Treatment with Lysin Eliminates Colonization and Prevents the Development of Otitis Media

<div><p>(A–B) Mice were colonized intranasally with 1 × 10⁵ CFU of S. pneumoniae and seven days later were infected with influenza virus. Four hours prior to viral infection, and then again at the time of viral infection, mice were either (A) treated intranasally with 1,000 μg of Cpl-1 lysin suspended in 20 μl of enzyme buffer⁴, or (B) mock treated with 20 μl of enzyme buffer. Mice were imaged for 60 seconds daily. (A) A representative mouse mock-treated with enzyme buffer develops bilateral otitis media 24 hours after infection with influenza virus, while (B) a representative mouse treated with lysin clears the colonizing pneumococci and does not develop a secondary bacterial infection.</p><p>(C) Although colonization of mice on day 7 did not differ between the groups (<i>p</i> = 1.00), significantly fewer mice treated with Cpl-1 remained colonized 24 hours later (<i>p</i> = 0.00012), and no treated mice developed otitis media compared to mock-treated animals, in which otitis media was seen in 80% (<i>p</i> = 0.00036). An asterisk indicates a significant difference between the groups using a 2-tailed Fisher's Exact test.</p></div

Visualization of Bioluminescent Bacteria Inside Live, Anesthetized Animals Shows the Induction of Otitis Media

<div><p>(A–B) Six- to eight-week-old female Balb/cJ mice (Jackson Laboratory, <a href="http://www.jax.org" target="_blank">http://www.jax.org</a>) were colonized intranasally with 1 × 10⁵ CFU of type 19F S. pneumoniae strain ST162^19F suspended in 100 μl of sterile PBS, then seven days later either (A) infected intranasally in a volume of 100 μl of sterile PBS with 100 TCID₅₀ (doses of virus infectious for 50% of tissue culture wells) of the Mount Sinai strain of influenza virus A/Puerto Rico/8/34 (H1N1), or (B) mock-infected with sterile PBS. Mice were imaged for 60 seconds daily using an IVIS Lumina Imaging System (Xenogen, <a href="http://www.xenogen.com" target="_blank">http://www.xenogen.com</a>), and images were analyzed using Living Image software (version 2.50.1, Xenogen). (A) Pneumococcal load in the nose of a representative mouse colonized with pneumococcus remained the same after mock-infection with PBS, while (B) a representative mouse infected with influenza virus developed otitis media in the left ear.</p><p>(C) Although colonization of mice on day 7 did not differ between the groups (<i>p</i> = 0.56), significantly more mice infected with influenza developed otitis media than did mock-infected animals (<i>p</i> = 0.00053). An asterisk indicates a significant difference between the groups using a 2-tailed Fisher's Exact test.</p></div