In early childhood, 7083 of children experience at least one episode of acute otitis media (AOM) ,. Streptococcus pneumoniae is the most common bacterial agent identified as the causative agent of these infections, although there is increasing evidence that a variety of respiratory viruses play a prominent role in the development and pathogenesis of AOM . Even though the heptavalent pneumococcal conjugate vaccine appears to be making an impact on the incidence of this disease among children in the United States, AOM (with more than 24 million diagnoses annually) remains the leading reason for physician visits and antibiotic prescriptions among preschool-aged children ,. Frequent use of antibiotics for AOM has led to a vicious cycle of diminishing returns: increased exposure has led to increasing drug resistance, which in turn makes the infections more difficult to treat, necessitating new drugs and more treatment. Recently, purified bacteriophage (phage) cell wall hydrolases, or lysins, have shown promise as novel anti-infectives due to their ability to eradicate nasal carriage of gram-positive pathogens, particularly S. pneumoniae ,. These highly active enzymes are produced by phages to disrupt the bacterial cell wall for the release of progeny phage. Here, we show that the Cpl-1 lysin, which is specific for S. pneumoniae , prevents AOM in a novel mouse model that mimics the natural pathogenesis of this common infection.
Current animal models for AOM have critical limitations. Modeling AOM in mice requires invasive and artificial procedures to establish infection, and sacrifice of the animals to determine outcomes. Larger animals such as chinchillas and ferrets may develop infection by more natural routes, but use of these models is limited by their size and complexity ,. Ideally, we wished to develop a non-invasive mouse model that was permissive of natural infection. We engineered a piliated strain of S. pneumoniae, known to efficiently colonize mucosal surfaces (a type 19F strain obtained from B. Henriques-Normark, ST16219F) , to express luciferase . Groups of five mice maintained in a BL2 facility were infected intranasally with 1 105 or 1 106 colony-forming units (CFU) of this bioluminescent strain under light anesthesia with 2.5 inhaled isoflurane using an established infection model approved by the St. Jude Children''s Research Hospital animal care and use committee . Animals were followed daily for development of infection for two weeks and thrice weekly for another four weeks. Within 72 hours of pneumococcal infection, 100 of mice (10/10) were visibly colonized with bacteria in the anterior portion of their nose, and 70 (7/10) had developed AOM. These infections of the middle ear all resolved by bioluminescent imaging within 48 hours, and no mice had evidence of AOM six days after challenge or later. Nasal colonization persisted for a median of 27 days (range 1734 days).
Around half of all children are colonized with S. pneumoniae . Alteration of eustachian tube function or disruption of mucosal surfaces through viral infection allows colonizing bacteria to ascend into the middle ear, triggering AOM . To model this phenomenon, we infected mice that had been stably colonized by pneumococcus with influenza virus and followed them for development of AOM. Although all mice had been colonized prior to infection with virus, 63 of virus-infected mice (19/30) developed AOM compared to 0 (0/10) of mice mock-infected with phosphate buffered saline (PBS) (). Twenty-one of thirty mice in the virus group had experienced AOM after introduction of the bacteria in the first 72 hours post colonization (with resolution before viral challenge), while eight of ten mice in the PBS control group had experienced AOM with resolution (unpublished data). Both de novo and recurrent infections were seen in the virus-infected mice, with no correlation to whether they had previously had AOM. This is the first mouse model of AOM in w