The gold standard of identification by bacterial culture has
The gold standard of identification by bacterial culture has been unable to meet the demand of bacterial biotin-LC-LC-tyramide analysis, because >90% of the clinical bacteria cannot be cultured. Next-generation sequencing (NGS) provides a new technology to study the microbiota and pathogenic mechanisms.
16S rDNA sequencing is a powerful tool for researching taxonomy and phylogeny of samples from a complex microorganism or environment. The generated operational taxonomic units (OTUs) can be analyzed at seven levels: kingdom, phylum, class, order, family, genus and species. Nasidze et al. found 39 bacteria that have never been reported in the mouth through 16S rDNA sequencing. Yang et al. analyzed salivary microorganisms in 19 individuals with active caries and 26 without caries with 16S rRNA and whole genome sequencing technology. They found that the microbial changes were greater in patients with active caries.
Materials and methods
Discussion The composition of upper respiratory tract flora in the healthy individuals was checked using 16S rDNA NGS. Upper respiratory tract flora is mainly composed of Firmicutes, Proteobacteria and Actinobacteria. We obtained a similar result in that a large proportion of flora in the upper respiratory tract were in four phyla: Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. Bacteroidetes is not unexpected because it is a group that inhabits the humanoral cavity. The similar results suggest that, although the upper respiratory tract contains heterogeneous sources of air- and food-borne microbes, the normal pharyngeal microbiota in different people is stable and large perturbations usually indicate infection. We found that Proteobacteria was the dominant phylum in H1N1 virus infection. The proportions of flora correlated with hypothermia and pneumonia, such as Pseudomonadaceae, Brucellaceae and Enterobacteriaceae in Proteobacteria increased in patients within H1N1 virus infection. We confirmed that Pseudomonas in Proteobacteria was the dominant genus in patients with H1N1 virus infection. Ochrobactrum, Brevundimonas, Caulobacter, Aquabacterium and Serratia genera in Proteobacteria significantly increased. More specifically, several Pseudomonas such as P. fluorescens, P. fulva, Pseudomonas lundensis and Pseudomonas peli were dominant species in patients with H1N1 virus infection. P. fluorescens has been associated with ventilator-associated pneumonia, damage to nerve and epithelial intestinal cells, and bacteremia. Nonetheless, careful interpretation is needed as P. fluorescens genomes are highly diverse and our results also showed large variation from the reference genome (P. fluorescens Pfl-5). It is reported that, during influenza virus infection, Moraxellaceae in Proteobacteria are more abundant than in healthy controls.. In contrast, Streptococcus in Firmicutes, Acinetobacter in Actinobacteria, Haemophilus, Moraxella and Rhizobium in Proteobacteria decreased in the G1 group. Streptococcus decreased significantly during H1N1 infection, which is in accord with Greninger et al. The increase in S. pneumoniae was not found in patients with H1N1 virus infection. In Non-H1N1 influenza infection, Pseudomonas of Proteobacteria was also the dominant genus. Proportions of Brevundimonas, Caulobacter, Aquabacterium, and Serratia in Proteobacteria, and Dolosigranulum in_Firmicutes also increased. Normal microbiota including Neisseria, Prevotella, Veillonella, Actinomyces, Porphyromonas and Streptococcus decreased in patients with Non-H1N1 influenza infection. We did not find any significant difference in P. aeruginosa between patients with H1N1 virus infection and healthy individuals. We did find a significant increase in P. aeruginosa in patients with Non-H1N1 influenza. The background of the immune dependent on needs further investigation in the future. In addition, there were two patients (S004 and S005) at an early stage of infection in the present study. Both of them only had symptoms of upper respiratory tract infection, but the bacterial compositions were similar to those in healthy controls. This indicates changes in the composition of dominant microflora with disease progression and influence by environmental factors, such as age, diet and antibiotic treatment. These data show that H1N1 virus infection perturbs the respiratory microbiome; presumably through its induction of host immune response. This tends to select flexible species capable of sensing and responding to a rapidly changing ecosystem, and translocating to the lower respiratory tract, which might result in secondary microbial infection. Microbial metagenomic analysis might help to define better the role of microflora during upper respiratory tract infection and lay a foundation to better safeguard human health.