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Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism by which this occurs is not well understood. This study evaluated the effect of urban particles (UP) on  Streptococcus pneumoniae  (pneumococcus)  in vitro  biofilm formation, colonization of human middle ear epithelium cells (HMEECs) as well as mouse nasal cavity and its transition to the middle ear and lungs. The  in vitro  biofilms and planktonic growth of  S. pneumoniae  were evaluated in metal ion free medium in the presence of UP. Biofilms were quantified by crystal violet (CV) microplate assay, colony forming unit (cfu) counts and resazurin staining. Biofilm structures were analyzed using a 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 were evaluated using fluorescent  in-situ  hybridization (FISH), cell viability was detected using the Ezcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using the Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization was evaluated using microarray.  In vivo  colonization of pneumococci in the presence of UP and transition to middle ear and lungs were evaluated using an intranasal mice colonization model. The UP exposure significantly increased (*p < 0.05) pneumococcal  in vitro  biofilms and planktonic growth. In the presence of UP, pneumococci formed organized biofilms with a matrix, while in absence of UP bacteria were unable to form biofilms. The  luxS, ply, lytA, comA, comB  and  ciaR  genes involved in bacterial pathogenesis, biofilm formation and quorum sensing were up-regulated in pneumococci biofilms grown in the presence of UP. The HMEECs viability was significantly decreased (p < 0.05) and bacteria colonization was significantly elevated (p < 0.05) in co-treatment (UP +  S. pneumoniae ) when compared to single treatment. Similarly, increased apoptosis and ROS production were detected in HMEECs treated with UP + pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, and immune response, were up-regulated in co-treatment and were unchanged or expressed in less fold in single treatments of UP or  S. pneumoniae . The  in vivo  study showed an increased pneumococcal colonization of the nasopharynx in the presence of UP and a higher transition of bacteria to the middle ear and lungs in the presence of UP. The UP exposure elevated  S. pneumoniae in vitro  biofilm and colonization of HMEECs, and  in vivo  mouse nasopharyngeal colonization, and increased dissemination to mouse middle ear and lungs.

scientific reports

Urban Particles Elevated Streptococcus pneumoniae Biofilms, Colonization of the Human Middle Ear Epithelial Cells, Mouse Nasopharynx and Transit to the Middle Ear and Lungs