16S rRNA amplicon sequencing of intestinal microbiota in Eurasian Wigeon around Osaka, Japan.

We report 16S rRNA gene amplicon data on the succession of intestinal microbiomes in migratory bird, Eurasian Wigeon (Mareca Penelope) wintering around Osaka, Japan. This study could serve as baseline data for comparison with microbiomes in migratory birds.

Impact of Different Attitudes toward Face-to-Face and Online Classes on Learning Outcomes in Japan.

During the coronavirus disease 2019 (COVID-19) pandemic, online-based learning has become mainstream in many countries, and its learning outcomes have been evaluated. However, various studies have shown that online-based learning needs to be optimized in the future, and the number of reports for this purpose is currently not sufficient. The purpose in this study was to determine the relationship between academic performance and attitudes toward face-to-face and remote formats among Japanese pharmacy students enrolled in a course designed for knowledge acquisition. A combination of face-to-face and remote formats was used in a practice course for sixth-year pharmacy students, designed to improve academic performance through knowledge acquisition. To evaluate learning outcomes, we used a questionnaire that was administered to the course participants and the results of examinations conducted before and after the course. Online-oriented and face-to-face-oriented groups differed in their attitudes toward the ease of asking questions of faculty and communicating with the faculty members and classmates in each format. In a knowledge acquisition course for Japanese pharmacy students, the study revealed that the same academic outcomes were achieved, regardless of the students' own perceptions of their aptitude for face-to-face or remote learning style.

Genome Sequence of Colistin-Resistant Escherichia coli CLR8, Isolated from the Feces of Larus argentatus.

Migratory birds are potential vehicles of antibiotic-resistant bacteria. In this study, a colistin-resistant Escherichia coli strain, CLR8, was isolated from the feces of Larus argentatus The draft genome sequence of the strain indicated that it hosts mcr-1, which is a plasmid-mediated colistin resistance gene.

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.

Draft Genome Sequence of Carbapenem-Resistant Pseudomonas fluorescens Strain BWKM6, Isolated from Feces of Mareca penelope.

Migratory birds are potential vehicles of antibiotic-resistant bacteria. Here, we isolated the multidrug-resistant Pseudomonas fluorescens strain BWKM6 from the feces of Mareca penelope The strain's draft genome sequence indicates that it harbors a metallo-beta-lactamase, a class C beta-lactamase, and several multidrug efflux pumps.

Draft Genome Sequence of Multidrug-Resistant Stenotrophomonas pavanii BWK1, Isolated from Mareca penelope Feces.

Migratory birds serve as vectors by transmitting antibiotic-resistant bacteria across large distances. Here, we isolated a multidrug-resistant Stenotrophomonas pavanii strain, BWK1, from Mareca penelope feces. Analysis of the draft genome sequence of the isolated strain indicated that BWK1 harbors a class A beta-lactamase, metallo-beta-lactamase, and several multidrug efflux pumps.

[Intestinal Microbiota in Migrating Barn Swallows around Osaka].

Migratory birds are considered as vectors of infectious diseases, owing to their potential for transmitting pathogens over large distances. The populations of barn swallow (Hirundo rustica) migrate from Southeast Asia to the Japanese mainland during spring and migrate back to Southeast Asia during autumn. This migratory population is estimated to comprise approximately hundreds to thousands of individuals per year. However, to date, not much is known about the gastrointestinal microbiota of the barn swallow. In this study, we characterized the fecal bacterial community in barn swallow. Using 16S rRNA gene metagenomic sequencing analysis, we examined the presence and composition of potentially pathogenic bacteria in the fecal samples, which were collected during spring season from Osaka. The number (±S.D.) of total bacteria was approximately 2.1(±3.4)×108 per gram of feces. In most samples, the bacterial community composition was dominated by families, such as Enterobacteriaceae, Pseudomonadaceae, Mycoplasmataceae, Enterococcaceae, Streptococcaceae, and Alcaligenaceae. However, no relationship was found between the bacterial community composition and geographical area in the fecal samples. Potentially pathogenic bacteria were detected at the rate of >0.1%, which included Pseudomonas spp., Escherichia/Shigella spp., Enterobacter spp., Yersinia spp., Mycoplasma spp., Enterococcus spp., Achromobacter spp., and Serratia spp. Our results suggested that barn swallow is instrumental in the transmission of these genera over large distances.

Draft Genome Sequence of Extended-Spectrum Beta-Lactamase-Producing Serratia fonticola BWK15 Isolated from Feces of Anas penelope.

Migratory birds have been postulated as potential vehicles of antibiotic resistance. Here we isolated the extended-spectrum beta-lactamase (ESBL)-producing Serratia fonticola strain BWK15 from the feces of Anas penelope The strain's draft genome sequence indicated that it harbors class A ESBL, class C beta-lactamase, and many multidrug efflux pumps.

Draft Genome Sequence of Multidrug-Resistant Cellulosimicrobium sp. Strain KWT-B, Isolated from Feces of Hirundo rustica.

Migratory birds have been postulated as potential spreaders of antibiotic resistance. Multidrug-resistant Cellulosimicrobium sp. strain KWT-B was isolated from the feces of Hirundo rustica A draft genome sequence indicated that the strain harbors multidrug-resistant transporters, multidrug efflux pumps, a vancomycin-resistant protein, and metallo-beta-lactamases.

Draft Genome Sequences of Amoeba-Resistant Aeromonas spp. Isolated from Aquatic Environments.

Amoeba-resistant Aeromonas veronii ARB3 and Aeromonas media ARB13 and ARB20, which may be important intracellular pathogens of eukaryotic hosts, were isolated from pond and river waters. The draft genome sequences indicate that the strains harbor multiple protein secretion systems and toxins that induce disruption of the actin cytoskeleton.

Draft Genome Sequence of an Antifungal Bacterium Isolated from the Breeding Environment of Dorcus hopei binodulosus.

Burkholderia sp. strain A1 was isolated from a decaying log present in the breeding environment of a stag beetle. The draft genome sequence indicates that strain A1 harbors many biosynthesis molecules, which have antimicrobial properties, and thus potentially eliminates the fungi by producing antifungal compounds, such as siderophores.

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.

High-frequency phage-mediated gene transfer in freshwater environments determined at single-cell level.

Lateral gene transfer by phages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency and range of phage-mediated gene transfer, it is important to understand the movement of DNA among microbes. Using an in situ DNA amplification technique (cycling primed in situ amplification-fluorescent in situ hybridization; CPRINS-FISH), we examined the propensity for phage-mediated gene transfer in freshwater environments at the single-cell level. Phage P1, T4 and isolated Escherichia coli phage EC10 were used as vectors. All E. coli phages mediated gene transfer from E. coli to both plaque-forming and non-plaque-forming Enterobacteriaceae strains at frequencies of 0.3-8 x 10(-3) per plaque-forming unit (PFU), whereas culture methods using selective agar media could not detect transductants in non-plaque-forming strains. The DNA transfer frequencies through phage EC10 ranged from undetectable to 9 x 10(-2) per PFU (undetectable to 2 x 10(-3) per total direct count) when natural bacterial communities were recipients. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viability in most cases. These results indicate that the exchange of DNA sequences among bacteria occurs frequently and in a wide range of bacteria, and may promote rapid evolution of the prokaryotic genome in freshwater environments.

Transfer of a phage T4 gene into Enterobacteriaceae, determined at the single-cell level.

The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10(-2) gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients.

Similarity of bacterial community structure between Asian dust and its sources determined by rRNA gene-targeted approaches.

The atmospheric movement of arid soil can play an important role in the movement of microorganisms attached to soil microparticles. Bacterial community structures in Asian dust collected at Beijing were investigated using the 16S rRNA gene sequence and compared to those in arid soil, a possible source of the dust. Asian dust samples contained 2.5×10(7) to 3.5×10(9) copies of the 16S rRNA gene gram(-1). Therefore, more than 10(13) bacterial cells (km)(-2) per month were estimated to arrive in Beijing via Asian dust. Terminal restriction fragment length polymorphism analysis revealed that the bacterial community structures in Asian dust samples differed greatly according to the scale of the dust event. The bacterial communities from major dust events were similar to those from an arid region of China.

Soil microbial community structure in an Asian dust source region (Loess plateau).

The bacterial community structure in four geographically isolated arid regions on the Loess plateau, China, a source of Asian dust, was investigated using a 16S rRNA gene. Denaturing gradient gel electrophoresis and sequencing demonstrated that community diversity in the Loess plateau was low, and a common Alphaproteobacteria phylotype was identified. Phylogenetic analyses of arid soils revealed that most phylotypes had low similarity with known strains in various phyla, suggesting that these regions contain phylogenetically divergent and unknown bacteria.

Application of real-time long and short polymerase chain reaction for sensitive monitoring of the fate of extracellular plasmid DNA introduced into river waters.

The precise estimation of extracellular DNA, long enough to encode a gene, is valuable for determining its potential involvement in genetic transformation. Here, the applicability of real-time long PCR was examined by using target DNA of different lengths and transformation with competent cells to monitor the fate of plasmid DNA released into rivers. Detection limits of the PCR were 7 and 30 copies reaction(-1) for a plasmid (4.1 kbp), and 30 and 3×10(4) copies reaction(-1) for lambda DNA (8.6 kbp and 15.5 kbp). The copy numbers of the plasmid obtained by the real-time long PCR were highly correlated with those determined by the transformation metod (R(2)=0.98). Real-time PCRs targeting a short fragment and full-length plasmid DNA were carried out to monitor fragmentation during 506 h of incubation. After 75 h, more than 100-fold larger amounts of the short fragments persisted compared to the full-length plasmid and the values remained constant in the following days. Real-time long PCR revealed that long DNA persisted in river water for prolonged periods of incubation and is thus useful to assess the fate of target DNA in natural water systems.

High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level.

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification.

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.

Genetic and physiological characterization of the intestinal bacterial microbiota of Bluegill (Lepomis macrochirus) with three different feeding habits.

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the host's feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.