一條 知昭

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.

This chapter characterizes the flight environment that is especially relevant to human performance. The first topic focuses on various acceleration and vibration environments (e.g., launch, orbital flight, re-entry). The next topic is the acoustic environment. Excessive noise can produce physiological and psychological effects, as well as the reduced ability to hear communications and alarms related acoustic requirements are discussed. The third topic is radiation. Various space environments (e.g., low-Earth orbit, suborbital flight, Moon or Mars missions) pose extreme levels of risk to humans and require shielding interventions based on radiation exposure limits. The final topic, microbial contamination, focuses on the relationship between human physiology and microbial ecology in space habitats. Concerns for effective control of contamination particularly in confined environments are discussed.

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.

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.

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.

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.

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.

Relocation of bacteria over long distances is a key issue in global bacterial inoculation. Certain of mobile bacteria then can adapt to their new location and affect the established ecosystem. Aeolian dust particles are thought to be carriers of microbes but definitive research is lacking. The contribution of aeolian dust to global migration of bacterial cells and their genes was therefore examined by culture-independent approaches. Asian dust particles were collected over the Japan Sea (10 km from coasts) at an altitude of 900 m to avoid contamination of soil particles lifted from ground, on 12 November 2010 (midst of the event, visibility: less than 10 km), 13 Nov. 2010, 16 Nov. 2010 (end of the event, visibility: 25 km) and 2 May 2011 (midst of the event, visibility: less than 10 km), with a sampler set in a small airplane. Microbial cells on dust particles were directly visualized by bio-imaging with laser scanning microscopy equipped with a microspectrophotometer, based on their specific fluorescence. 16S rRNA gene was directly extracted and it was then confirmed by quantitative PCR that bacterial abundance on collected dust particles drastically declined from 105cells/m3to less than 1/100 as the dust event subsided. Taxonomically diverse bacteria were found by sequencing of 16S rRNA genes of clones obtained from each collected Asian dust sample, such as Actinobacteria, Bacilli and Sphingobacteria. Some of these bacteria retained growth potential despite the long-range transportation. These results demonstrate that bacteria attach to aeolian dust particles and migrate globally during dust events thus may contribute to the diversity of the bacterial gene pool.

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 "realtime" 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 mm. 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.

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 (105 cells [cm2]-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.

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/m(3) 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.

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 I pm) 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.

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.

Humans have changed their environment to survive and to achieve a safer and more comfortable life. For example, drinking water and wastewater infrastructures are indispensable for civilized societies to flourish and to prevent water-borne infectious diseases. However, excessive loading on environments might disturb microbial ecosystems, resulting in outbreaks of pathogenic microbes and the expansion of infectious diseases. Clarifying the relationship between environmental alterations and changes in microbial ecosystems is thus important to prevent further outbreaks of infectious diseases. The present study aims to understand the links between the following factors: environmental alterations; ecosystem disturbance and the occurrence of infectious disease; and impact on society. We focus on legionellosis and nontuberculous mycobacterial diseases from the viewpoint of their environmental linkage. While Legionella spp. are ubiquitous in aquatic environments, Legionella pneumophila often increases in anthropogenic environments, such as cooling towers or spas, and can cause outbreaks of legionellosis. Recently, travel-associated Legionnaires' disease has caused concern in many countries. The numbers of patients infected with nontuberculous Mycobacteria (NTM) have increased worldwide since the 1990s. Disturbances to microbial ecosystems caused by changes in water usage might be one cause of NTM diseases. Clarifying the dynamics of Legionella pneumophila and NTM in aquatic environments should help prevent outbreaks of diseases associated with these bacteria.

A new method for rapid enumeration of physiologically active mycobacteria was developed by acid-fast bacilli staining (Auramine O) following formazan reduction. Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the widely used culture-dependent approach. (C) 2010 Elsevier B.V. All rights reserved.

Waterborne diseases were globally occurred, and many people were suffered from and often killed by them. In order to prevent outbreaks of waterborne diseases, rapid detection of pathogenic microbes in aquatic environment is the important strategy in addition to the construction of water supply and vaccination. The 16S ribosomal RNA (rRNA) gene is often used as a target gene for bacterial detection using hybridization techniques. In this study, we aimed to design the oligonucleotide probes that could be used for the detection of waterborne bacteria with hybridization techniques because design of specific probes is important to assure for the precise detection of target bacteria. We then evaluated the specificities of designed probes by using an oligonucleotide microarray. In conclusion, we confirmed that seven designed oligonucleotide probes were suitable for the specific detection of waterborne bacteria. These probes appear to be used for 16S rRNA targeted hybridization techniques such as fluorescence in situ hybridization (FISH) and oligonucleotide microarray.

Staphylococcus epidermidis (S. epidermidis) often cause sepsis and related diseases by transfusion of contaminated platelet concentrates (PCs). The proliferation process of this bacterium in PCs has been unclear, thus, bio-imaging system was applied for analyzing the dynamics of S. epidermidis in PCs. S. epidermidis were spiked into PCs or Luria Bertani (LB) broth. These samples were collected at each sampling time during incubation (up to 7 days), and colony-forming-units were counted. Bacterial number and their size distribution in each sample were also determined with a new bio-imaging system. The morphological characters of S. epidermidis growing in the samples were observed precisely by scanning electron microscopy (SEM). The numbers of S. epidermidis were stable for 48 hr after the spiking as lag-phase, while the bio-imaging analysis also showed that aggregates proliferated during "lag-phase." The aggregates were also observed in LB media, however, their sizes were much smaller than those in PCs. SEM suggested that the aggregates were micro-colonies (MCs) of staphylococcal cells and cores of the MCs are composed with platelets (PLTs). Out results suggested that S. epidermidis formed floating MCs in PCs during "lag-phase." Therefore, the term of lag phase of S. epidermidis in PCs should be called as "pseudo-lag phase." The initial processes of forming MCs in PCs are thought to be an interaction between bacterial cells and PLTs. Floating MCs would be the source of biofilms on the inside of PC storage bags. New information obtained in this study would be useful for understanding the dynamics of growing bacteria in PCs.

We applied transcription in vitro following multiplex PCR in order to improve the sensitivity and rapidity of microbial detection in aquatic environment with an oligonucleotide microarray. Transcripts of 16S rRNA gene were fluorescently labeled and hybridized on fabricated oligonucleotide chip. The assay sensitivity was evaluated by detecting cultured bacteria inoculated into natural river water. By using our procedure, the assay was completed more rapidly (6 hr) than conventional oligonucleotide microarray assays (> 12 hr), and its sensitivity was improved: detection limit was decreased by one order of magnitude. This method might be useful for monitoring pathogenic bacteria in aquatic environments.

Three space agencies, JAXA, ESA and NASA, have announced space exploration and utilization plans, and space habitation has become more realistic. After full-scale space habitation is started, environments adequate for human needs must be created and maintained. Comfortable environments for people, however, inevitably provide microorganisms’ favorite conditions for growth and survival. The Russian manned space station “Mir” has experienced fungal damages, and also many researchers demonstrated that bacteria changed their characters, such as pathogenicity, growth rate and resistance to high salt and acidic condition, under microgravity conditions. Investigation of microorganisms in space stations is required to assure not only health but also hardware reliability. In this review, we summarized published researches on microorganisms in space habitation environments.

The digital image analysis software BACS II was developed to enumerate multicolor stained cells rapidly and easily by fluorescence microscopy with multiple excitation. Image analysis requires the following: (i) acquisition of RGB images of the same microscopic fields under different excitations, (ii) subtraction of background, (iii) smoothing and edge detection, (iv) particle recognition and measurement of characteristics (area, length, RGB intensity, etc.), (v) discrimination of cells from other particles, (vi) classification of each cell, and (vii) enumeration. Using the algorithm of BACS II, bacterial cells can be counted based on more than three excitations. The accuracy of enumeration by BACS II was examined in a mixture of three cultured strains (Escherichia coli O157:H7, E. coli K-12, and Staphylococcus epidermidis) triple stained with DAPI, FITC-labeled anti-E. coli O157:H7 antibody, and Cy3-labeled rRNA-targeted probes for specific detection of E. coli cells. Pond water containing indigenous bacteria, algae, and genetically engineered E. coli with a green fluorescent protein-producing gene was stained with DAPI and evaluated as well. There was a strong linear correlation between the counts determined by microscopic visual counting and image analysis for each microbial population (r²=0.94-0.98) over the range of 10²-10⁷ cells/ml. Enumeration of multicolor stained microbial cells by BACS II required less than 15 min from image acquisition to obtain results, while conventional microscopic visual counting took more than 30 min. BACS II should prove useful for any research using fluorescence microscopy, especially when multicolor analysis is needed.