**1. Introduction**

The application of DNA sequence technology covers a wide range of fields. As next-generation sequencing (NGS) has progressed, it has become more widely used in an array of practical applications [1, 2]. One direction of use includes the field of precision medical care. In cases of cancer caused by genetic mutations, molecular targeted drugs can be discovered by investigating gene mutations. In addition, the application of NGS is advancing in industrial fields; for instance, genotyping of animals and plants can be performed at low cost and the genetic markers can be screened with NGS.

NGS enables profiling of complex microbial communities in nature as well as that of indigenous microbiota associated with living organisms [3, 4]. Higher forms of life coexist with huge numbers of microorganisms, including bacteria, viruses, fungi, and, in some cases, protozoa and parasites—albeit bacteria are the most important microorganisms in terms of their numbers and host interactions. There are as many viruses as numbers, but many of them are viruses (phages) that infect bacteria.

Bacteria are present on the surface bodies of living organisms, such as their skin, gastrointestinal tracts, respiratory systems, and oral cavities, and they are colonized with an inherent balance in each place. This balance constitutes a stable complex ecosystem through crosstalk between bacteria and hosts. Among these places of localization, the digestive tract has the most abundant localization in terms of both the number and the type. In humans, 90% of established bacteria inhabit the digestive tract.

The intestinal microbial community includes not only enormous numbers and types of microorganisms but also active metabolic activity. The genes of the intestinal microbial community are present in proportion of at least 100-fold more than that of the genome of the host and various metabolites are produced, which are absorbed into the host body. NGS is now an indispensable item in the study of intestinal microbial communities and is applied not only to humans but also to other living organisms, such as domestic animals, insects, poultry, and wild birds [5].

Understanding the transboundary movement of microorganisms is an important requirement from the perspective of public health and environmental science [6, 7]. Microorganisms travel geographically over distant areas on the earth via ocean currents and atmospheric movement. In addition, migratory birds carry pathogenic microorganisms when traveling over long distances to several parts of the world [8–10]. Numerous pathogenic bacteria, such as pathogenic *Escherichia coli*, *Salmonella* spp., *Listeria monocytogenes*, *Pseudomonas aeruginosa*, *Botulinum* spp., *Listeria* spp., and *Campylobacter jejuni*, have been isolated from bird feces [11–13].

Research efforts have been performed to identify the bacterial communities contained in the feces of migratory birds (Bar-headed goose, shorebirds, swallow, etc.) by a novel culture-independent method [14, 15]. We attempted to explore the stability of the intestinal bacterial communities in migratory birds, the difference in the intestinal bacterial communities among birds at the individual and species levels, and the potential of long-distance movement of antibiotic-resistant bacteria associated with migratory birds [16–19]. Research on the spread of bacterial populations over vast distances has led to the elucidation of the roles of migratory birds regarding human health risks, thereby enabling the prediction of potential outbreaks based on their migratory patterns. NGS is useful for understanding bacterial diversity and for discovering novel bacteria [20]. The present review considers the potential role of wild birds in the transmission of intestinal microbiota, including antibiotic-resistant microorganisms, and our current knowledge of microbiota associated with migratory birds using NGS technologies.

### **2. Methodology for analysis of bacterial community composition**

Since the scientific study on bacteria began in the 19th century, pure culture methods supported the progress of microbiology in a wide range of fields such as medicine, pharmacy, biology, agriculture, and fermentation engineering. In the latter half of the 1970s, however, the method of total direct counting was developed, in which bacteria were stained with fluorescent dye and directly observed or counted under a fluorescence microscope. The use of this method revealed that several environmental bacteria cannot yet be cultivated by the conventional laboratory techniques [21, 22]. Therefore, new bacterial detection methods that were independent of culturing began to be developed in succession [23–26]. From the latter half of the 1990s, the method to directly extract DNA from the sample without culturing the bacteria and using a universal primer to target the conserved region of the 16S rRNA gene or PCR amplification with genus-specific primers to decode the DNA sequence became widespread [15, 27, 28]. Since the obtained gene information

#### *Dissemination of Intestinal Microbiota by Migratory Birds across Geographical Borders DOI: http://dx.doi.org/10.5772/intechopen.82707*

depended on the number of bacterial clones that the researcher could handle at a time, these methods were found to be limited to about tens to thousands.

In the past few years, comprehensive analysis of DNA sequences using NGS has spread rapidly [5]. NGS is a powerful fundamental technology that is capable of concurrently determining the nucleotide sequences of tens of millions to hundreds of millions of DNA fragments. It is also capable of advanced and high-speed processing, such as multiple determinations of multiple samples. Moreover, the expenditure on equipment and operations for this method has also been reduced. The use of NGS can help acquire genetic information of tens of thousands to hundreds of thousands of bacterial species in a short time. NGS can also aid in the understanding of the entire picture, thereby enabling a greater focus on specific interesting bacteria based on the phylogenetic taxonomic information. This, in turn, would lead to further qualitative and quantitative analyses in detail.

Because 16S rRNA gene contains both highly conserved regions for primer design and hypervariable regions to identify the phylogenetic characteristics of microorganisms, 16S rRNA gene sequence has become the most widely used marker gene for profiling bacterial communities [29, 30]. Full-length 16S rRNA gene sequences consist of nine hypervariable regions that are separated by nine highly conserved regions [31, 32]. Study with bioinformatics tools attempted to evaluate the phylogenetic sensitivity of the hypervariable regions in comparison with the corresponding full-length sequences and revealed that the V4–V6 regions represented the optimal subregions for bacterial phylogenetic studies of the new phyla [33]. Since the 16S rRNA gene differs from 1 to 16 in the number of copies per cell depending on the genus [34], the relative proportion obtained by NGS does not necessarily agree with the ratio of actual community composition, although the dominant populations can be ascertained.
