Characterization of Tonsil Microbiota and Their Effect on Adenovirus Reactivation in Tonsillectomy Samples

The adenoviral DNA is prevalent in adenotonsillectomy specimens from pediatric patients, though the virus seems to be in latent state. The tonsils are at the forefront of airway entry point and are the first line of defense against airway viral and bacterial infections. We hypothesized that tonsil microbiota plays a role in human adenovirus (HAdV) latency and reactivation. In this study, we surveyed the presence of HAdV in tonsillectomy samples from 81 patients and found that HAdV DNA was in 85.2% of the tonsil samples. We then determined the microbiota of the samples. Taxonomic profiling showed that Proteobacteria, Firmicutes, Fusobacteriota, and Bacteroidota accounted for approximately 70% of the total phyla in tonsil samples. A correlation analysis showed that the HAdV-positive samples had significantly higher abundance of Neisseria and Bifidobacterium and lower abundance of Streptococcus, Ochrobactrum, and Lactobacillus than that of the HAdV-negative samples. Culture-based isolation followed by 16S rRNA sequencing identified Staphylococcus aureus, Streptococcus pneumoniae, Veillonella, Prevotella, Capnocytophaga sputigena, Pseudomonas aeruginosa, Neisseria, and Moraxella catarrhalis from the samples. Gas chromatography-mass spectrometry (GC-MS) profiling of short-chain fatty acids in bacterial cultures of minced tonsillectomy tissues or representative isolates showed the cultures contained various amounts of short-chain fatty acids (SCFAs). Treatment of isolated tonsil lymphocytes with bacterial lipopolysaccharide (LPS) or with SCFAs promoted HAdV reactivation. The compounds also promoted HAdV reactivation in a xenograft model with implanted tonsil fragments. This study shows a potential interplay between tonsil microbiota and HAdV reactivation that may lead to recurrent virus infection of respiratory tract disease. IMPORTANCE Human adenovirus infection is common among pediatric patients and can be life-threatening among organ transplant recipients. Adenovirus is transmitted by close contact, but it is believed that a majority of invasive events appear to arise from viral reactivation. The human tonsil is a reservoir for virus latency and has a high prevalence of latently infected adenovirus. Also, tonsils are located at the gateway of the respiratory tracts and are commonly exposed to bacterial pathogens. Here, we uncovered adenoviral DNA-positive and -negative samples that appeared to harbor distinct distribution patterns of microorganisms. SCFAs, primary metabolites of microbiota on tonsils, could induce the adenovirus reactivation in tonsil lymphocytes, resulting in adenovirus replication and production of infectious virions. The study suggests that viral-bacterial interaction plays a role in virus reactivation from latency and could be a contributing factor for recurrent viral infection in pediatric patients.