Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

The identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved by using phenotype-based techniques and eventually molecular tools. These techniques remain time-consuming, expensive, and technically demanding. We used a method based on matrixassisted 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 greater than 0.1 were retained. The spectra of 559 clinical isolates were then compared to that of each of the 58 reference strains as follows: 400 Pseudomonas aeruginosa, 54 Achromobacter xylosoxidans, 32 Stenotrophomonas maltophilia, 52 Burkholderia cepacia complex (BCC), 1 Burkholderia gladioli, 14 Ralstonia mannitolilytica, 2 Ralstonia pickettii, 1 Bordetella hinzii, 1 Inquilinus limosus, 1 Cupriavidus respiraculi, and 1 Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine BCC strains and one R. mannnitolilytica strain 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 from 94 to 100% of cases, respectively. Altogether, these data demonstrate that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting gram-negative bacilli.

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A study was performed to compare matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), linked to a recently engineered microbial identification database, and two rapid identification (ID) automated systems, BD Phoenix (Becton Dickinson Diagnostic Systems, France) and VITEK-2 (bioMérieux, Marcy L'Etoile, France), for the ID of coagulase-negative staphylococci (CoNS). Two hundred and thirty-four clinical isolates of CoNS representing 20 species were analyzed. All CoNS isolates were characterized by sodA gene sequencing, allowing interpretation of the ID results obtained using the respective database of each apparatus. Overall correct ID results were obtained in 93.2%, 75.6% and 75.2% of the cases with the MALDI-TOF-MS, Phoenix and VITEK-2 systems, respectively. Mis-ID and absence of results occurred in 1.7% and 5.1% of the cases with MALDI-TOF-MS, in 23.1% and 1.3% with the Phoenix, and in 13.7% and 0.9% with the VITEK-2 systems, respectively. In addition, with the latter automate, 10.3% of the IDs were proposed with remote possibility. When excluding the CoNS species not included in the databases of at least one of the three systems, the final percentage of correct results, Mis-ID and absence of ID were 97.4%, 1.3% and 1.3% with MALDI-TOF-MS, 79%, 21% and 0% with the Phoenix, and 78.6%, 10.3% and 0.9% with the VITEK-2 system, respectively. The present study demonstrates the robustness and high sensitivity of our microbial identification database used with MALDI-TOF-MS technology. This approach represents a powerful tool for the fast ID of clinical CoNS isolates.

We report eight cases of airway colonization by Geosmithia argillacea in patients with cystic fibrosis. This filamentous fungus, resembling members of the genera Penicillium and Paecilomyces, was identified by molecular analysis. All patients carried a mutation on each CFTR (cystic fibrosis transmembrane conductance regulator) allele, with at least one copy of the F508del mutation. The first isolation of this fungus occurred from F508del-homozygous patients at a younger age than in F508del-heterozygous patients. Before recovery of G. argillacea, all patients were treated with itraconazole; two of them had also received voriconazole for an Aspergillus fumigatus infection. However, antifungal susceptibility patterns showed high MICs of voriconazole for all isolates, and high MICs of amphotericin B and itraconazole for the majority of them, but mostly low minimum effective concentrations (MECs) of caspofungin. The appearance and persistence of G. argillacea in the airways were not associated with exacerbation of the disease. However, the clinical implications of G. argillacea, particularly in immunocompromised patients, remain a concern, particularly given recent observations suggesting that this fungus may also cause disseminated infections.

S. Tubiana). y Bruno Hoen and Xavier Duval contributed equally. z The members of COMBAT study group are listed at the Acknowledgments section. Contents lists available at ScienceDirect Clinical Microbiology and Infectionj o u r n a l h o m e p a g e : w w w . c l i n i c a l m i c r o b i o l o g y a n d i n f e c t i o n . c o m

Matrix-assisted laser desorption ionization–time of flight mass spectrometry is not widely used to identify bacteria directly from positive blood culture bottles (BCBs) because of overlong protocols. The objective of this work was to develop and evaluate a simple extraction protocol for reliable identification from BCBs. The 10-min protocol was applied over a 5-month period. Direct identifications on day 0 were compared with those obtained from colonies on day 1 [log(score) of ≥2]. We evaluated a range of seven log(score) thresholds on day 0 from 1.4 to 2.0 to find the lower confidence score that provides the higher percentage of direct identifications without loss of accuracy. With a log(score) threshold of ≥1.5 at day 0, our protocol allowed us to identify 80% of bacteria in 632 BCBs (96% of Enterobacteriaceae, 95% of Staphylococcus aureus, 92% of enterococci, and 62% of streptococci). At least one bacterial species of the mixture was identified in 77% of the polymicrobial samples. The rapidity and reliability of the protocol were factors in its adoption for routine use, allowing us to save up to 24 h in identifying 80% of the bacteria in the BCBs and, thus, to supply useful information to adapt antibiotic therapy when necessary. We currently provide reliable daily direct identifications of staphylococci, enterococci, Enterobacteriaceae, Pseudomonas aeruginosa, and beta-hemolytic streptococci.

The incidence-density rate of carbapenemase-producing isolates per 1000 hospital-days was low and 30-fold lower than that of carbapenem-NS isolates (0.125) and almost 300-fold lower than that of ESBL-producing isolates (1.104) in these French hospitals.