Invasions of the oropharynx: microbiome of healthy and infected respiratory tissue

Research aiming to understand pathogens in infection is shifting rapidly towards considering not only the individual pathogen but the whole microbial community. Therefore, understanding microbial communities through exploring the key questions in community ecology, such as the relationship between diversity and stability, are relevant here also. Research has made considerable progress in characterising microbial communities of different body sites but the human oropharynx microbiome is still among the less well known despite its importance in hosting various commensal bacteria and being an important entry site for pathogenic intrusion. Determining the healthy oropharynx microbiome will allow comparison to various disease scenarios and the attributes that change a community from a healthy to diseased state. This thesis represents the most comprehensive survey of looking at the longitudinal bacterial community structure in the oropharynx. Here, analysis was done on the bacterial oropharynx microbiome composition, its natural fluctuations and stability, and relating these to the changes that occur to the microbiome before, during and after an infection. This involved initial swabbing of the oropharynx of eighteen baseline-healthy, non-smoking participants weekly for a total period of 9 months and sequencing the V1-V2 region of the 16S rRNA gene using Illumina MiSeq sequencing. This would determine the community make up that is representative of a healthy state. This was then directly compared to oropharyngeal samples taken weekly from 12 smokers within the same age range for a total period of 6 months to observe the community differences between smokers and non-smokers. Looking at the healthy participants (non-smokers) alone, the key taxa recovered were Firmicutes at phylum level and Streptococcus, Prevotella and Veillonella at genus level; these were the most abundant taxa in healthy samples. There was variation in taxa within and between participants, but this variability in microbial community structure occurred more at genus and OTU level. Variability was influenced by changes in health status, although environmental factors were also likely to play a role even though they were not investigated here. Disturbances to the oropharynx microbiome were shown in participants that had cold-related symptoms (negative for viruses) and antibiotic treatment. These communities had decreased diversity (as opposed to high diversity healthy communities) and changes in abundances of certain taxa. However, participants recovered quickly from these disturbances (within one week after the disturbance) in that the microbiome returned to a state similar in community composition prior to the disturbance. This showed the oropharynx microbiome of baseline-healthy participants to be relatively resilient and stable as samples from the same participants were similar on a weekly basis. Looking at smokers, they had distinct changes in the bacterial community of the oropharynx in comparison to non-smoking healthy participants. This included changes in abundance of taxa with increased Bacteroidetes, Proteobacteria and Actinobacteria at phylum level and Streptococcus at genus level and increased abundances in pathogenic microorganisms such as S. pneumoniae which overall affected the functions associated with the bacterial community. These communities also appeared stable (regardless of having an altered state) in that samples from smoking participants were also similar on a weekly basis, but interestingly, were only disrupted during antibiotic treatment and not during an infection from samples with cold related symptoms. Therefore this thesis provides insight into the oropharynx microbiome of healthy participants (non-smokers) and smokers. It examines the stability and resilience of the oropharynx microbiome during specific scenarios and identifies the key and important taxa in a healthy and unhealthy community. By continuing to develop this research it may be possible to identify, treat and restore respiratory diseases by examining the oropharynx microbiome through identification of taxa and functions