Changes in bacterial diversity and community structure in drinking water distribution system revealed by high throughput sequencing.

For microbiological management of water quality, it is important to identify bacteria and to understand the community structure. To analyze the community structure during water purification and distribution, we selected a distribution system in which water from other water treatment facilities was not mixed with the target water. Changes in the bacterial community structure during treatment and distribution processes in a slow filtration water treatment facility were analyzed using 16S rRNA gene amplicon sequencing with a portable sequencer MinION. The microbial diversity was reduced by chlorination. The genus level diversity increased during distribution and this diversity was maintained through to the terminal tap water. Yersinia and Aeromonas were dominant in the intake water, and Legionella was dominant in the slow sand filtered water. Chlorination greatly reduced the relative abundance of Yersinia, Aeromonas, and Legionella, and these bacteria were not detected in the terminal tap water. Sphingomonas, Starkeya and Methylobacterium became dominant in the water after chlorination. These bacteria could be used as important indicator bacteria to provide useful information for microbiological control in drinking water distribution systems.

Microbial Water Quality Assessment of Private Wells Using 16S rRNA Gene Amplicon Sequencing with a Nanopore Sequencer.

Private wells are used daily worldwide as convenient household water sources. In Japan, where water supply coverage is high, well water is occasionally used for non-potable purposes, such as irrigation and watering. Currently, the main microbiological test of well water is designed to detect Escherichia coli, which is an indicator of fecal contamination, using culture methods. Water use such as watering generates bioaerosols, which may cause airborne infection. However, many causative bacteria of aerosol-derived infections, such as Legionella spp., are difficult to detect using culture methods. Thus, more comprehensive modern assessment is desirable for securing the microbiological quality of well water. Here, the bacterial community structure of five private wells located in different environments was examined using the rapid and portable MinION sequencer, which enabled us to identify bacteria to the species level based on full-length 16S ribosomal RNA (rRNA) gene sequences. The results revealed the differences in the bacterial community structures of water samples from the five wells and detected Legionella pneumophila and Aeromonas hydrophila as new candidate microbial indicators. The comprehensive analysis method used in this study successfully detected bacteria causing opportunistic infections, which are difficult to detect by conventional methods. This approach is expected to be routinely applied in the future as a highly accurate method for assessing the microbiological quality of private well water.

Bacterial bioburden and community structure of potable water used in the International Space Station.

The control of microbes in manned spaceflight is essential to reducing the risk of infection and maintaining crew health. The primary issue is ensuring the safety of a potable water system, where simultaneous monitoring of microbial abundance and community structure is needed. In this paper, we develop a flow cytometry-based counting protocol targeting cellular flavin autofluorescence as a tool for rapid monitoring of bacterial cells in water. This was successfully applied to estimate the bacterial bioburden in the potable water collected from the International Space Station. We also demonstrate the efficacy of the MinION nanopore sequencer in rapidly characterizing bacterial community structure and identifying the dominant species. These monitoring protocols' rapidity and cost effectiveness would contribute to developing sustainable real-time surveillance of potable water in spaceflight.

Microbial Monitoring in the International Space Station and Its Application on Earth.

The space habitat is a confined environment with a simple ecosystem that consists mainly of microorganisms and humans. Changes in the pathogenicity and virulence of bacteria, as well as in astronauts' immune systems, during spaceflight may pose potential hazards to crew health. To ensure microbiological safety in the space habitat, a comprehensive analysis of environmental microbiota is needed to understand the overall microbial world in this habitat. The resulting data contribute to evidence-based microbial monitoring, and continuous microbial monitoring will provide information regarding changes in bioburden and microbial ecosystem; this information is indispensable for microbiological management. Importantly, the majority of microbes in the environment are difficult to culture under conventional culture conditions. To improve understanding of the microbial community in the space habitat, culture-independent approaches are required. Furthermore, there is a need to assess the bioburden and physiological activity of microbes during future long-term space habitation, so that the "alert" and/or "action" level can be assessed based on real-time changes in the microbial ecosystem. Here, we review the microbial monitoring in the International Space Station-Kibo, and discuss how these results will be adapted to the microbial control in space habitation and pharmaceutical and food processing industries.

Genetic relatedness of Mycobacterium avium subsp. hominissuis isolates from bathrooms of healthy volunteers, rivers, and soils in Japan with human clinical isolates from different geographical areas.

Japan reportedly has high incidence rate of nontuberculous mycobacterial lung disease (14.7 cases per 100,000 person in 2014). In Japan, the most common etiology is Mycobacterium avium subsp. hominissuis (MAH). MAH is a typical inhabitant of the environment, especially bathrooms, which are considered as a potential source of infection. To corroborate this hypothesis, we determined the detection rate of MAH in bathrooms of healthy volunteers by an ordinary culture method and we analyzed the genetic relatedness of these isolates with those from patients and other sources. We collected swabs of bathtub inlets, showerheads, bathroom drains, and shower water from 180 residences throughout Japan. The overall MAH detection rate was 16.1%, but the rate varied among regions: it was high in Kanto (9/34, 26.5%) and Kinki (9/33, 27.3%), but low in Kyushu (0/11, 0%), Tohoku (1/23, 4.3%), and Hokkaido (2/23, 8.7%). MAH was detected primarily in bathtub inlet samples (25 out of 170 residences). Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of 57 MAH isolates from bathrooms of the healthy volunteers with human clinical isolates. A minimum spanning tree generated on the basis of the VNTR data indicated that isolates from the bathrooms of the healthy volunteers had a high degree of genetic relatedness with those from Japanese patients, bathrooms of patients, and river water, but not with those from Russian patients and Japanese pigs. These results showed that bathtub inlets in Japan provide an environmental niche for MAH and suggest that bathrooms are one of the important infection sources of MAH in Japan. Understanding country-specific lifestyle habits, such as bathing in Japan, as well as the genetic diversity of MAH, will help in elucidating the sources of this pathogen.

Culture independent approach reveals domination of human-oriented microbes in a pharmaceutical manufacturing facility.

Strict microbial control is required in pharmaceutical manufacturing facilities, for which environmental microbial monitoring is fundamental. Appropriate microbial control is based on understanding the abundance and community structure of the microbes in the target environment, but most microbes are not culturable by conventional methods. Here, we determined the bacterial abundance and assessed the environmental microbiome in a pharmaceutical manufacturing facility using rRNA gene-targeted quantitative PCR (qPCR) and high-throughput sequencing of rRNA gene fragments. A commercially available microbial particle counter was also used for real-time measurements. In the air of the first gowning room and the passageway of the facility, the microbial particle number determined by both the particle counter and qPCR was ca. 104/m3; the number of microbial particles was about 100 times the number of culturable bacteria. Thus, the measurement of microbes using the particle counter was accurate. In the second gowning room of the facility, managed by a HEPA filter, the number of particles in the air was dependent on human movement, and was below the detection limit around 10 min after movement. Bacteria of the phyla Proteobacteria, Firmicutes, and Actinobacteria were frequently detected in samples from the facility; these bacteria are constituents of the human microbiota. Among fungi, Aspergillus and Cladosporium were detected in the air, and Malassezia was dominant on the walls. Our results provide fundamental data for the evaluation and control of microbes in pharmaceutical and food industry facilities.

Effects of Asian dust events on atmospheric bacterial communities at different distances downwind of the source region.

Aeolian dust particles arising from arid and semiarid zones are known to carry microbes by air currents. The effect of wind-borne bacteria on atmospheric bacterial population at various downwind distances from the dust source regions must be clarified, but has not yet been reported. This study monitored the bacterial abundance and community composition in outdoor aerosol samples in Beijing, China, which is close to the Asian dust source regions, and compared them with the results obtained in a distant region (Osaka, Japan). The Asian dust collected in Beijing contained (4±3)×104bacterial cells/m3, approximately 4 times higher than in Osaka. On 15 April 2015, Beijing experienced severe Asian dust events with a 1000-fold increase in bacterial abundance, relative to non-Asian dust days. Dominant bacterial phyla and classes in Asian dust collected in Beijing were Actinobacteria, Bacilli and Acidobacteria, and the bacterial community composition varied more widely than in Osaka. The bacterial community compositions differed between the Beijing and Osaka dusts, even for the same Asian dust events. These results indicated that aerosol bacterial communities nearer the dust source are more affected by eolian dust than their distant counterparts.

Positive Selection in F-Box Domain (lpp0233) Encoded in Legionella pneumophila Strains.

　Recent studies have shown that the genome of Legionella pneumophila is characterized by many foreign genes from a variety of eukaryotes. The eukaryotic like proteins are known to play a role in its multiplication within host cells; however, their evolutionary genetics of L. pneumophila in environments is unknown. In this study, we examined the nonsynonymous/synonymous substitution rate of eukaryotic like domain encoding genes among L. pneumophila strains. In silico analysis revealed that the nonsynonymous/synonymous substitution rate in F-box domain gene (lpp0233) was higher than those in other eukaryotic like domain and protein encoding genes and housekeeping genes. The F-box domain gene sequences in L. pneumophila strains isolated from a natural hot spring were more diversified than those of previously known strains, owing to preferential positive selection.

Investigation of bacterial effects of Asian dust events through comparison with seasonal variability in outdoor airborne bacterial community.

Atmospheric bacterial dispersion with aeolian dust has been reported to have a potential impact on public health and ecosystems. Asian dust is a major aeolian event that results in an estimated 4 million tons of Asian dust particles falling in Japan annually, 3,000-5,000 km away from their source regions. However, most studies have only investigated the effects of Asian dust during dust seasons. Therefore, in this study, outdoor bacterial abundance and community composition were determined by 16S rRNA quantitative PCR and amplicon sequencing, respectively, and compared on Asian and non-Asian dust days (2013-2015; 44 samples over four seasons). Seasonal variations in bacterial abundance of non-Asian dust days were not observed. Bacterial abundance of individual samples collected on non-Asian dust days changed dynamically relative to Asian dust days, with bacterial abundance occasionally reaching those of Asian dust days. The bacterial community composition on non-Asian dust days was rather stable seasonally, and did not differ from that on Asian dust days. These results indicate that bacteria in Asian dust does not immediately influence indigenous bacterial communities at the phylum/class level in distant downwind areas; accordingly, further studies of bacterial communities in downwind areas closer to the dust source are warranted.

Abundance and Community Structure of Bacteria on Asian Dust Particles Collected in Beijing, China, during the Asian Dust Season.

Approximately 180 t/km(2) of Asian dust particles are estimated to fall annually on Beijing, China, and there is significant concern about the influence of microbes transported by Asian dust events on human health and downwind ecosystems. In this study, we collected Asian dust particles in Beijing, and analyzed the bacterial communities on these particles by culture-independent methods. Bacterial cells on Asian dust particles were visualized first by laser scanning microscopy, which demonstrated that Asian dust particles carry bacterial cells to Beijing. Bacterial abundance, as determined by quantitative polymerase chain reaction (PCR), was 10(8) to 10(9) cells/g, a value about 10 times higher than that in Asian dust source soils. Inter-seasonal variability of bacterial community structures among Asian dust samples, as compared by terminal restriction fragment length polymorphism (T-RFLP), was low during the Asian dust season. Several viable bacteria, including intestinal bacteria, were found in Asian dust samples by denaturing gradient gel electrophoresis (DGGE). Clone library analysis targeting 16S ribosomal RNA (rRNA) gene sequences demonstrated that bacterial phylogenetic diversity was high in the dust samples, and most of these were environmental bacteria distributed in soil and air. The dominant species in the clone library was Segetibacter aerophilus (Bacteroidetes), which was first isolated from an Asian dust sample collected in Korea. Our results also indicate the possibility of a change in the bacterial community structure during transportation and increases in desiccation-tolerant bacteria such as Firmicutes.

Four-year bacterial monitoring in the International Space Station-Japanese Experiment Module "Kibo" with culture-independent approach.

Studies on the relationships between humans and microbes in space habitation environments are critical for success in long-duration space missions, to reduce potential hazards to the crew and the spacecraft infrastructure. We performed microbial monitoring in the Japanese Experiment Module "Kibo", a part of the International Space Station, for 4 years after its completion, and analyzed samples with modern molecular microbiological techniques. Sampling was performed in September 2009, February 2011, and October 2012. The surface of the incubator, inside the door of the incubator, an air intake, air diffuser, and handrail were selected as sampling sites. Sampling was performed using the optimized swabbing method. Abundance and phylogenetic affiliation of bacteria on the interior surfaces of Kibo were determined by quantitative PCR and pyrosequencing, respectively. Bacteria in the phyla Proteobacteria (γ-subclass) and Firmicutes were frequently detected on the interior surfaces in Kibo. Families Staphylococcaceae and Enterobacteriaceae were dominant. Most bacteria detected belonged to the human microbiota; thus, we suggest that bacterial cells are transferred to the surfaces in Kibo from the astronauts. Environmental bacteria such as Legionella spp. were also detected. From the data on bacterial abundance and phylogenetic affiliation, Kibo has been microbiologically well maintained; however, the microbial community structure in Kibo may change with prolonged stay of astronauts. Continuous monitoring is required to obtain information on changes in the microbial community structure in Kibo.

[Risk Management of Teratogenic Drugs ~The Current States of Practice in Europe, US and Japan~].

Thalidomide was approved for the treatment of multiple myeloma in Japan under a risk management program, named TERMS, in 2008. Since then, we have been conducting a survey of the stakeholders to assess the effectiveness of TERMS. These surveys showed patients had enough knowledge of the risks of thalidomide. In the USA, legislation in 2007 granted its U.S. Food and Drug Administration (FDA) the authority to require Risk Evaluation and Mitigation Strategies (REMS) when necessary to ensure that a drug's benefits outweigh its risks. As of 2015, more than 70 drugs, including thalidomide, have REMS programs. In Germany, in the early 1960s, over 5000 children were born with deformities. Therefore, the safety regulations in Germany go far beyond the European Medicines Agency (EMA) safety regulations at the time of thalidomide re-approval; thalidomide can be prescribed by a special prescribing form, including both proof of the patients' awareness of information about the risks, and participation in a pregnancy prevention program. While Japan has taken similar safety measures, a portion of thalidomide is still privately imported there. By March 2013, 594 patients have been registered to Japan's Safety Management System for Unapproved Drugs (SMUD), which was introduced by the Ministry of Health, Labour and Welfare (MHLW) in 2009. The number of females of child bearing potential (FCBPs) was 33 and the fraction (33/594=5.6%) was higher than that (48/7370=0.7%) in the case of TERMS. Risk management of thalidomide is described in this review.

Expression of gyrB and 16S ribosomal RNA genes as indicators of growth and physiological activities of Legionella pneumophila.

To determine whether the DNA gyrase (gyrB) and 16S ribosomal RNA (16S rRNA) genes can be used as indicators of the biological activities of Legionella pneumophila, the expression levels were estimated. The ratio of mRNA/DNA in gyrB was 0.7 in mid log phase and decreased drastically after the log phase. For 16S rRNA, the ratio was highest in mid log phase (7.0×10(3)), and the value that was about 10% of that in the log phase was maintained for six days. The rRNA may be vital in the resting or active but nonculturable cells that are not growing but physiologically active. The expression levels of gyrB mRNA and 16S rRNA can be used as indicators of the growth activity and the physiological activity of L. pneumophila, respectively. Therefore, by measurement of these indicators, we can evaluate the activities of Legionella cells in various environments.

Distribution and respiratory activity of mycobacteria in household water system of healthy volunteers in Japan.

The primary infectious source of nontuberculous mycobacteria (NTM), which are known as opportunistic pathogens, appears to be environmental exposure, and it is important to reduce the frequency of exposure from environmental sources for preventing NTM infections. In order to achieve this, the distribution and respiratory activity of NTM in the environments must be clarified. In this study, we determined the abundance of mycobacteria and respiratory active mycobacteria in the household water system of healthy volunteers using quantitative PCR and a fluorescent staining method, because household water has been considered as one of the possible infectious sources. We chose healthy volunteer households in order to lessen the effect of possible residential contamination from an infected patient. We evaluated whether each sampling site (bathroom drain, kitchen drain, bath heater pipe and showerhead) have the potential to be the sources of NTM infections. Our results indicated that drains in the bathroom and kitchen sink are the niche for Mycobacterium spp. and M. avium cells were only detected in the bathtub inlet. Both physicochemical and biologic selective pressures may affect the preferred habitat of Mycobacterium spp. Regional differences also appear to exist as demonstrated by the presence (US) or absence (Japan) of Mycobacterium spp. on showerheads. Understanding of the country specific human activities and water usage will help to elucidate the infectious source and route of nontuberculous mycobacterial disease.

Microbial monitoring of crewed habitats in space-current status and future perspectives.

Previous space research conducted during short-term flight experiments and long-term environmental monitoring on board orbiting space stations suggests that the relationship between humans and microbes is altered in the crewed habitat in space. Both human physiology and microbial communities adapt to spaceflight. Microbial monitoring is critical to crew safety in long-duration space habitation and the sustained operation of life support systems on space transit vehicles, space stations, and surface habitats. To address this critical need, space agencies including NASA (National Aeronautics and Space Administration), ESA (European Space Agency), and JAXA (Japan Aerospace Exploration Agency) are working together to develop and implement specific measures to monitor, control, and counteract biological contamination in closed-environment systems. In this review, the current status of microbial monitoring conducted in the International Space Station (ISS) as well as the results of recent microbial spaceflight experiments have been summarized and future perspectives are discussed.

Asian dust particles induce macrophage inflammatory responses via mitogen-activated protein kinase activation and reactive oxygen species production.

Asian dust is a springtime meteorological phenomenon that originates in the deserts of China and Mongolia. The dust is carried by prevailing winds across East Asia where it causes serious health problems. Most of the information available on the impact of Asian dust on human health is based on epidemiological investigations, so from a biological standpoint little is known of its effects. To clarify the effects of Asian dust on human health, it is essential to assess inflammatory responses to the dust and to evaluate the involvement of these responses in the pathogenesis or aggravation of disease. Here, we investigated the induction of inflammatory responses by Asian dust particles in macrophages. Treatment with Asian dust particles induced greater production of inflammatory cytokines interleukin-6 and tumor necrosis factor- α (TNF- α ) compared with treatment with soil dust. Furthermore, a soil dust sample containing only particles ≤10 µ m in diameter provoked a greater inflammatory response than soil dust samples containing particles >10 µ m. In addition, Asian dust particles-induced TNF- α production was dependent on endocytosis, the production of reactive oxygen species, and the activation of nuclear factor- κ B and mitogen-activated protein kinases. Together, these results suggest that Asian dust particles induce inflammatory disease through the activation of macrophages.

Changes in the airborne bacterial community in outdoor environments following Asian dust events.

Bacterial abundance and community compositions have been examined in aeolian dust in order to clarify their possible impacts on public health and ecosystems. The influence of transcontinentally transported bacterial cells on microbial communities in the outdoor environments of downwind areas should be determined because the rapid influx of a large amount of bacterial cells can disturb indigenous microbial ecosystems. In the present study, we analyzed bacteria in air samples (approximately 100 m(3) d(-1)) that were collected on both Asian dust days and non-Asian dust days over 2 years (between November 2010 and July 2012). Changes in bacterial abundance and community composition were investigated based on their 16S rRNA gene amount and sequence diversity. Seasonal monitoring revealed that airborne bacterial abundance was more than 10-fold higher on severe dust days, while moderate dust events did not affect airborne bacterial abundance. A comparison of bacterial community compositions revealed that bacteria in Asian dust did not immediately disturb the airborne microbial community in areas 3,000-5,000 km downwind of dust source regions, even when a large amount of bacterial cells were transported by the atmospheric event. However, microbes in aeolian dust may have a greater impact on indigenous microbial communities in downwind areas near the dust source. Continuous temporal and spatial analyses from dust source regions to downwind regions (e.g., from the Gobi desert to China, Korea, Japan, and North America) will assist in estimating the impact of atmospherically transported bacteria on indigenous microbial ecosystems in downwind areas.

Bacterial communities in pigmented biofilms formed on the sandstone bas-relief walls of the Bayon Temple, Angkor Thom, Cambodia.

The Bayon temple in Angkor Thom, Cambodia has shown serious deterioration and is subject to the formation of various pigmented biofilms. Because biofilms are damaging the bas-reliefs, low reliefs engraved on the surface of sandstone, information about the microbial community within them is indispensable to control biofilm colonization. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of biofilm samples from the pigmented sandstone surfaces showed that the bacterial community members in the biofilms differed clearly from those in the air and had low sequence similarity to database sequences. Non-destructive sampling of biofilm revealed novel bacterial groups of predominantly Rubrobacter in salmon pink biofilm, Cyanobacteria in chrome green biofilm, Cyanobacteria and Chloroflexi in signal violet biofilm, Chloroflexi in black gray biofilm, and Deinococcus-Thermus, Cyanobacteria, and Rubrobacter in blue green biofilm. Serial peeling-off of a thick biofilm by layers with adhesive sheets revealed a stratified structure: the blue-green biofilm, around which there was serious deterioration, was very rich in Cyanobacteria near the surface and Chloroflexi in deep layer below. Nitrate ion concentrations were high in the blue-green biofilm. The characteristic distribution of bacteria at different biofilm depths provides valuable information on not only the biofilm formation process but also the sandstone weathering process in the tropics.

Microchip-based terminal restriction fragment length polymorphism for on-site analysis of bacterial communities in freshwater.

Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is important for a healthy life. Bacterial number and their community structure in freshwater should be determined as quickly as possible, and "real-time" and "on-site" microbiological methods are required. In this study, we examined the protocol for microchip-based terminal restriction fragment length polymorphism (T-RFLP) analysis, which uses microchip electrophoresis for rapid microbial community analysis. The availability of microchip-based T-RFLP was compared with conventional T-RFLP analysis, which uses a capillary electrophoresis system, with freshwater samples (spring water, river water, groundwater, and hydroponics solution). The detection limit of targeted bacteria by on-chip T-RFLP analysis was 1% (10(3) cells/mL). The fragment sizes determined by the two analysis methods were highly correlated (r(2)=0.98). On-chip T-RFLP analysis was completed within 15 min. T-RFLP profiles of nine hydroponics solution samples were analyzed by multidimensional scaling. Considerable changes and stability in bacterial community structure during hydroponic culture were detected by both analyses. These results show that on-chip T-RFLP analysis can monitor changes in bacterial community structure, as well as conventional T-RFLP analysis. The present results indicate that on-chip T-RFLP analysis is an effective tool for rapid and "on-site" bacterial community profiling in freshwater environments, as well as freshwater used for medical and industrial purposes.

16S ribosomal RNA gene-based phylogenetic analysis of abundant bacteria in river, canal and potable water in Bangkok, Thailand.

In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze-thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR-DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR-DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.