Antibiotic modulation of mucins in otitis media; should this change our approach to watchful waiting?

Abstract

Background: Gel-forming mucins (GFMs) play important roles in otitis media (OM) pathogenesis. Increased mucin expression is activated by pathogens and proinflammatory cytokines. Bacterial biofilms influence inflammation and resolution of OM and may contribute to prolonged mucin production. The influence of specific pathogens on mucin expression and development of chronic OM with effusion (OME) remains an area of significant knowledge deficit.

Objectives: To assess the relationship between GFM expression, specific pathogens, middle ear mucosal (MEM) changes, biofilm formation, and antibiotic utilization.

Methods: Mixed gender chinchillas were inoculated with nontypeable Haemophilus influenzae (NTHi) strain 86028NP or Streptococcus pneumoniae (SP) strain TIGR4 via transbulla injection. Antibiotic was administered on day 3-5 post inoculation. GFM expression was measured by quantitative PCR. Biofilm formation was identified and middle ear histologic changes were measured.

Results: SP infection resulted in higher incidence of biofilm and ME effusion compared with NTHi infection. However, NTHi persisted in the ME longer than SP with no substantive bacterial clearance detected on day 10 compared with complete bacterial clearance on day 10 for 50-60% of the SP-infected chinchillas. Both infections increased MEM inflammatory cell infiltration and thickening. NTHi upregulated the Muc5AC, Muc5B and Muc19 expression on day 10 (p = 0.0004, 0.003, and 0.002 respectively). SP-induced GFM upregulations were trended toward significant. In both NTHi and SP infections, the degree of GFM upregulation had a direct relationship to increased MEM hypertrophy, inflammatory cell infiltration and biofilm formation. Antibiotic treatment reduced the incidence of ME effusion and biofilm, limited the MEM changes and reversed the GFM upregulation. In NTHi infection, the rate of returning to baseline level of GFMs in treated chinchillas was quicker than those without treatment.

Conclusions: In an animal model of OM, GFM genes are upregulated in conjunction with MEM hypertrophy and biofilm formation. This upregulation is less robust and more quickly ameliorated to a significant degree in the NTHi infection with appropriate antibiotic therapy. These findings contribute to the understanding of pathogen specific influences on mucin expression during OM pathogenesis and provide new data which may have implications in clinical approach for OM treatment.

Keywords: Antibiotic treatment; Gel-forming mucins; Otitis media.

Figure 1.. Bacterial counts recovered from chinchilla middle ear cavities.

The viable NTHi-86028NP and SP-TIGR4 were detectable in the middle ear of infected chinchillas up to day 17 post inoculation. NTHi-86028NP displayed some clearance from chinchilla ME on day 17. Of the surviving SP-TIGR4 infected animals, however, demonstrated a clearance in 50% of affected chinchillas starting on day 10 with the significant lower average number of viable cells than those of day 3 and day 6. (#) indicated p<0.05 vs day 3, (*) indicated p< 0.05 vs day 6.

Figure 2.. Histologic changes in the middle ear of OM induced chinchillas.

(A). The opening access to chinchilla middle ear cavity (MEC) from top of the bulla. Blue arrow indicated gross white biofilm mass, often accompany by effusion, detected in the affected chinchilla as compared to the ME cavity without biofilm. (B). The representation image of NTHi-infected chinchilla bulla, sectioned on sagittal plane, on day 10 post inoculation. The infection caused the inflammatory cell infiltration into the middle ear cavity. MEM lined along the bone structure. Yellow tag indicated area where MEM thickness was measured. High magnification of inset (C) demonstrated MEM and significant aggregate of immune cells in exudate (blue arrow) in the middle ear cavity. (D). The change in chinchilla MEM thickness and impact of antibiotic treatment during OM. Each data point represented an average of repeated measurements. Log Transformation was applied and the Student’s t-test was used to compare the group. D3; day 3 post inoculation, D10; day 10 post inoculation, (*) indicated p<0.05.

Figure 3.. Mucin expression in the middle ear mucosa of NTHi-86028NP infected chinchillas.

NTHi-86028NP caused upregulation of Muc5AC, Muc5B and Muc19 in the ME from day 6 post inoculation. The elevated chinchilla Muc5AC, Muc5B, and Muc19 reached significant level on day 10 (P<0.05). On day 17, all GFM expression were back to baseline level in half of the animals. High variation in GFMs levels were displayed in this outbred animal model. (*) indicated p<0.05.

Figure 4.. Impact of antibiotic treatment on mucin expression in the NTHi-86028NP infected chinchilla MEMs.

When treated with ceftriaxone, NTHi-86028NP infected chinchillas MEM demonstrated significant decrease in Muc5B on day 10 compared to animals without treatment. The reduction of Muc5AC and Muc19 levels were toward significance. The rates of returning to baseline level of all GFMs were quicker in animals given antibiotic treatment. D6, D10, D17 indicated day 6, day 10, day 17 post inoculation, respectively. (*) indicated p<0.05.