Modulation of biofilm formation by Stenotrophomonas maltophilia during chronic lung infection in cystic fibrosis patients: a longitudinal population study

Introduction The isolation of the multi-drug resistant Stenotrophomonas maltophilia from the lungs of people with cystic fibrosis (CF) is increasing. In the diseased CF lung, pathogens are exposed to a complex range of selection pressures (including host physiological factors, therapeutic antimicrobials, and competing microorganisms) that are thought to drive genetic and phenotypic diversity in the pathogen over time. In a series of studies, we found that CF S. maltophilia isolates can grow as biofilms - sessile communities inherently resistant to antibiotics and host immune response - not only on abiotic surfaces, but also on CF-derived epithelial monolayer, probably because of a selective adaptation to CF airways. The present study was carried out to understand how biofilm forming ability evolves during chronic colonization of CF lung. For this purpose, temporally isolated strains from several CF patients were comparatively evaluated for biofilm formation ability according to their clonal relatedness. Variations in biofilm formation were also related to growth rate, motility, and in vivo virulence. Materials and Methods Ninety-three S. maltophilia isolates were collected, over a 11-year period, from 10 chronically infected CF patients. A comparative genomic analysis based on Whole Genome Sequencing was performed. Pooled DNA sequencing libraries were 300 bp paired-end sequenced on the Illumina MiSeq platform. Biofilm formation onto polystyrene and growth rate were spectrophotometrically evaluated, whereas bacterial motility (swimming, swarming, and twitching) were assessed using dedicated agar media. In vivo virulence was evaluated in a Galleria mellonella model of systemic infection. Results Genome-wide SNPs analysis revealed 20 sequence types (STs), 11 of which “unknown” for a total of 47 isolates. Strains belonging to 5 STs were shared by at least two patients. Most of strains (91 out of 93, 97.8%) were able to form biofilm, although with striking differences. STs were comparable for biofilm biomass formed. In ST1002 only, biofilm amount was negatively correlated with growth rate, but positively with both swimming and twitching motilities (p<0.05, Spearman r). Biofilm formation was significantly modulated over time towards a direction (increased or decreased) depending on the patient and ST considered. Principal Component Analysis showed a positive association between biofilm biomass formed and virulence as measured in wax moth model. Conclusions Our results showed, for the first time, that the highly stressful environment such as CF lung significantly modulates the ability of S. maltophilia to form biofilm over chronic infection, depending on allelic profile and lung considered. The improved understanding of the “biological cost” this bacterium has to pay will be helpful to design new therapeutic strategies in the future.