Biofilm induction by glucose and/or sodium chloride in S. aureus isolates from the airways of Spanish people with cystic fibrosis

Background: In the cystic fibrosis (CF) airways, S. aureus adapts and persists by the emergence of small-colony-variants (SCV) or mucoid phenotypes (PT) with increased biofilm-formation. Mucoid-PTs have shown to be associated with mutations in the intercellular-adhesin (ica) operon. The aim was the assessment of the SCV and/or mucoid-PTs, and the biofilm formation of a collection of S. aureus isolates from Spanish people with cystic fibrosis (pwCF). Methods: Sixty-nine S. aureus positive-samples were collected from 11 pwCF over time from a Spanish hospital. All samples were grown on Columbia-blood-agar and Congo-Red-agar (CRA). Isolates were studied by spatyping and selected ones by MultiLocus-Sequence-Typing. All isolates were examined for biofilm-formation by the microtiter-plate-assay. Glucose and/or sodium chloride were tested as possible inductors of biofilm formation. In biofilm producer isolates, a detachment assay was performed to differentiate the biofilm nature. In addition, Sanger sequencing of parts of the ica operon (icaR and the intergenic region, IGR) was performed on selected isolates. Results: By growth on CRA, some initial strains consisted of a mixture of isolates. Therefore, 99 different isolates were analyzed from the initial 69 S. aureus positive-samples. Ten isolates from 4 patients (P) were SCVs (CC398t571/t108, CC121-t1994) and 5 isolates from 2 P (MSSA-CC398-t108 and MSSA-CC5-t306) showed the mucoid-PT. In P1 (MRSA-CC1-t127), P5 (MSSA-CC8-t008; MSSA CC15-t084) and P8 (MSSA-C121-t1994; MSSA-CC7-t796) biofilm formation was induced by glucose and NaCl. As determined by the detachment assay, these biofilms were polysaccharide-dependant with only silent mutations in icaR and the IGR. In P3 (MRSA-CC398-t011), P9 (MSSA-CC398-t571) and P10 (MSSA-CC398-t108), biofilm was only induced by glucose. Remarkably, 4 mucoid isolates were detected in P10. The nature of these biofilms will be determined in further analysis. Conclusions: Since the presence of mucoid and SCV PTs in airways of pwCF are associated with more severe lung disease, the culture of these PTs should be improved in clinical microbiology laboratories. Further investigations will be carried out to determine the biofilm nature and the presence of possible mutations in the ica operon.