Investigating bacterial interaction of Upper Respiratory Tract microbiota

Abstract

Background. The microbiome of the upper respiratory tract (URT) has received considerably less research attention than other body sites. There is potential for the URT to harbour opportunistic pathogens, such as Staphylococcus aureus, and so understanding the population dynamics at this site is important. This study aims to investigate the microbial ecology of the human upper airway with a focus on antagonism between the corynebacteria and staphylococci. Methods. Mucosal swabs were collected separately from the anterior nares and nasal turbinates of 20 healthy adult subjects. Genomic DNA extraction and PCR amplification targeting the hypervariable region (V4) of the 16S rRNA gene was conducted (MiSeq) and analysed using QIIME. In addition, nasal swab isolates were cultured and identified using near full-length sequencing of the 16S rRNA gene. Isolates identified as corynebacteria or staphylococci were typed using repetitive element sequence-based PCR. Antagonism was determined using an agar-based inhibition assay. Where antagonism was observed, further imaging was conducted using electron microscopy (FEI Tecnai 12 Biotwin). Intra-volunteer clones of both Corynebacterium pseudodiphtheriticum and Staphylococcus aureus were grown in broth co-cultures in a ratio of 1:1. Viable plating and quantification were performed at different time points (24, 48h). S. aureus colonies were assessed for morphological changes to the parent and screened for changes in genome, virulence and growth metrics. Results. Upper airway samples were generally dominated by either Staphylococcus or Corynebacterium, with relative abundance analysis suggesting a clear inverse correlation between these two genera. Ordination of amplicon datasets did not suggest inter volunteer profiles to cluster by sample site, with differences in beta diversity driven by the presence or absence of Corynebacterium spp. as the dominant genus (FDR P = 0.001). Five subjects yielded bacterial cultures (from all sampled locations) identified as S. aureus, while eight subjects yielded positive bacterial cultures (from all sampled locations) of Corynebacterium spp. A total of 23 S. aureus and 40 corynebacterial isolates were characterised using (GTG)5 rep-PCR and suggested that intra-individual strain diversity was limited, with the nares and turbinates of individuals dominated by a single clone. Despite the apparent inverse correlation between these genera, cell-cell antagonism was limited when investigated on agar. Of 920 pairwise interactions, observable zones of inhibition were only reported between a single strain of C. pseudodiphtheriticum and S. aureus. Imaging under EM revealed this effect to be bactericidal with clear lytic effects on staphylococcal cell morphology. The co-culture of isolates explanted from two separate volunteers suggested S. aureus to dominate systems within 24h incubation, resulting in a near loss of viable corynebacteria. These systems yielded S. aureus colonies with reduced colony diameter and missense mutations within the trpB gene. Conclusion. Together, these data suggest that the nasal microbiota is complex, but culturable staphylococci and corynebacteria were limited in terms of clone type. Analysis of generated nasal microbiota profiles suggested an inverse correlation in terms of relative abundance. Whilst detectable zones of inhibition were not observed in agar assays, SCVs were observed and the biological significance of this phenomenon requires further investigation.