Middle ear infections (otitis media) are a significant burden on children’s health. They are one of the more common childhood ailments and are responsible for about a third of children’s general practitioner visits. Three bacteria of the nasopharynx have been well documented as true pathogens of otitis media. These are Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. A newer bacterium, Alloiococcus otitidis, which is more difficult to culture than the initial three pathogens, was discovered in 1989 by Faden & Dryja.
The pathogenicity of A. otitidis in otitis media is controversial, as is the association of two other bacteria, Turicella otitidis and Corynebacterium auris. These three bacteria are regarded as commensals of the outer ear canal, and how they move into the middle ear is subject to much debate. My hypothesis is that during a primary middle ear infection, commensal bacteria of the external ear canal may be able to move through the inflamed tympanic membrane and into the middle ear where they act as opportunistic pathogens. This may create a more persistent bacterial infection, with greater resistance to antibiotic therapies.
DNA was extracted directly from swabs of the outer ear canal and nasopharynx of children with otitis media and otitis-free controls, and from the middle ear exudates of the otitis media study group. This DNA was then amplified using primers specific for each of the organisms of interest. The presence of a specific PCR product, confirmed by DNA sequencing, indicated a positive result.
Swabs from each of the sites of interest were also grown on 10% Columbia blood agar, 10% Columbia chocolate agar and in BHI broth supplemented with foetal calf serum. These results gave further information on bacterial presence in New Zealand children beyond the six studied.
Sau-PCR microbial community profiling was used to demonstrate the diversity of species present in the polymicrobial samples. DNA was restriction digested and the resulting banding patterns were compared between study sites, and study groups.
Results and Conclusions:
Moraxella species colonised the outer ear canal of control participants more regularly than the outer ear canal of otitis media patients (58% vs. 33%). This difference was found to be statistically significant (p= 0.0023). Differences between the colonisation of the outer ear canal and nasopharyngeal body sites were found at a statistically significant level in control participants with A. otitidis (p=0.0433), C. auris (p=0.0000), T. otitidis (p=0.0050) and Moraxella spp. (p=0.0005). Of the same body sites in otitis media patients, only the colonisation of C. auris (p=0.0002) and T. otitidis (p=0.0004) were found to be statistically significant. This shows that four of the six studied bacteria colonise both anatomical sites in otitis media patients in a similar manner (without statistical significance), as opposed to only two in the control participants.
Of the six bacterial species analysed using non-culture techniques, S. pneumoniae, H. influenzae and C. auris failed to be cultured. This may show that storage prior to analysis made samples less suitable for culture based research. A number of other species were cultured, with subsequent sequencing identifying them. This included species thought to be associated with a decreased risk of developing otitis media, found in both of the control participant nasopharynxes that were studied, and in only one nasopharynx from the otitis media group. A. otitidis was cultured regularly in outer ear samples, and one middle ear effusion.
Sau-PCR microbial profiling was an effective but crude indication of microbial diversity. Otitis media patient nasopharyngeal profiles resembled each other, with resemblance extending to the middle ear effusion profile. Control participant banding patterns within sites were similar, but this resemblance did not extend to the otitis media group profiles. Comparisons between the banding patterns from known bacterial species to those of unknown polymicrobial species were useful if the identifying species had a unique banding pattern, as with A. otitidis. Sau-PCR therefore proved useful in analysing the complexities that are polymicrobial samples, however identifications of these species by cloning proved unsuccessful.
Further research using all the techniques above with increased numbers of participants is necessary to substantiate results of this study. Information on antibiotic resistance, viral presence, and biofilm status could prove useful information in helping treat infection.||