(536) Not only 5bp-deletions in the intergenic region of the ica operon, but also mutations in icaR Staphylococcus aureus residing in the airways of people with cystic fibrosis

Background: Staphylococcus aureus has been identified as a major respiratory pathogen isolated from the airways of people with cystic fibrosis (pwCF). It is well-known that persistent bacterial colonization/infection of the lungs allows adaption of this organism to the CF-environment. However, less is known about the presence of S. aureus with a mucoid phenotype in CF airways. Recently, we recovered S. aureus isolates with mucoid phenotypes (PTs) from respiratory specimens of pwCF, that constitutively overproduced biofilms consisting of polysaccharide intercellular adhesin (PIA), which was due to a 5bp-deletion (5bp-del) in the intergenic region (IGR) of the intercellular adhesion (ica) locus [1]. Neither the prevalence nor a possible impact of such mucoid S. aureus PTs on lung disease is known so far.

Methods: We initiated a prospective multicenter study to determine the prevalence of mucoid S. aureus in pwCF with chronic S. aureus colonization/infection. Respiratory specimens were sent to the central laboratory in Münster, Germany, and streaked on Columbia blood (CB) and CB agar with colistin and nalidixic acid (CNA), which suppresses growth of Gram-negative rods. From these plates, 10 S. aureus isolates, which were supposed to represent all different PTs, were picked and sub-cultured on CB and Congo Red (CR) agar, which allows to distinguish mucoid from non-mucoid S. aureus [1]. All isolates were tested for biofilm formation in a static 96-well plate assay and were subjected to spa-sequence typing and to sequencing of the IGR to screen for the 5bp-del. If no mutation in the IGR was observed in mucoid isolates, the entire ica-operon was sequenced.

Results: Thirteen German and one Austrian CF centers recruited 611 pwCF. Mucoid S. aureus strains were isolated from 41 of 435 S. aureus positive specimens (9.43%). So far, spa-typing was performed for 835 S. aureus isolates of pwCF with mucoid isolates with t091 being the most prevalent spa-type (12%). Most mucoid isolates were high biofilm producers. Until now, the analysis of the 5bp-del was performed for isolates of 19 pwCF and was detected in mucoid S. aureus isolates of 9 pwCF from 7 different centers, while the 5bp-del could not be determined in mucoid isolates of 10 pwCF. So far, unrecognized mutations in icaR, the repressor of the ica-operon, were identified in mucoid isolates of 6 patients from 6 centers. Mucoid isolates of 6 patients carried different mutations in icaR, while mucoid isolates of 6 patients neither carried the 5bp-del nor mutations in icaR.

Conclusions: Using more intense culture conditions, we were able to identify an unexpected high prevalence of mucoid S. aureus PTs in pwCF. No specific S. aureus clone was associated with mucoidy. In about 50% cases, the 5bp-del was identified indicating that this mutation is common during chronic S. aureus infection in pwCF. In addition, we identified new mutations in icaR and other so far unidentified mutations, which most likely lead to mucoidy of S. aureus and which will be elucidated in future studies.

Acknowledgements: Ethical vote 2017-457-f-S; ClinicalTrials.govNCT04171583
Funded by the German Research Foundation KA2249/7-1

References: 1. Schwartbeck B, Birtel J, Treffon J, Langhanki L, Mellmann A, Kale D, et al. Dynamic in vivo mutations within the ica operon during persistence of Staphylococcus aureus in the airways of cystic fibrosis patients. PLoS Pathog. 2016;12(11):e1006024; doi: 10.1371/journal.ppat.1006024 [doi];PPATHOGENS-D-16-01700 [pii].