Multiplex Hybrid Capture Improves the Deep Detection of Antimicrobial Resistance Genes from Wastewater Treatment Plant Effluents to Assess Environmental Issues.

Metagenomic sequencing (mDNA-seq) is one of the best approaches to address antimicrobial resistance (AMR) issues and characterize AMR genes (ARGs) and their host bacteria (ARB); however, the sensitivity provided is insufficient for the overall detection in wastewater treatment plant (WWTP) effluents because the effluent is well treated. This study investigated the multiplex hybrid capture (xHYB) method (QIAseq × HYB AMR Panel) and its potential to increase AMR assessment sensitivity. The mDNA-Seq analysis suggested that the WWTP effluents had an average of 104 reads per kilobase of gene per million (RPKM) for the detection of all targeted ARGs, whereas xHYB significantly improved detection at 601,576 RPKM, indicating an average 5,805-fold increase in sensitivity. For instance, sul1 was detected at 15 and 114,229 RPKM using mDNA-seq and xHYB, respectively. The blaCTX-M, blaKPC, and mcr gene variants were not detected by mDNA-Seq but were detected by xHYB at 67, 20, and 1,010 RPKM, respectively. This study demonstrates that the multiplex xHYB method could be a suitable evaluation standard with high sensitivity and specificity for deep-dive detection, highlighting a broader illustration of ongoing dissemination in the entire community.

Rapid On-Site Monitoring of Bacteria in Freshwater Environments Using a Portable Microfluidic Counting System.

Freshwater environments and natural water parks are important as recreation areas; however, people enjoying recreation at the river- or lake-side are sometimes infected with pathogenic microbes. Microbiological monitoring is fundamental for the routine evaluation of water quality. Fluorescent staining techniques are regarded as among the most useful rapid microbiological methods; however, preparation of samples for fluorescence microscopy is often labor-intensive, and one usually has to take the samples to a laboratory for measurement, which often alters the culturability of bacteria in the samples. These factors have created demand for a rapid and simple method of bacterial quantification in freshwater that can be performed on-site. In this study, we applied our microfluidic device, which was originally designed for on-chip fluorescent staining and semi-automated counting of target microbial cells with fluorescent antibody-staining, to enumerate bacterial cells in freshwater. This was combined with a self-made portable system for rapid on-site monitoring of the bacterial cells. Numbers of both esterase-active bacteria and total bacteria in pond water samples could be successfully determined by on-chip staining with 6-carboxyfluorescein diacetate and SYBR Green II, respectively, using the portable microfluidic counting system. The counting was completed within 1 h (30 min for pre-filtration of freshwater and 30 min for on-chip staining and counting). These results indicate that rapid and accurate counting of bacterial cells in freshwater can be performed and this technique could be applied for "on-site first screening" purposes in microbial quality control of freshwater.

Rapid On-Site Detection and Quantification of Foodborne Pathogens Using Microfluidic Devices.

The potential for foodborne infectious disease outbreaks has increased not only on a local scale but also on a regional and international scale. Simple, rapid, and accurate methods to enumerate pathogenic bacteria in food and drink are required to prevent the spread of these bacteria. Here, I describe applications of a microfluidic device for on-chip fluorescent staining and semiautomated counting of target bacteria in food samples.

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.

Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system.

Legionnaires' disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently-labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 104 cells/ml, but lower numbers of L. pneumophila cells (101 to 103 cells/ml) could be detected following concentration of 0.5-3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.

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.

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.

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.

Bacterial monitoring with adhesive sheet in the international space station-"Kibo", the Japanese experiment module.

Microbiological monitoring is important to assure microbiological safety, especially in long-duration space habitation. We have been continuously monitoring the abundance and diversity of bacteria in the International Space Station (ISS)-"Kibo" module to accumulate knowledge on microbes in the ISS. In this study, we used a new sampling device, a microbe-collecting adhesive sheet developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. We first validated the adhesive sheet as a sampling device to be used in a space habitat with regard to the stability of the bacterial number on the sheet during prolonged storage of up to 12 months. Bacterial abundance on the surfaces in Kibo was then determined and was lower than on the surfaces in our laboratory (10(5) cells [cm(2)](-1)), except for the return air grill, and the bacteria detected in Kibo were human skin microflora. From these aspects of microbial abundance and their phylogenetic affiliation, we concluded that Kibo has been microbiologically well maintained; however, microbial abundance may increase with the prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in Kibo is required.

Global dispersion of bacterial cells on Asian dust.

The atmospheric dispersion of bacteria over long distances is an important facet of microbial ecology. Certain groups of dispersed bacteria can adapt to their new location and affect established ecosystems. Aeolian dust particles are known to be carriers of microbes but further research is needed to expand our understanding of this field of microbiology. Here we showed the potential of aeolian dust to global migration of bacterial cells. We demonstrated the presence of microbial cells on dust particles directly by bio-imaging. Bacterial abundance on dust particles declined from 10(5) to less than 10(3) cells/m3 as the dust event subsided. Taxonomically diverse bacteria were identified by 16S rRNA gene sequencing and some of these bacteria retained growth potential. Our results confirm that bacteria can attach to aeolian dust particles and they have the potential to migrate globally during dust events and thus can contribute to the diversity of downwind ecosystems.

High prevalence of qnr and aac(6')-Ib-cr genes in both water-borne environmental bacteria and clinical isolates of Citrobacter freundii in China.

We investigated the prevalence of qnr and aac(6')-Ib-cr genes in water-borne environmental bacteria and in clinical isolates of Enterobacteriaceae, as well as the subtypes of qnr. Environmental bacteria were isolated from surface water samples obtained from 10 different locations in Hangzhou City, and clinical isolates of Citrobacter freundii were isolated from several hospitals in four cities in China. qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes were screened using PCR, and the genotypes were analyzed by DNA sequencing. Ten of the 78 Gram-negative bacilli isolated from water samples were C. freundii and 80% of these isolates carried the qnrB gene. qnrS1 and aac(6')-Ib-cr genes were detected in two Escherichia coli isolates and qnrS2 was detected in one species, Aeromonas punctata. The qnr and aac(6')-Ib-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. freundii, respectively. Of the clinical C. freundii isolates with the qnr gene, 65 isolates (63.1%) carried qnrB, but only three (2.9%) and one (1.0%) carried qnrA1 and qnrS2, respectively, while five isolates carried both qnrA1 and qnrB, and one isolate carried both qnrS1 and qnrB. The qnrB9 gene was the dominant qnrB subtype, followed by qnrB8 and qnrB6. Southern hybridization studies indicated that the qnr genes are located on different plasmids. Plasmids isolated from both environmental and clinical C. freundii isolates appeared to be homogenous.

Break down of asian dust particle on wet surface and their possibilities of cause of respiratory health effects.

Asian dust (called 'Kosa' in Japan) is comprised of a large number of soil particles originating from the arid regions and deserts of China and Mongolia and dispersed long-range to Japan. A major public concern about Asian dust is its impact on human health. We collected Asian dust particles over the Japan Sea at an altitude of 900 m to directly estimate their effects on health. We examined the properties of the collected particles on wet surfaces. Through size distribution measurements and scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis, we demonstrated that small dust particles (less than 1 µm) form aggregations with water-soluble salts such as calcium and sodium and they are transported to Japan as aggregates. These aggregates probably break down into small particles on nasal mucous membranes and may cause adverse respiratory health effects.