Author details

less-degraded host DNA [56]. If the DNA within the sample contains a small number of interesting sequences in relatively high concentrations of non-interesting sequences, less sequences are often not amplified. In this case, the use of annealing inhibiting primers which overlap with the 3<sup>0</sup> end of one of the universal primers is effective [57]. The inclusion of primers to block host

As fungi contained in the intestinal tract of seabirds, Blastocladiomycota, Chytridiomycota, Entomophthoromycotina, Ascomycota, Mucoromycotina, and Basidiomycota have been detected [58, 59]. Nebela spp., Alveolata, Stramenopila, Rhizaria, Amoebozoa, Excavata, Choanoflagellatea, Glaucophyta, Cryptophyceae, Chlorophyceae, Trebouxiophyceae, Ulvophyceae, Prasinophyceae, and Mamiello-

The use of culture-independent methods for studying bird-associated microbial communities could prove invaluable in the expansion of our current knowledge. NGS targeting the 16S rRNA gene allows comprehensive clarification of the sampled bacterial communities and their associated movement with migratory birds. This methodology also is clarifying the details of bacterial communities, which are moving long distances with migratory birds. Since the 16S rRNA gene differs from 1 to 16 in the number of copies per cell depending on genus [60], the relative proportion obtained by NGS does not necessarily agree with the ratio of actual community composition, but the dominant populations can be ascertained. Applications of NGS will lead to a better understanding of the whole picture of the bacterial communities in migratory birds. Narrowing down the target bacteria using NGS will enable us to identify unknown pathogens or reveal the potential migration status of known pathogens that have not

The dynamics of individual pathogenic bacteria and drug-resistant bacteria need to be examined in detail in the future. By conducting community composition investigations in parallel with functional investigations (e.g., drug resistance), these methods will lead to an understanding of the mechanisms by which multidrug-resistant bacteria spread around the world. Addressing the current implications of birds as potential vectors of pathogenic bacteria is of great interest. Analysis of the indigenous bacterial flora of migratory birds may highlight the importance of human hygiene and the environmental significance of microbial transfer associated with natural avian migratory patterns. When wild birds are vectors of disease, it is important to identify the true source of the infectious organisms. NGS, being a cultureindependent method, will facilitate further understanding of the complexities and interactions of the genera inherently present in the avian gut and of those acquired from the environment.

This work was supported by the JSPS Grant-in-Aid for Scientific Research (C) (15K00571) and Grant-in-Aid for Scientific Research on Innovative Areas (15H05946). We thank Takashi Fujimitsu, Kenji Kataoka, and Yuina Ishimoto for technical assistance during this work.

DNA amplification can increase the number of nonhost sequences significantly.

phyceae have been detected as protozoa contained in the intestinal tract of seabirds.

8. Conclusion and future perspectives

46 Metagenomics for Gut Microbes

been noticed thus far due to methodological constraints.

Acknowledgements

Takehiko Kenzaka\* and Katsuji Tani

\*Address all correspondence to: kenzat@osaka-ohtani.ac.jp

Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan
