Size resolved characteristics of urban and suburban bacterial bioaerosols in Japan as assessed by 16S rRNA amplicon sequencing

To study the size-resolved characteristics of airborne bacterial community composition, diversity, and abundance, outdoor aerosol samples were analysed by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing with Illumina MiSeq. The samples were collected using size-resolved samplers between August and October 2016, at a suburban site in Toyama City and an urban site in Yokohama City, Japan. The bacterial communities were found to be dominated by Actinobacteria, Firmicutes, and Proteobacteria. At the genus level, we found a high abundance of human skin-associated bacteria, such as Propionibacterium, Staphylococcus, and Corynebacterium, in the urban site. Whereas, a high abundance of bacteria associated with soil and plants, such as Methylobacterium and Sphingomonas, was observed in the suburban site. Furthermore, our data revealed a shift in the bacterial community structure, diversity, and abundance of total bacteria at a threshold of 1.1-µm diameter. Interestingly, we observed that Legionella spp., the causal agents of legionellosis in humans, were mainly detected in > 2.1 µm coarse particles. Our data indicate that local environmental factors including built environments could influence the outdoor airborne bacterial community at each site. These results provide a basis for understanding the size-resolved properties of bacterial community composition, diversity, and abundance in outdoor aerosol samples and their potential influence on human health.

Scientific RepoRtS | (2020) 10:12406 | https://doi.org/10.1038/s41598-020-68933-z www.nature.com/scientificreports/ composition of outdoor aerosol samples collected using size-resolved samplers at a suburban site in Toyama City and an urban site in Yokohama City, Japan. The elevation and distance from the sea are remarkably similar between the two locations. To study the size-resolved characteristics of airborne bacterial community composition, diversity, and abundance, we used quantitative PCR and Illumina MiSeq sequencing.

Results
Sequencing. We obtained 2,291,974 raw sequence reads from 54 samples collected at a suburban site in Toyama City and an urban site in Yokohama City, Japan ( Supplementary Fig. S1, Supplementary Table S1 and S2). A total of 1,647,648 reads (30,512 reads per sample) were clustered into 1,158 operational taxonomic units (OTUs) (97% similarity). Good's coverage values were greater than 99% for all samples.
Comparison of bacterial community structure. The Shannon alpha-diversity indexes of bacteria associated with the suburban samples were greater than those associated with the urban samples at both > 1.1 µm and < 1.1 µm (Fig. 1). A principal coordinates plot and hierarchical clustering of the bacterial community showed that air samples from suburban (> 1.1 µm; red colour) grouped separately from those of other sample groups in most cases (Figs. 2,3). Linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed 23 genera with an LDA score of at least 2.0 that were significantly more abundant in the four sample groups (Fig. 4). Specifically, we found five genera to be enriched in the samples: Staphylococcus and Propionibacterium in the urban samples (< 1.1 µm; purple colour), Corynebacterium in the urban samples (> 1.1 µm; blue colour), and Methylobacterium and Sphingomonas in the suburban samples (> 1.1 µm; red colour). All of these were fairly abundant in the samples (at least 4% of the population).
Comparison of bacterial abundance. The total bacterial gene copy number in samples > 1.1 µm was 3.1-fold higher than in samples < 1.1 µm ( Supplementary Fig. S3). The abundance of total bacteria in samples of > 1.1 µm ranged from 1.3 × 10 3 to 5.0 × 10 4 copies m −3 (1.5 × 10 4 copies m −3 on average). Whereas, the abundance of total bacteria in samples of < 1.1 µm ranged from 1.0 × 10 3 to 1.7 × 10 4 copies m −3 (5.0 × 10 3 copies m −3 on average). From the perspective of the sampling area, the total bacterial gene copy number in the suburban samples was 1.7-fold higher than in the urban samples ( Supplementary Fig. S3). The abundance of total bacteria in the suburban samples ranged from 1.6 × 10 3 to 5.0 × 10 4 copies m −3 (1.5 × 10 4 copies m −3 on average). Whereas, the abundance of total bacteria in the urban samples ranged from 1.0 × 10 3 to 3.7 × 10 4 copies m −3 (8.9 × 10 3 copies m −3 on average).
Legionella-assigned OTUs. Interestingly, the bacterial pathogen Legionella spp. was detected. The results provide valuable data for hazard evaluation of the effects of bioaerosols on human health. We examined Legionella-assigned OTUs in air samples (Fig. 5, Supplementary Table S3). Among nine samples, Legionella spp. were mainly detected in coarse particle samples (> 2.1 µm; Stage 1 to 5 of size-resolved sampler). The detection rate of the Legionella-assigned OTUs ranged from 0.004 to 1.421% (0.479% on average). Phylogenetic analysis of the genus Legionellae showed that some OTUs were closely related to the sequences from cooling tower water samples in Japan 18 .

Discussion
In the present study, we examined the bacterial community composition and diversity of outdoor aerosol samples using size-resolved samplers and Illumina MiSeq sequencing at a suburban site in Toyama City and an urban site in Yokohama City, Japan. At any point in time, different sites have their own unique bacterial communities, and local sources of bacteria and other environmental factors may be involved in the assembly of the communities. Bacterial composition, diversity, abundance, and predominant genera showed size-resolved characteristics. The bacterial composition was congruent with those reported in other bioaerosol studies: with the airborne bacterial community dominated by Proteobacteria, Actinobacteria, and Firmicutes at the phylum level, and Actinobacteria, Alphaproteobacteria, Bacilli, Gammaproteobacteria, and Betaproteobacteria at the class level 16,17,[19][20][21][22] .
As shown in Fig. 4, LEfSe analysis showed that the high abundance of human skin-associated bacteria, such as Propionibacterium, Staphylococcus, and Corynebacterium, may be a feature of urban site 23,24 . This situation could indicate that outdoor bacterial communities were influenced by bacteria in the built environment. Whereas, the high abundance of bacteria associated with soil and plants, such as Methylobacterium and Sphingomonas, may be a feature of suburban site 24 . These five bacterial genera have been frequently detected in outdoor and indoor air [23][24][25] . The genus Propionibacterium is a Gram-positive, anaerobic, rod-shaped bacteria that produces propionic acid as its end product of fermentation. This genus in the family Propionibacteriaceae consists of species from various habitats, including mature cheese, cattle rumen, and human skin 26 . The commensal bacterium Cutibacterium acnes (formerly Propionibacterium acnes) is involved in the maintenance of human skin, and it is also the pathogen responsible for acne vulgaris and other diseases 27  www.nature.com/scientificreports/ harmless and reside normally on the skin and mucous membranes of humans and other organisms. In humans, S. epidermidis is the most frequently recovered staphylococcal species 28 . The genus Corynebacterium comprises Gram-positive, non-sporulating (although they have club-like ends), aerobic, pleomorphic bacilli that are isolated from a range of environments including soil, water, blood, and human skin. Pathogenic strains of Corynebacterium can infect animals or humans 29,30 . The genus Methylobacterium comprises Gram-negative, obligately aerobic, rod-shaped bacteria. This genus displays pink pigmentation and the bacteria are facultative methylotrophs. They are ubiquitous in nature and are commonly found in the atmosphere, soil, water, and in the phylloplane, where some may produce plant growth-promoting substances 31 . Rarely, Methylobacterium species are found in clinical samples as opportunist pathogens. The genus Sphingomonas comprises Gram-negative, non-spore-forming, chemoheterotrophic, strictly aerobic bacteria. This genus typically produces yellow-pigmented colonies and has been detected in various environments, including soil, water, clinical specimens, the plant phyllosphere and rhizosphere, air, and other locations 32 .
Airborne bacteria vary in size from 0.1 to 5.0 µm 33,34 . Smaller sized bacteria more easily attach to fine particles such as PM 2.5 , or even smaller particles 15 . It has been suggested that the bacterial concentration and size distribution vary with sampling site. Interestingly, our data revealed a shift in the bacterial community structure, diversity, and abundance in PM at a threshold of 1.1 µm diameter in size. Similarly, Wei et al. 35 reported a disparity in bacterial communities according to the abundance of rare species, such as Bacilli being higher in PM 1.0 (2.4%) than in PM 2.5 (1.8%), and Defluviicoccus being higher in PM 2.5 (2.5%) than in PM 1.0 (0.5%), which may be associated with cell size and cell growth patterns. Blais Lecours et al. 36 showed that bacteria mainly attached Interestingly, we observed that Legionella spp., the causal agents of legionellosis including a pneumonia-type illness known as Legionnaires' disease and a mild flu-like illness known as Pontiac fever, were mainly detected in > 2.1-µm coarse particles. Assuming the average of rRNA gene copy number of 4 per bacterial genome and 3 per Legionella genome (https ://rrndb .umms.med.umich .edu/), our results suggest that Legionella spp. represented less than 1% of the total bacterial community. Mathieu et al. 37 observed that Legionella bacteria represented a small fraction 0.05-0.9% of the total airborne biocontaminants above the fan of the cooling towers and close to an industrial sludge water treatment basin. To our knowledge, this is the first study to detect Legionella spp. in outdoor air samples in Japan. In addition, Fig. 5 shows that some OTUs were closely related to the sequences from cooling tower water samples in Japan 18 . Therefore, we hypothesized that outdoor aerosol samples in Japan often contain Legionella derived from cooling tower water. However, the short fragments generated by 16S rRNA amplicon sequencing on the illumina miseq platform (~ 400 bp in this study) limit their use for 16S rRNA gene-based bacterial identification. Currently, the genus Legionella comprises more than 60 different bacterial species and 70 serogroups that live in many environments, both natural (e.g., rivers, lakes, soil, and ponds) and artificial (e.g., swimming pools, showers, cooling towers, fountains, and waste water treatment plants). Within the genus Legionella, several species can cause clinical disease in humans, such as L. pneumophila, L. dumoffii, L. bozemanii, L. longbeachae, and L. micdadei 38,39 . Legionella pneumophila serogroup 1 is the most virulent strain causing the vast majority of Legionella infections. In the present study, common causative agents of legionellosis were not detected. However, all species of the genus Legionella are potentially pathogenic in humans 40 . According to the nationwide sentinel surveillance system 41 , the peak season for legionellosis was July in Japan; although more patients were reported from more populated prefectures as expected, the number of patients per 100,000 population was high in Toyama, Ishikawa, Okayama, and Tottori Prefectures. Kanatani et al. 42 found that puddles on asphalt roads could serve reservoirs for L. pneumophila in the environment, which can increase potential opportunities for exposure.
In conclusion, this study provides useful information on the size-resolved bacterial communities in outdoor aerosol samples. The results showed that size-resolved differences occurred in terms of airborne bacterial community composition, diversity, and abundance at a suburban site in Toyama City and an urban site in Yokohama City, Japan. The most likely source of airborne bacteria in the urban site was humans. Furthermore, we detected Legionella spp., the causal agents of legionellosis in humans, and these organisms could often be derived from cooling tower water. These findings could provide a foundation for understanding the transmission and health effects of bioaerosols.    45 and sickle 46 with a minimum Sanger quality of 20 and a minimum length of 150. Paired sequence reads were assembled using FLASH 47 with a minimum overlap of 10. The obtained sequence data were then processed using USEARCH version 10.0.240 48 and analysed with the software package Quantitative Insights into Microbial Ecology (QIIME) version 1.9.1 49 . Sequences were clustered into operational taxonomic units (OTUs) using the Greengenes 13_8 reference OTU database 50 (97% similarity). For 16S rRNA gene fragment analysis, singleton, chloroplast and mitochondrial OTUs were removed. All samples were rarefied to even sequencing depth based on the sample having the lowest sequencing depth of 17,478 reads (sample: Oct-U7) before total sum normalization. Statistical analysis was conducted using the R software, version 3.5.2 51 . We used ggplot2 package 52 and the phyloseq package 53 . Betadiversity was explored by principal coordinate analysis (PCoA) of Bray-Curtis dissimilarity among sample groups with different locations and size. Statistical significance was calculated by the permutational multivariate analysis of variance (PERMANOVA) in vegan package 54 . LEfSe was applied to identify specific bacterial genera among sample groups 55 . Hierarchical cluster analysis was performed based on Bray-Curtis dissimilarity matrices of relative abundance of bacterial OTU with "stats" in R package 51 . Taxa were considered significant based on LDA score of > 2 and p-value < 0.05. A phylogenetic tree was constructed using the neighbour-joining method with Kimura 2 parameter distances in MEGA X software. All sequences have been deposited in the DNA Data Bank of Japan (DDBJ) under the accession number DRA009183.

Real-time TaqMan PCR.
Real-time TaqMan PCR reactions were performed using a Thermal Cycler Dice Real Time System (TP-850, Takara Bio, Otsu, Japan). Quantification of the 16S rRNA gene of the total bacteria was performed as previously described 56 . Each reaction mixture was prepared in a total volume of 25 µL with 12.5 µL Premix Ex Taq (Probe qPCR, Takara Bio), 0.2 µM forward primer 1055f, 0.2 µM reverse primer 1392r, 0.25 µM TaqMan probe 16Staq1115, and 2 µL of standard or extracted DNA. For the assay, the PCR program was 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, and 30 s at 60 °C. DNA standards were prepared from serial dilutions of pGEM-T Easy Vector (Promega) containing the 16S rRNA gene from Escherichia coli K-12 strain W3110. Duplicate aliquots of the standards and the samples were included in each PCR run. All assays included a negative control in which no template was present.