Inquilinus limosus was characterized in 2002. Since then, colonization and infections by this opportunistic species, which usually displays a multidrug-resistant phenotype, have been increasingly reported in cystic fibrosis patients (CFPs). A mucoid phenotype of I. limosus is usually reported, but the existence of non-mucoid isolates has been recognized. Biofilms infections have emerged as a major public health concern because biofilm-growing bacteria are highly resistant to antibiotics and host immune defenses. A biofilm is a community of microorganisms encased
within a matrix of extracellular polymeric substance (EPS) and attached to a surface. Our aim was to compare the capacity of biofilm formation and EPS production of I. limosus isogenic clinical isolates with mucoid and non-mucoid phenotypes. Isolates used in this study were: 1) six sequential clinical isolates recovered from sputum samples of a pediatric CFP chronically colonized by a mucoid I. limosus between 2006-2013 denominated I. limosus MP06 (draft genome sequenced), MP07, MP10, MP12, and MP13-M (mucoid) and MP13-NM (nonmucoid).
The last two isolates were recovered from the same sputum specimen. All of them were previously characterized as isogenic isolates by BcuI and XbaI-PFGE. From cultures of MP06, a non-mucoid morphotype (MP06-NM) was recovered during this work. 2) 161-13, a mucoid isolate recovered from an hemoculture of a non-CFP, considered as a contaminant. Two reference strains, I. limosus LMG 20952 and Inquilinus sp. LMG 20953, were included. Biofilms were prepared using a static microtitre plate model. After 48 or 72h-incubation in TSB-glucose 0.2%, the
final culture density was determined by measuring the OD540. Then, plates were stained with Crystal Violet (CV) or with calcofluor white to quantify the total biomass (attached cells and extracellular matrix) and EPS production, respectively. Each isolate was evaluated by octuplicate in three different assays. There were differences in bacterial growth between sequential clinical strains, being the final culture density significantly higher in those with non-mucoid phenotype (P < 0.0001). Furthermore, mucoid strains were deficient in biofilm formation while
MP13-NM formed weak biofilms as well as Inquilinus sp. LMG 20953. In contrast, I. limosus LMG 20952 and 161-13 strains were more proficient at forming biofilms. Curiously, EPS production detected with calcofluor was very poor in both, mucoid and non-mucoid sequential strains. In the other hand, I. limosus LMG 20952 and 161-13 strains produced higher amounts of EPS (P < 0.0001). Interestingly, even though MP13-M and MP13-NM were isolated from the same sputum specimen, the isogenic non-mucoid strain showed improved growth and produced higher amounts of biofilm biomass, suggesting an adaptive strategy to the lung of the CFP. There is a need to attain greater understanding of the pathogenicity of this emergent microorganism in CFP, and thereby guide effective management.