MALDI-TOF SPECTROMETRY FOR THE IDENTIFICATION OF BURKHOLDERIA CEPACIA COMPLEX SPECIES RECOVERED FROM PATIENTS WITH CYSTIC FIBROSIS

MINAN A; LASCH P; BOSCH; NOSEDA DIEGO GABRIEL; DEGROSSI J; MONTANARO P; YANTORNO OSVALDO; NAUMANN D

San Pablo

Workshop; Spec 2008. Shedding Light on Disease: Optical Diagnosis for the New Millennium.; 2008

Sociedad Internacional de Espectroscopía

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) is a useful technique for the identification of bacteria on the basis of their characteristic protein mass spectrum fingerprint. This new technology has emerged as a prompt identification methodology of microorganisms starting either from whole cells, cell lysates or bacterial extracts. The rapid and accurate diagnosis of respiratory infections in cystic fibrosis (CF) patients is critical not only in the early implementation of appropriate antimicrobial therapy and infection control measures, but also in epidemiological surveys. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, other non-fermenting (NF) Gram negative rods as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. can colonize CF patient lungs. Particularly the BCC is an important group of organisms whose complex taxonomy has evolved dramatically in the last years. Although BCC comprised 9 distinct species (genetic types or genomovars), recently., genetic methods such as 16 S rRNA recA gene sequence analysis and multilocus sequence typing (MLST) (2,3), have included 1 additional species to the complex and have described 5 novel BCC species and a group of bacteria preferred to as Taxon K which comprises at least two species (4,5). In routine clinical laboratories the identification of BCC members and their differentiation from the other NF rods is still a challenge due to misidentifications that often occur. The aim of the present study was to apply MALDI-ToF MS and multivariate analysis for the rapid identification of whole-cells of BCC bacteria at the species level, and their discrimination from the most common NF rods isolated from sputum samples of CF patients. We investigated 126 BCC clinical isolates (B. cepacia, B. multivorans, B. cenocepacia, B. stabilis, B. ambifaria and Taxon K bacteria), and 27 NF isolates, representing the 6 most clinically relevant NF rods (P. aeruginosa, S. maltophilia, A. xylosoxidans, Pandoraea spp, R. pickettii and Inquilinus limosus), recovered from patients hospitalized in different Cystic Fibrosis Centers and Hospitals of Argentina between 2004 and 2006. In addition 26 reference strains were analysed. The clinical isolates were phenotypically identified according to the guidelines for clinical microbiology practices for CF respiratory tract specimens. BCC bacteria were further identified by recA gene sequence analysis and the non-BCC bacteria were identified by 16S rRNA gene sequencing. MALDI-ToF spectrometry measurements were performed on 24 h-growth cultures L grown on tryptone soya agar.