Epidemiology of Burkholderia contaminans in cystic fibrosis lung infection by molecular typing and FTIR-based phenotyping

PABLO MARTINA; ALEJANDRA BOSCH; CONSTANZA MANNINO; ANTONIO LAGARES; JUEGEN SCHMIT; DIETER NAUMANN; OSVALDO YANTORNO

Manchester, Reino Unido.

Workshop; FT-IR Spectroscopy in Microbiological and Medical Diagnostics; 2010

University of Manchester, Manchester, Reino Unido.

Burkholderia cepacia Complex (BCC) species have the capacity to spread within populations of patients with cystic fibrosis (CF) and to cause serious epidemic outbreaks. Besides, they may also be responsible for various infections in immuno-compromised non-CF patients (1, 2). BCC bacteria used to be mainly patient-to-patient transmitted. Nevertheless, due to the implementation of strict infection control practices in hospitals and to the education of patients the transmission of bacteria among them has been significantly reduced in the last years. However, these stringent infection control measures do not prevent the acquisition of BCC organisms from industrial settings, industrial products, or the natural environments (2, 3). Therefore, the correct identification and typing of BCC organisms is essential for determining the epidemiological relationships among clinical and environmental isolates and for the implementation of successful infection control systems. Among the 17 bacterial species that have been described for BCC, in the last years, B. contaminans has been isolated from sputum cultures of CF patients, in the UK, Italy, Portugal, USA, Canada, Brazil and Argentina, and has also been recovered from environmental samples such as river water, soil, roots, animals, industrial pharmaceutical products, personal care products, and domestic products (2, 3, 4). In this work, multiple genomic typing systems including, information on strain type obtained by recA sequence type (ST) acquired at http://pubmlst.org/bcc/, the genomic profiles based on gyrB restriction fragment length polymorphism (gyrB-RFLP-PCR), and the molecular types defined by repetitive intergenic consensus PCRs (ERIC and BOX PCRs) fingerprinting patterns, were compared with whole cell FT-IR spectroscopy fingerprinting phenotyping. The study was performed with 105 B. contaminans clinical isolates (12 belonging to non-CF patients) and 10 industrial setting isolates. Bacterial Identification was achieved sequencing recA gene (one of the housekeeping genes in the Bcc multilocus sequence typing -MLST- scheme) and searching against the public MLST database the strain typing (recA ST) and species identification outcome (2). Samples for FT-IR spectroscopy analysis were prepared as previously reported under strict standardized culture conditions (growth medium, incubation time and temperature) and spectra were acquired avoiding polyhidroxy acids (such as PHB) accumulation and cell protein appendages interferences (4). Here, we report that nor molecular typing methodologies, neither FT-IR typing schemes showed evidences of similarities among most of the B. contaminans isolates recovered from industrial settings and sputum samples of CF patients. Although most of the CF clinical isolates did not show recA polymorphisms, almost 80 % were recA ST 71; PCR fingerprinting patterns (BOX-PCR) revealed a relatively high level of genotypic diversity within them showing at least 6 different genotypes. On the other hand, FT-IR spectroscopy combined with hierarchical cluster analysis, using first derivative spectra in 3 IR ranges as input data, was also capable to distinguish distinct phenotypes within the recA ST 71 CF-clinical isolates, adding even additional levels of discrimination than PCR fingerprinting.We conclude that as both PCR fingerprinting and FT-IR spectroscopy phenotyping showed excellent typeability they could be helpful tools for global epidemiological studies of B. contaminans. Particularly, FT-IR spectroscopy can be considered a simple and rapid methodology for differentiating strains in sets of isolates obtained during outbreaks, or answering questions of clonality among microbial isolates.   References (1) Drevinick et al. 2010. Clin Microbiol. 48: 1888-1891. (2) Mahentthiralingam, et al. 2008, J Appl Microbiol. 104:1539-1551. (3) Baldwin et al 2005, J Clin Microbiol 43: 4665-4673. (4) Bosch, et al.  2008, J Clin Microbiol 46: 2535–2546.