, bacterial identification, Gram negative bacilli * Corresponding author Agnès Ferroni Laboratoire de Microbiologie Hôpital Necker-Enfants Malades 149 rue de Sèvres 75015 Paris, France Tel: 33 1 44 49 49 62 Fax: 33 1 44 49 49 60 E-mail: agnes.ferroni@nck.aphp.fr  1   Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00569-08 JCM Accepts, published online ahead of print on 6 August 2008  Abstract 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 The identification of nonfermenting Gram-negative bacilli isolated from cystic fibrosis (CF)  patients is usually achieved using phenotypic based techniques and eventually molecular tools. These techniques remain time consuming, expensive, and technically demanding. We used a method based on Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting Gram-negative bacilli was used. To identify peaks discriminating between these various species, the  profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity above 0.1 were retained. The spectra of 559 clinical isolates were then compared with that of each of the 58 reference strains : 400 Pseudomonas aeruginosa ,   54  Achromobacter xylosoxydans , 32 Stenotrophomonas maltophilia , 52  Burkholderia cepacia  complex (Bcc), 1  Burkholderia gladioli, 14  Ralstonia mannitolilytica , 2  Ralstonia picketti , 1  Bordetella hinzii, 1  Inquilinus limosus, 1 C upriavidus respiraculi, 1  Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine Bcc and 1  R. mannnitolilytica  strains were identified as  belonging to the appropriate genus but not the correct species. We subsequently engineered Bcc and  Ralstonia  specific databases using additional reference strains: using these databases, correct identification for these species increased from 83 to 98% and 94 to 100 % of cases, respectively. Altogether, these data demonstrates that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting Gram-negative bacilli. 2  Introduction 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Pseudomonas aeruginosa is the main bacterial pathogen isolated from the sputum of patients suffering from Cystic Fibrosis (CF). Other non fermenting Gram negative bacilli of clinical importance that can be isolated from this location are  Achromobacter xylosoxydans , Stenotrophomonas maltophilia  or species belonging to the  Burkholderia cepacia  complex (Bcc). Other non fermenting bacteria belonging to the genus  Ralstonia or  Burkholderia  are less likely to be isolated. Recently, novel bacterial species have been isolated from CF  patients as  Ralstonia mannitolilytica  (6,10), Pandoraea   apista  (4,22),  Inquilinus limosus  (5,25). Bacteria isolated during CF do not have the same virulence, thus pointing out the importance of bacterial identification by routine clinical microbiology laboratories for the management of CF patients. Moreover, the emergence of new bacterial species requires accurate identification tools in order to understand their clinical relevance and distribution in CF. Conventional phenotypic methods and commercial kits are sometimes not suitable for strains isolated from CF patients. These pathogens often lack key phenotypic characters required for their identification (1,10,20,23,24,26). In addition, in some circumstances, misidentification is due to the fact that the species are not in the database of commercial kits (10,23). Molecular tools such as 16S rRNA gene sequencing provide reliable results (10,16,26). Other techniques such as FISH (27) and amplified ribosomal DNA restriction assays are available (22). Despite their good accuracy, these molecular techniques cannot be used routinely as they are expensive, time consuming and technically demanding. Several studies have reported the use of Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) for bacterial identification (8,9,13-15,17). MALDI-TOF-MS can examine the profile of proteins detected directly from intact  bacteria. This technique, based on relative molecular masses, is a soft ionization method 3  allowing desorption of peptides and proteins from whole different cultured microorganisms. Ions are separated and detected according to their molecular mass and charge. For a given bacterial strain, this approach yields reproducible spectrum within minute,64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 consisting of a series of peaks corresponding to m/z ratios of ions released from  bacterial proteins during laser desorption. Recently, we engineered a strategy to identify bacteria belonging to the  Micrococaceae   family (2). The aim of this present work is to extend this strategy to non fermenting bacilli recovered in CF patients, thus opening the path towards rapid, accurate and cheap means of  bacterial identification in routine laboratories. Our first step was to build a complete database for all species belonging to the group of non fermenting Gram negative bacilli recovered in human, including those isolated in CF patients. We then validated this database using the identification by MALDI-TOF MS of clinical strains recovered from CF  patients. Material and methods Bacterial strains. The reference strains used to engineer the MALDI-TOF MS database  belonged to 58 species of non fermenting Gram negative bacilli   that can possibly be recovered from CF patients. Three databases were engineered, and the 87 strains used to complete these databases are described in tables 1, 2 and 3. The tested strains used to validate the databases were : (i) 512 clinical isolates of non fermenting Gram negative bacilli recovered from the sputum of CF children attending the pediatric department of the Necker-Enfants malades hospital (Paris, France) between 01/01/06 and 31/12/06 : 400 P. aeruginosa  strains (101 patients), 54  Achromobacter xylosoxydans (12 patients), 32 S. maltophilia  (12 patients), 9  R. 4  mannitolilytica  (1 patient), 14 Bcc (2 patients ), 1  Burkholderia gladioli , 1  Bordetella hinzii  and 1  I. limosus . These strains were identified by phenotypical tests or molecular method as  previously described (10). Briefly, isolates displaying Green or Yellow-Green pigmentation,  positive oxidase test, growth at 42°C, growth on cetrimide agar and susceptibility to colimycin (disk diffusion method) were identified as Pseudomonas aeruginosa . Isolates that did not express those criteria were identified using the API 20NE system (bioMerieux, Marcy-l'Etoile, France). The results of the API 20NE tests were interpreted using the APILAB PLUS software package (bioMerieux). When the API 20NE system did not identify a P. aeruginosa  or a  A. xylosoxydans or a S. maltophilia , the identification was further  pursued by sequencing an internal fragment of the 16Sr RNA gene as previously described (10). Sixteen of the 400 P. aeruginosa  strains and 26 of the 112 non P. aeruginosa  strains required molecular methods for identification.  B. cepacia  strains were identified by 16SrDNA sequencing. Identification was confirmed by the Observatoire National des Cepacia using ARDRA (amplified rDNA restriction analysis) (21,22). Species-specific recA PCR and/or  Burkholderia cepacia  complex-recA restriction analysis was used to circumvent the limitations of ARDRA within the  Burkholderia cepacia  complex (19). 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 (ii) 47 clinical strains obtained from the Observatoire National des Cepacia (Toulouse, France): 38 Bcc strains (11  Burkholderia vietnamiensis , 10  Burkholderia multivorans , 8  B. cenocepacia , 5  Burkholderia stabilis , 1  Burkholderia dolosa , 1  Burkholderia pyrrocinia, 2  B. cepacia )  , 1  Burkholderia thailandensis, 5  R. mannitolilytica, 2  Ralstonia picketti, 1 Cupriavidus respiraculi . All bacterial strains used in the study were stored at -80°C in trypticase soy broth supplemented with 15% glycerol. MALDI-TOF-MS.  The strains were grown on Mueller-Hinton agar and incubated for 24h at 37°C. Most of the isolates grew after 24h but some strains that didn’t grow after 24h were 5
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