Abstract
Since 2005, advances in next-generation sequencing (NGS) technologies have revolutionized biological science. One particular
application of NGS technologies is to elucidate microbiomes in built environments. We are currently conducting a series of studies on the
elucidation and control of mass infection mechanisms based on dynamic measurement of environment microbiomes. The objective of this
study is to clarify the dispersion characteristics of oral bacteria in built environment.
Bacterial communities from occupants’ hands and oral cavities, doorknobs, desk and keyboard surfaces, and air in a university
laboratory were investigated. For each sample, the variable region 4 (V4) of the bacterial 16S ribosomal RNA (rRNA) gene was amplified
by polymerase chain reaction (PCR) using the primer set 5’-acactctttccctacacgacgctcttccgatct-GTGCCAGCMGCCGCGGTAA-3’
(1st_515F) and 5’-gtgactggagttcagacgtgtgctcttccgatct-GGACTACHVGGGTWTCTAAT-3’ (1st_806R). The 16S rRNA amplification
protocol version 4_13 was used as a pretreatment with the Next-Generation Sequencer (NGS, Illumina MiSeq system v2). DNA quality
was checked using the Agilent 2200 TapeStation. All samples that contained the necessary quality and quantity of nucleic acid
concentration for analysis were then analyzed. The produced sequence library was mixed. To improve the quality of the mixed sequence
library, a refining processing using the AMPureXP PCR purification system (Beckman Coulter, Inc.) was carried out.
About data analysis, the leads for all samples obtained in the sequence analysis (lead 1 and lead 2) were unified, and then
assemblies of lead 1 and lead 2 were extracted, followed by clustering and representation arrangement. CD-HIT-OTU was used for
alignment extraction and representation arrangement. Basic Local Alignment Search Tool (BLAST) search was performed using the DDBJ
16S rRNA database (version 2016 01 12) by querying representation arrangements. For sequencing, alignment (PyNAST) and
genealogical tree creation (FastTree) were performed using a phylogenetic system (RDP classifier), and template alignment was
accomplished using the QIIME pipeline. Rarefaction analysis and comparison with a bacillus solution were performed using the QIIME
pipeline.
The main results obtained by this study are shown as follows.
1) Higher rank 6 phyla of 98% of rates of all 18 phyla being detected and occupying to the whole on a bacterial phyla level more than at
composition ratio 1% were Firmicutes (44.9%), Proteobacteria (30.2%), Actinobacteria (9.5%), Bacteroidetes (8.4%), Fusobacteria
(3.3%) and Cyanobacteria (1.7%).
2) Higher rank 15 genera of 70% of rates of all 149 genera were Streptococcus (28.4%), Haemophilus (9.0%). Prevotella (5.0%),
Staphylococcus (4.5%), Neisseria (4.2%), Corynebacterium (3.8%), Pseudomonas (2.8%), Rothia (2.8%), Fusobacterium (2.1%),
Enhydrobacter (1.6%), Veillonella (1.4%), Leptotrichia (1.2%), Granulicatella (1.2%), Acinetobacte (1.1%), Porphyromonas (1.0%).
3) On the species level, reads of one or more larger than 3 orders had 33 species from each sampling point, and about 30% of bacteria
were pathogenic bacterium or opportunistic infection bacterium. In particular, P. melaninogenica which is a disease germ in a mouth,
and R. dentocariosa and R. mucilaginosa which is an opportunistic infection bacillus in a mouth were detected from all parts.
4) By the analysis of species intersection of each group of bacteria, reads larger than 3 orders from all samples were P. melaninogenica
and R. aeria and both bacteria are causative organism of respiratory tract infection. That is, the fact that oral cavity bacteria of this
human associated were detected from all surfaces and indoor air showed clearly that oral cavity bacteria had dispersed in all parts
among indoor environment.
