Urban Particles increased Streptococcus pneumonaie colonization to human middle ear epithelia cells

Background: Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism underline is not well understood. In this study, we evaluated the effect of urban particles (UP) on S. pneumoniae in-vitro biofilm formation, colonization on Human middle ear epithelium cells (HMEECs) and in mouse nasal cavity and transition to middle ear and lungs. Methods: S. pneumoniae in vitro biofilms and planktonic growth was evaluated in metal ion free medium in presence of UP, and biofilms were quantified by CV-microplate assay, cfu counts and resazurin staining. Biofilm structures were analyzed using scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs was evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected by EZcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization were evaluated using microarray. In vivo colonization of pneumococci in presence of UP and transition to middle ear and lungs were evaluated using intranasal mice colonization model. Results: UP exposure significantly (*p< 0.05) increased pneumococcal in vitro biofilms and planktonic growth. In presence of UP pneumococci formed organized biofilms with matrix, while in absence of UP bacteria was unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilms formation and quorum sensing were up-regulated in pneumococci biofilms grown in presence of UP. The HMEECs viability was significantly (p<0.05) decreased and bacteria colonization was significantly (p<0.05) elevated in co-treatment (UP+S. pneumoniae) in compare to single treatment. Similarly, increased apoptosis and ROS produce were detected in HMEECs treated with UP+ pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, immune response were up-regulated in co-treatment, those genes were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. In vivo study showed increased pneumococcal colonization to nasal cavity in presence of UP and higher transition of bacteria to middle ear and lungs in presence of UP.

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Yadav MK, Go YY, Jun I, Chae SW, Song JJ