Dynamic in vivo mutations within the ica operon during persistence of Staphylococcus aureus in the airways of cystic fibrosis patients

Abstract

Cystic fibrosis (CF) is associated with chronic bacterial airway infections leading to lung insufficiency and decreased life expectancy. Staphylococcus aureus is one of the most prevalent pathogens isolated from the airways of CF patients. Mucoid colony morphology has been described for Pseudomonas aeruginosa, the most common pathogen in CF, but not for S. aureus. From the airways of 8 of 313 CF patients (2.5%) mucoid S. aureus isolates (n = 115) were cultured with a mean persistence of 29 months (range 1 month, 126 months). In contrast to non-mucoid S. aureus, mucoid isolates were strong biofilm formers. The upstream region of the ica operon, which encodes the proteins responsible for the synthesis of the polysaccharide intercellular adhesin (PIA), of mucoid isolates was sequenced. Spa-types of mucoid and non-mucoid strains were identical, but differed between patients. Mucoid isolates carried a 5 bp deletion in the intergenic region between icaR and icaA. During long-term persistence, from two patients subsequent non-mucoid isolates (n = 12) with 5 bp deletions were cultured, which did not produce biofilm. Sequencing of the entire ica operon identified compensatory mutations in various ica-genes including icaA (n = 7), icaD (n = 3) and icaC (n = 2). Six sequential isolates of each of these two patients with non-mucoid and mucoid phenotypes were subjected to whole genome sequencing revealing a very close relationship of the individual patient’s isolates. Transformation of strains with vectors expressing the respective wild-type genes restored mucoidy. In contrast to the non-mucoid phenotype, mucoid strains were protected against neutrophilic killing and survived better under starvation conditions. In conclusion, the special conditions present in CF airways seem to facilitate ongoing mutations in the ica operon during S. aureus persistence.

Author Summary

Staphylococcus aureus is one of the most common pathogens isolated from the airways of cystic fibrosis (CF) patients. In this study, we identified unusual mucoid S. aureus isolates in 8 of 313 (2.5%) CF patients. All mucoid isolates carried a 5 bp deletion upstream of the ica operon, which resulted in increased expression of PIA/PNAG biofilm. In three patients, mucoid isolates were recovered for extended periods up to 126 months. Surprisingly, later sequential non-mucoid isolates (n = 12) of two patients also carried the 5 bp deletion. Sequencing of the entire ica operon identified compensatory mutations in different ica genes ( icaA, icaD, icaC) in these isolates. A close relationship of these isolates and of the first mucoid and closest non-mucoid isolate without 5 bp deletion were confirmed by whole genome sequencing. Transformation with expression vectors with respective wild-type genes restored mucoidy. Mucoid isolates were protected against neutrophil killing and survived better under starvation conditions. In conclusion, the special conditions present in CF airways seem to facilitate ongoing mutations in the ica operon during persistence of S. aureus.