Comparative Analysis of Microbiome in Nasopharynx and Middle Ear in Young Children With Acute Otitis Media

Abstract

Acute otitis media (AOM) is the most common pediatric infection for which antibiotics are prescribed in the United States. The role of the respiratory tract microbiome in pathogenesis and immune modulation of AOM remains unexplored. We sought to compare the nasopharyngeal (NP) microbiome of children 1 to 3 weeks prior to onset of AOM vs. at onset of AOM, and the NP microbiome with the microbiome in middle ear (ME). Six children age 6 to 24 months old were studied. Nasal washes (NW) were collected at healthy visits 1 to 3 weeks prior to AOM and at onset of AOM. The middle ear fluids (MEF) were collected by tympanocentesis at onset of AOM. Samples were stored in Trizol reagents or phosphate-buffered saline (PBS) at −80°C until use. The microbiome was characterized by 16S rRNA gene sequencing. Taxonomic designations and relative abundance of bacteria were determined using the RDP classifier tool through QIIME. Cumulative sum scaling normalization was applied before determining bacterial diversity and abundance. Shannon diversity index was calculated in Microsoft excel. The relative abundance of each bacteria species was compared via Mann-Whitney U test. We found that the NW microbiome of children during healthy state or at baseline was more diverse than microbiome during AOM. At AOM, no significant difference in microbiome diversity was found between NW and MEF, although some bacteria species appear to differ in MEF than in NW. The microbiome of samples stored in PBS had significant greater diversity than samples stored in Trizol reagent.

Upper Respiratory Tract Microbiome and Otitis Media Intertalk: Lessons from the Literature

“…Xu et al compared the MEF microbiota during AOM episode to the NP microbiota analyzed on nasal wash (NW) samples: A significantly higher abundance of A. otitidis was detected in MEF during AOM, compared with NW in health and disease; authors concluded that the ME could harbor a resident microbiome that becomes different from NP after the onset of an infection. Moreover, NP microbiome was analyzed prior to the onset of AOM vs. at AOM onset: In line with previous data, NP microbiome during health was significantly more diverse than during AOM [60].…”

Viral Inactivation Impacts Microbiome Estimates in a Tissue-Specific Manner

“…The combined effect of phenol and guanidine isothiocyanate components of TRIzol disrupts cell membranes and denatures proteins, making this reagent effective at inactivating viruses ( 49 , 72 – 74 ). Buffers containing TRIzol are commonly used for sample storage, as TRIzol also denatures DNase and RNase enzymes, thus protecting DNA and RNA from degradation ( 75 ). However, in our study, TRIzol treatment had important effects across samples from different body sites.…”

“…In a recent study, incubation of samples containing SARS-CoV-2 with 10% TRIzol for 10 min was shown to reduce the levels of viral RNA detection by digital PCR ( 38 ). Further, it has been previously shown that microbiome samples stored in TRIzol had lower diversity than storage in phosphate-buffered saline (PBS), suggesting that this reagent may deplete part of the nucleic acids in the samples and thus may not be suitable for microbiome studies ( 75 ).…”

Nasopharyngeal carriage rates and serotype distribution of Streptococcus pneumoniae among school children with acute otitis media in Central Java, Indonesia

“…The microbiome during AOM was similar to that of the middle-ear fluid microbiome at the onset of AOM. Haemophilus , Moraxella and Streptococcus constitute the most abundant microbiota in both healthy and AOM-affected children [ 36 ]. This indicates that AOM is a consequence of the otopathogens ascending from the nasopharynx to the middle ear.…”