RISA–HPLC analysis of lung bacterial colonizers of cystic fibrosis children

Microbiological analysis of sputum samples, from children affected by cystic fibrosis (CF) and showing signs of acute or chronic infections, is routinely performed by culture-dependent approaches involving selective media and biochemical tests. These identification schemes are time-consuming, and may lead to false negative results. The aim of this work was to evaluate the efficacy of a Ribosomal Intergenic Spacer Analysis (RISA) coupled to high performance liquid chromatography (HPLC) for the detection and monitoring of CF lung microbial colonizers including Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, the Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. These RISA–HPLC analyses were performed over a 10-months period on infants (below 18 months) and children that were or were not yet known to be colonised by P. aeruginosa. The RISA–HPLC profiles were found specific of the patients' microbial communities. A specific P. aeruginosa RISA–HPLC peak corresponding to 550 bp PCR products was recorded, and used to investigate P. aeruginosa persistence through time and after various therapeutic treatments. The RISA–HPLC profiles showed the CF children to be colonized by few bacterial species, and sometimes revealed peaks corresponding to bacterial species that were not detected by the selective plating approaches. Significant RISA–HPLC infra-specific variations were observed for most bacterial colonizers of CF lungs except P. aeruginosa. These species could yield as much as 5 distinct RISA–HPLC peaks, with some of these profiles being strain-specific. RISA–HPLC shows a great potential for revealing colonization by novel emerging pathogens, and for evaluating the efficacy of therapeutic treatments on the global bacterial community of CF lungs.