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.

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.

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.

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.

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.

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.

A ten years (2000-2009) surveillance of resistant Enterobacteriaceae in Zhejiang Province, China.

OBJECTIVE: In Zhejiang Province, there are several highly developed cities near the coast and several relatively under-developed mountain areas. Analysis of the composition of bacteria isolated from patients as well as their antibiotic resistance profile from various areas of this province, and tracing of such data year-by-year, will help to delineate the bacterial resistance profile of these areas and to understand how the stage of socio-economical development impacts on the composition of clinical micro-flora and their resistance profile. METHODS: In order to investigate variation in resistance rates and isolation rates of Enterobacteriaceae, from 2000 to 2009 in Zhejiang Province, China, Enterobacteriaceae isolated from 15 hospitals located in different regions of the province were surveyed. RESULTS: The total numbers of the Enterobacteriaceae isolated increased more than 20-fold from 2000 to 2009. Among the Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae were the dominant isolates. The percentage of E. coli and K. pneumoniae that produced detectable extended-spectrum ß-lactamases (ESBLs) increased from 2000 to 2007, and then declined slightly in 2008 and 2009. The percentages of K. pneumoniae and E. coli that were resistant to ceftazidime increased sharply from 2000 to 2009. There were remarkable increases in the carbapenem resistant rates during the decade, but they were much higher for the isolates from the developed cities than from the rural areas. In 2002, carbapenem-resistant E. coli was first found in Hangzhou, one of the highly developed cities in Zhejiang Province. By 2009, carbapenem-resistant bacteria were found for all species of Enterobacteriaceae surveyed in almost all areas of the province, although they were more frequently identified in developed areas than in rural areas. CONCLUSION: Much restrictive actions have to be taken in terms of rational use of antibiotics and nosocomial control to prevent the further spread of the drug-resistant pathogens.

Rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting.

A microfluidic device-based system for the rapid and semiautomated counting of bacteria in freshwater was fabricated and examined. Bacteria in groundwater and in potable water, as well as starved Escherichia coli O157:H7 spiked in pond water, were able to be on-chip stained and enumerated within 1 h using this system.

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.

Rapid and sensitive detection of viable bacteria in contaminated platelet concentrates using a newly developed bioimaging system.

BACKGROUND: Rapid and sensitive methods for the detection of bacteria in platelet concentrates (PCs) are required as well as inactivation techniques to decrease the transfusion-associated risk of infection from bacterially contaminated PCs. In this study, a rapid microbiologic method for the sensitive counting of viable bacteria in PCs was developed by combining a fluorescent staining technique and a bioimaging system. STUDY DESIGN AND METHODS: An esterase indicator, carboxyfluorescein diacetate, was used to detect physiologically active bacteria. Treatment was optimized to selectively remove platelets (PLTs). Bacterial cells trapped on a filter were automatically discriminated from other particles or PLT debris and counted by a bioimaging system. The sensitivity, rapidity, and recovery rates were evaluated using PCs spiked with 14 reference bacterial strains and clinical isolates. RESULTS: Lysis treatment with enzyme and detergent was effective to remove PLTs and white blood cells. Two buffers for fluorescent vital staining were needed for highly sensitive detection of pathogenic bacteria. Fewer than 100 cells spiked in 5-mL PCs were detected by the bioimaging system after treatment and fluorescent staining, and this result shows that PLTs are selectively digested by the treatment. Bacterial cells spiked in 25-mL PCs were detected within 45 minutes (treatment, 15 min; filtration and fluorescent staining, 15 min; automated counting and precise image analysis, 10-15 min). CONCLUSION: The microbiologic method described here is rapid and sensitive, and this method has potential for the screening of PCs contaminated with bacterial cells. Furthermore, this method could contribute to further evaluation of inactivation techniques.

Rapid and automated enumeration of viable bacteria in compost using a micro-colony auto counting system.

The micro-colony method was used to enumerate viable bacteria in composts. Cells were vacuum-filtered onto polycarbonate filters and incubated for 18 h on LB medium at 37 degrees C. Bacteria on the filters were stained with SYBR Green II, and enumerated using a newly developed micro-colony auto counting system which can automatically count micro-colonies on half the area of the filter within 90 s. A large number of bacteria in samples retained physiological activity and formed micro-colonies within 18 h, whereas most could not form large colonies on conventional media within 1 week. The results showed that this convenient technique can enumerate viable bacteria in compost rapidly for its efficient quality control.

Rapid quantification of bacterial cells in potable water using a simplified microfluidic device.

A simplified microfluidic device for quantification of bacteria in potable water was fabricated and examined. Comparisons of counts of Escherichia coli by the microfluidic system and by epifluorescence microscopy closely correlated (r2=0.99). Bacteria in natural mineral water and in purified household tap water were accurately enumerated by using this system within 15 min after fluorescent staining.

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.

Simultaneous enumeration of viable Enterobacteriaceae and Pseudomonas spp. within three hours by multicolor fluorescence in situ hybridization with vital staining.

A new means of rapidly and simultaneously counting viable phylogenetically different bacteria was developed. The cyanine dimer dye, BOBO-3 that selectively stains bacteria with damaged membranes were used to evaluate bacterial viability based on membrane integrity. Viable Enterobacteriaceae and Pseudomonas spp. could be selectively detected within three hours using multicolor fluorescence in situ hybridization (FISH) following BOBO-3 staining (BOBO3-FISH).

Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system.

A rapid bead assay for detecting pathogenic bacteria with a simple microfluidic chip-based system was developed. Five oligonucleotide probes corresponding to the 16S rRNA of the targeted bacteria were coupled covalently to fluorescent beads. Four species of bacteria (Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, Yersinia enterocolitica, and Bacillus cereus) were used as representative food-borne pathogenic bacteria. The RNAs extracted from pure cultures of these microorganisms were fluorescently labeled and hybridized to the oligonucleotide probes-immobilized fluorescent beads (Bead assay). The duplexes of RNAs and the probes-immobilized beads were analyzed with the commercially available microfluidic chip-based system. This bead assay provided results within 3 h following RNA extraction from bacterial cells.

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.

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.