DOI: 10.6084/m9.figshare.26600054.v1
title: Mice fatal pneumonia model induced by less-virulent <i>Streptococcus pneumoniae</i> via intratracheal aerosolization

description: <b>Aim:</b> Animal models of fatal pneumonia caused by <i>Streptococcus pneumoniae</i> (<i>Spn</i>) have not been reliably generated using many strains of less virulent serotypes. <b>Materials & methods:</b> Pulmonary infection of a less virulent <i>Spn</i> serotype1 strain in the immunocompetent mice was established via the intratracheal aerosolization (ITA) route. The survival, local and systemic bacterial spread, pathological changes and inflammatory responses of this model were compared with those of mice challenged via the intratracheal instillation, intranasal instillation and intraperitoneal injection routes. <b>Results:</b> ITA and intratracheal instillation both induced fatal pneumonia; however, ITA resulted in better lung bacterial deposition and distribution, pathological homogeneity and delivery efficiency. <b>Conclusion:</b> ITA is an optimal route for developing animal models of severe pulmonary infections. <i>Streptococcus pneumoniae</i> (<i>Spn</i>), a type of bacteria, can cause serious illness and death in otherwise healthy people. One way that we study pneumonia is using animals. However, pneumonia in animals infected with <i>Spn</i> in the laboratory does not mimic that in humans very well. To study this illness, we need a new way to set up a proper animal model. This study set up a method called intratracheal aerosolization (ITA). In ITA, bacteria can form small droplets called aerosols and reach the deepest parts of a mouse’s lung. ITA can cause deadly illness in mice infected with <i>Spn</i>, even if the mice are healthy. The ITA method could be a useful tool to set up animal models of serious pneumonia with less virulent bacteria. <i>Streptococcus pneumoniae</i> (<i>Spn</i>) is the most commonly isolated bacterial pathogen in severe community-acquired pneumonia. Many serotypes of <i>Spn</i> do not cause reliable pulmonary infections and deaths in animal models through intranasal instillation (IN), the most frequently used pneumonia modeling method. Intratracheal instillation (ITI) can inoculate &gt;99% pathogens into of the low respiratory tract of animals but it often causes unilateral pneumonia, stress, hypoxia and even death in mice. Intraperitoneal injection (IP), the common systemic infection method, is possible to induce secondary pneumonia, but there is a lack of detailed research. Intratracheal aerosolization (ITA) delivers pathogens directly into the lungs in the form of aerosol particles, enabling a precise dose and widespread lung distribution, but it is rarely used in <i>Spn</i> infections. <i>Spn</i> strain ATCC 33400, which belongs to serotype 1 (S1) was used in this study. The <i>Spn</i> aerosol conducted by Micro Sprayer was measured by volume, bioactivity and aerodynamic particle size distribution. The IN, ITI, ITA and IP group mice were compared by the survival, local and systemic bacterial spread, pathological changes and inflammatory responses. The micro sprayer produces S1 aerosol with fine particle size, full bioactivity and precise dose. S1 is a highly invasive but less virulent strain of <i>Spn</i>, and only pathogenic to the primary infection organ (the lung) and blood-filtering organs (the liver and spleen). IN cannot achieve satisfied mortalities (50%) even under the high-dose (10<sup>7</sup> CFU) S1 challenge, and causes transient bacteremia with no sequelae under the medium-dose (10<sup>5</sup> CFU) S1 challenge. The two intratracheal routes (ITA and ITI) lead S1 deposition in the alveoli and improve the mortalities to 100% under the medium-dose (10<sup>5</sup> CFU) S1 challenge, and both present as lobar pneumonia. ITA presents better lung bacterial distribution, pathological homogeneity and animal-to-animal reproducibility compared with ITI. IP fails to develop secondary pneumonia but causes fatal abdominal sepsis. ITA induces more severe injuries and inflammatory responses in the lungs than in the blood and <i>vice versa</i> for IP. Necessary conditions for developing fatal <i>Spn</i> pneumonia are as below: enough pathogen evade mucociliary clearance and enter alveoli; abundant proinflammatory cytokines TNF-α and IL-1β are secreted. ITA makes the less virulent S1 strain colonize the mice’s alveoli massively and dispersedly, and develop a representative fatal pneumonia. ITA might be a promising methodological approach for improving the virulence of less-virulent and avirulent strains in the lungs and developing animal models of pneumococcal pneumonia, providing a valuable tool for the study of <i>Spn</i> pathogenic mechanisms and therapeutic interventions.