Development of a non-invasive murine infection model for acute otitis media

Abstract

Otitis media (OM) is one of the most frequent diseases in childhood, and Streptococcus pneumoniae is among the main causative bacterial agents. Since current experimental models used to study the bacterial pathogenesis of OM have several limitations, such as the invasiveness of the experimental procedures, we developed a non-invasive murine OM model. In our model, adapted from a previously developed rat OM model, a pressure cabin is used in which a 40 kPa pressure increase is applied to translocate pneumococci from the nasopharyngeal cavity into both mouse middle ears. Wild-type pneumococci were found to persist in the middle ear cavity for 144 h after infection, with a maximum bacterial load at 96 h. Inflammation was confirmed at 96 and 144 h post-infection by IL-1b and TNF-a cytokine analysis and histopathology. Subsequently, we investigated the contribution of two surface-associated pneumococcal proteins, the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA), to experimental OM in our model. Pneumococci lacking the slrA gene, but not those lacking the ppmA gene, were significantly reduced in virulence in the OM model. Importantly, pneumococci lacking both genes were significantly more attenuated than the DslrA single mutant. This additive effect suggests that SlrA and PpmA exert complementary functions during experimental OM. In conclusion, we have developed a highly reproducible and non-invasive murine infection model for pneumococcal OM using a pressure cabin, which is very suitable to study pneumococcal pathogenesis and virulence in vivo.

Influenza-Induced Inflammation Drives Pneumococcal Otitis Media

“…Preliminary experiments showed that a pressure increase of 40 kPa delivered an intranasally administered inoculum to the middle ear (see Fig. S1 in the supplemental material), consistent with previous studies (20). The administration of inactivated Udorn/72 to the middle ear did not induce inflammation (Fig.…”

“…We then sought to assess if virus-induced middle ear inflammation was dependent on active viral replication. Udorn/72 was thus inactivated by ␤-propiolactone (BPL) treatment, and a pressure cabin system (20) was used to deliver the virus from the nasal cavity to the middle ear of 14-day-old mice. Preliminary experiments showed that a pressure increase of 40 kPa delivered an intranasally administered inoculum to the middle ear (see Fig.…”

“…The pressure cabin was used essentially as described previously (20). Briefly, 10 l BPLinactivated Udorn/72 diluted in PBS was applied to both nostrils of anesthetized mice and the pressure was raised to 40 kPa.…”

Two DHH Subfamily 1 Proteins Contribute to Pneumococcal Virulence and Confer Protection against Pneumococcal Disease

“…Intranasal infection was performed as described above in the colonization model with the following exception: methylcellulose (1%) was added to the inoculum for all OM experiments in order to minimize leakage of inoculum to the lungs (67). Mice were placed in a supine position in the pressure cabin after infection as described previously (62). Briefly, an initial pressure rise was set at 10 kPa, and when the mouse started to regain consciousness and the first swallowing movements occurred, pressure was raised at the rate of 5 kPa per 15 s until a pressure of 40 kPa was reached, enabling the inoculum to reach the middle ear cavity.…”

“…Groups of mice were sacrificed at 24, 48, and 96 h postinfection, where mice were bled out by retro-orbital puncture, followed by cervical dislocation. The bullas enclosing the middle ears (ME) were dissected from the temporal bone and homogenized in the presence of 1 ml sterile PBS per ear, as previously described (62,67). Bacteria were also recovered from the nasopharynx by performing an NPL using 1 ml of sterile PBS, and the lungs were extracted and homogenized in 2 ml sterile PBS.…”