Asian Sand Dust Particles Increased Pneumococcal Biofilm Formation and Colonization in Human Middle Ear Epithelial Cells and Rat Middle Ear Mucosa.

INTRODUCTION

Air pollutants such as Asian sand dust (ASD) and are risk factors for otitis media (OM). In this study, we evaluate the role of ASD in pneumococcal biofilm growth and colonization on human middle ear epithelium cells (HMEECs) and rat middle ear using the rat OM model.

METHODS

D39 biofilm growth in the presence of ASD (50-300 μg/ml) was evaluated in metal ion-free BHI medium using CV-microplate assay, colony-forming unit (cfu) counts, resazurin staining, scanning electron microscopy (SEM), and confocal microscopy (CF). Biofilm gene expression analysis was performed using real-time RT-PCR. The effects of ASD or individually or on co-treatment on HMEECs were evaluated by detecting HMEEC viability, apoptosis, and reactive oxygen species (ROS) production. colonization of in the presence of ASD was evaluated using the rat OM model, and RNA-Seq was used to evaluate the alterations in gene expression in rat middle ear mucosa.

RESULTS

biofilm growth was significantly ( < 0.05) elevated in the presence of ASD. SEM and CF analysis revealed thick and organized pneumococcal biofilms in the presence of ASD (300 μg/ml). However, in the absence of ASD, bacteria were unable to form organized biofilms, the cell size was smaller than normal, and long chain-like structures were formed. Biofilms grown in the presence of ASD showed elevated expression levels of genes involved in biofilm formation (, competence (, , , and toxin production ( and . Prior exposure of HMEECs to ASD, followed by treatment for pneumococci, significantly ( < 0.05) decreased cell viability and increased apoptosis, and ROS production. experiment results showed significantly ( < 0.05) more than 65% increased bacteria colonization in rat middle ear mucosa in the presence of ASD. The apoptosis, cell death, DNA repair, inflammation and immune response were differentially regulated in three treatments; however, number of genes expressed in co-treatments was higher than single treatment. In co-treatment, antimicrobial protein/peptide-related genes (S100A family, Np4, DEFB family, and RATNP-3B) and OM-related genes (CYLD, SMAD, FBXO11, and CD14) were down regulated, and inflammatory cytokines and interleukins, such as IL1β, and TNF-related gene expression were elevated.

CONCLUSION

ASD presence increased the generation of pneumococcal biofilms and colonization.