Microbial growth and importance of flushing inside closed-type infusion devices during administration of lipid emulsion in vitro setting.

Background: We investigated the extent of growth of microorganisms with simultaneous administration of lipid emulsions with infusions for Total Parenteral Nutrition (TPN), assuming that the lipid emulsions contaminated with microorganisms are stagnant in a closed-type infusion device. We also investigated if bacterial growth can be prevented in the infusion device by flushing the inside of the infusion device with saline solution after the administration of lipid emulsion from the side tube in vitro setting. Methods: We made a preparation by adding Escherichia coli to the lipid emulsion and started the infusion simultaneously with the infusion solution for TPN and lipid emulsion with the piggyback method. Immediately after the completion of lipid emulsion infusion, we conducted flushing with saline solution. The volume of saline solution was none, 5, 10, or 20 mL at a flow rate of 1 mL/s. Infusion solution that was stagnant in the infusion device was collected immediately before completing the lipid emulsion infusion and 20 h after flushing, i.e., 24 h after starting the infusion for TPN, and the number of viable bacteria was determined. Results: The number of viable E. coli increased in the infusion device of all three species used in this experiment 24 h after starting the lipid emulsion infusion without flushing. We found that bacterial growth could be prevented through flushing with saline solution after the completion of lipid emulsion infusion and flushing out the stagnant infusion solution in the closed-type infusion device. Conclusions: We found that if E. coli was present in the closed-type infusion device, it would multiply. We also found that the number of viable bacteria varied according to the variety and internal structure of the closed-type infusion device as well as the liquid volume used for flushing, although flushing can prevent the growth of microorganisms. Proper management and manipulation of infusion is required to prevent infection.

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.

Bactericidal effects of deep ultraviolet light-emitting diode for solutions during intravenous infusion.

Background: Ultraviolet irradiation is effectively used as a disinfection method for inactivating microorganisms. Methods: We investigated the bactericidal effects by irradiation with a deep-ultraviolet light-emitting diode (DUV-LED) on the causative microorganisms of catheter related blood stream infection contaminating the solution for intravenous infusion. For irradiation, prototype modules for water disinfection with a DUV-LED were used. Experiments were conducted on five kinds of microorganisms. We examined the dependence of bactericidal action on eleven solutions. Administration sets were carried out three types. Results: When the administration set JY-PB343L containing the infusion tube made of polybutadiene was used, the bactericidal action of the DUV-LED against all tested microorganisms in the physiological saline solutions was considered to be effective. We confirmed that the number of viable bacteria decreased in 5% glucose solution and electrolyte infusions with DUV-LED irradiation. Conclusions: These results indicate that the DUV-LED irradiation has bactericidal effects in glucose infusion and electrolyte infusions by irradiating via a plasticizer-free polybutadiene administration set. We consider DUV-LED irradiation to be clinically applicable.

[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.

Water Soluble Vitamins Enhance the Growth of Microorganisms in Peripheral Parenteral Nutrition Solutions.

Peripheral parenteral nutrition (PPN) solutions contain amino acids, glucose, and electrolytes, with or without some water soluble vitamins. Peripheral venous catheters are one of the causes of catheter related blood stream infection (CRBSI), which requires infection control. In Japan, PPN solutions have rarely been prepared under aseptic conditions. However, in recent years, the necessity of adding vitamins to infusions has been reported. Therefore, we investigated the effects of water soluble vitamins on growth of microorganisms in PPN solutions. AMINOFLUID® (AF), BFLUID® (BF), PARESAFE® (PS) and PAREPLUS® (PP) PPN solutions were used. Water soluble vitamins contained in PP were also used. Causative microorganisms of CRBSI were used. Staphylococcus epidermidis decreased after 24 hours or 48 hours in all solutions. On the other hand, Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans increased, especially in PP. When each water soluble vitamin was added to BF and PS, growth of S. aureus was greater in solutions that contained nicotinamide than in solutions that contained other vitamins. As for C. albicans, they grew in all test solutions. C. albicans grew especially well in solutions that contained biotin. When commercial amino acids and glucose solutions with electrolytes are administered, in particular those containing multivitamins or water soluble vitamins, efforts to control infection must be taken to prevent proliferation of microorganisms.

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.

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.

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.

Inactivation of bacteria in freshwater by momentary decompression following high pressurization.

Rapid and continuous pressure treatment was realized using a hydraulic pump and the momentary decompression following high pressurization was used to inactivate bacteria. The number of colony-forming E. coli decreased to 1/1000 in response to 10 cycles of pressure treatment. In groundwater samples, repeated pressure treatment led to a two-log decrease in the number of colony-forming bacteria. These findings suggest that repeated cycles of momentary decompression following high pressurization enabled a marked decrease in bacterial growth activity. The results presented herein may contribute to microbiological quality control and the safety of freshwater.

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.

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.

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.

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.