Mutation frequency of Escherichia coli isolated from river water: potential role in the development of antimicrobial resistance.

Bacteria acquire genetic variations that help them to adapt to stressful environmental conditions, and these changes may be associated with the development of antimicrobial resistance. In this study, we investigated the mutation frequencies of 270 isolates of Escherichia coli from river water, which represents a relatively unstressful environment. As we predicted, mutation frequencies of the E. coli isolates ranged from <1 × 10-11 to 6.3 × 10-8 (median, 1.7 × 10-9), and a strong mutator (≥ 4 × 10-7) was not detected. To better understand the role of mutation frequency in the development of antimicrobial resistance, we assessed antimicrobial sensitivity after exposure of the E. coli isolates to subinhibitory concentrations of ciprofloxacin, as a surrogate for stress. We found that antimicrobial resistance increased in bacteria with a low mutation frequency after exposure, and the relative increase in antimicrobial resistance generally increased, depending on the mutation frequency. Thus, mutation frequency may contribute to the development of antimicrobial resistance of bacteria in natural environments.

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis.

We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.

Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.

Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.

Chlamydia pneumoniae enhances Interleukin 8 (IL-8) production with reduced azithromycin sensitivity under hypoxia.

Obligate intracellular bacterium Chlamydia pneumoniae causes respiratory tract infections such as community-acquired pneumonia. During infection, C. pneumoniae induces inflammatory responses in host cells and the oxygen concentration at the infection sites subsequently decreases. Because hypoxic conditions influence further inflammatory responses and reduced antibacterial effects, this may exacerbate the C. pneumoniae infection. Here, we show inflammatory responses and drug sensitivity in C. pneumoniae-infected cells under hypoxic conditions. First, we confirmed the enhanced growth of C. pneumoniae under hypoxia, which indicates that the hypoxic condition we used could adequately reproduce past reports. We then demonstrated a significant increase in production of the pro-inflammatory cytokine Interleukin 8 (IL-8) in C. pneumoniae-infected cells under hypoxic conditions. Furthermore, hypoxia decreased the antibacterial effects of azithromycin against C. pneumoniae compared with normoxic conditions. Together, our data suggest that inflammatory responses and drug sensitivity may have been underestimated in C. pneumoniae infection in previous studies. Thus, to accurately understand the Chlamydia infection, it may be necessary to perform in vitro experiments under hypoxic conditions.

A simple and short microbiology practical improves undergraduate nursing students' awareness of bacterial traits and ability to avoid spreading infections.

BACKGROUND: Nurses are responsible for implementing appropriate measures to reduce hospital infections, especially with multidrug resistant bacteria, so nursing students should learn about microbiology. This helps them to understand bacterial dissemination and infectious disease control. Because of tight schedules, however, its teaching is limited in undergraduate nursing classes in Japan. We therefore tested whether a simple short practical session in a microbiology class could help to improve undergraduate nursing students' awareness of bacterial traits and how to prevent infections. METHODS: This study involved second-grade nursing students (n = 76). Two short practical sessions (a total of 3 h, across 2 days) were used to assess the effectiveness of washing or disinfection on hand bacteria in a 16-class microbiology course (total class time was 24 h, plus an exam). Hand bacteria were sampled on LB agar plates with orientation during the first half-day, and the plates examined for colonies with distinct color or morphological traits, and discussed, in the second session, a week later. Questionnaires before and after the exercise were used to assess changes in awareness of unseen bacteria inhabiting around us connecting bacterial traits and how to prevent infections. RESULTS: The results showed that the practical increased the nursing students' awareness of fomites (utensils) (p = 0.0115), fomites (contact-based) (p = 0.0016), habitats (body surface) (p = 0.0127), action facilitating hospital infection (p = 0.0166), and changes in physical condition caused by bacterial infections (p = 0.0136). There were no changes in word associations (p = 0.627) or habitats (inside body) (p = 0.308). Difficulty score, which is an element in questionnaire psychometric properties, tended to be close to the expected score through the practical, but not statistical significant. In addition, regardless of before or after practical, Cronbach α score, which is an indicator of the reliability among items of multi-choice questions, showed > 0.8, indicating validity of evaluation items. Thus, the student's awareness of unseen bacteria inhabiting around us was significantly increased as compared to those before practical in microbiology class. CONCLUSIONS: The simple short practical effectively improved nursing students' awareness of unseen bacteria inhabiting around us in microbiology course, useful for even tight teaching schedules.

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.

The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.

Analysis of adult damselfly fecal material aids in the estimation of antibiotic-resistant Enterobacterales contamination of the local environment.

Because damselflies are ubiquitously but focally present in natural environments and play a critical role as predators of other insect species, the fecal matter of damselflies may be useful for investigating antibiotic-resistant bacterial populations, including human pathogens, in local environments. We therefore examined the prevalence of antibiotic-resistant bacteria, including Enterobacterales, in fecal material from 383 damselflies (adults and larvae) collected from seven locations around Sapporo City, Japan, in 2016 and 2017. Fecal samples were plated on soybean casein digest (SCD) agar plates with and without antibiotics (SCD-A and SCD-w/o, respectively) to identify environmental bacteria and gut bacteria, respectively, and on MacConkey agar plates with antibiotics (MacConkey-A) to select for Gram-negative bacteria, including human pathogenic Enterobacterales species. The prevalence of colonies on each of the plates was compared, and representative colonies on MacConkey-A plates were identified to the species level using an API 20E kit and the MALDI Biotyper system. Overall, SCD-w/o plates showed a gut bacterial load of approximately 108 colony-forming units per adult damselfly or larva. There was a significant difference between the prevalence of colonies on the SCD-A and MacConkey-A plates, and a significantly increased prevalence of antibiotic-resistant bacteria on MacConkey-A plates was observed in samples collected from Shinoroshinkawa. Cluster analysis based on minimum inhibitory concentration values of 59 representative isolates from MacConkey-A agar plates revealed that samples from Shinoroshinkawa contained a higher prevalence of Enterobacterales than those from other sampling locations. Thus, fecal materials discharged by adult damselflies could be used in future studies as a simple tool for estimating antibiotic-resistant bacteria, including Enterobacterales species, in the local environment.

Tetrahymena promotes interactive transfer of carbapenemase gene encoded in plasmid between fecal Escherichia coli and environmental Aeromonas caviae.

Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP-1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid-acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10-4 to 10-5 . All plasmid-acquired bacteria showed a MIC against CAZ of >128 µg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP-1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co-localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple-antibiotic resistant bacteria.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Impact of bacterial traces belonging to the Enterobacteriaceae on the prevalence of Chlamydia trachomatis in women visiting a community hospital in Japan.

We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n = 257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n = 27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n = 20) and -negative samples (n = 27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae.

Obligate intracellular chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. While pathogenic chlamydiae have adapted to a wide range of vertebrates, environmental chlamydiae inhabit unicellular amoebae, the free-living Acanthamoeba. However, how and why this divergence occurred remains unclear. Meanwhile, giant viruses consisting of protozoa-related and protozoa-unrelated viruses have been discovered, with the former group being suggested to have more influenced environmental chlamydiae during their evolution while cohabiting host amoebae. Against this background, we attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were significantly shared in both the chlamydia genomes depending on the giant virus type. In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high. Meanwhile, the prevalence of protozoa-related giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae; the actual number of sequences similar to giant virus was also significantly predominant compared with those in the environmental chlamydial genomes. Among them, the most prevalent of giant virus was in the case of chlamydiae with Megavirus chiliensis; total of 1338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) showed the presence of orthologs between these with several clustered. In addition, Pearson's single regression analysis revealed that almost the prevalence of the genes from the giant viruses in chlamydial genomes was negatively and specifically correlated with the number of chlamydial open reading frames (ORFs). Thus, these results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of a putative linkage between chlamydiae and the giant viruses, providing us with a hint to understand chlamydial evolution.

Acanthamoeba S13WT relies on its bacterial endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid media.

Soil-borne amoeba Acanthamoeba S13WT has an endosymbiotic relationship with an environmental Neochlamydia bacterial strain. However, regardless of extensive experiments in liquid media, the biological advantage of the symbiosis remained elusive. We therefore explored the role of the endosymbiont in predator-prey interactions on solid media. A mixed culture of the symbiotic or aposymbiotic amoebae and GFP-expressing Escherichia coli or Salmonella Enteritidis was spotted onto the centre of a LB or B-CYE agar plate preinoculated with a ring of mCherry-expressing Legionella pneumophila (Legionella 'wall'). The spread of the amoebae on the plate was assessed using a fluorescence imaging system or scanning electron microscopy. As a result, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacked these GFP-expressing bacteria and formed flower-like fluorescence patterns in an anticlockwise direction. Other bacteria (Pseudomonas aeruginosa and Stenotrophomonas maltophilia), but not Staphylococcus aureus, were also backpacked by the symbiotic amoebae on LB agar, although lacked the movement to anticlockwise direction. Furthermore, in contrast to the aposymbiotic amoebae, the symbiotic amoebae backpacking the E. coli broke through the Legionella 'wall' on B-CYE agar plates. Thus, we concluded that Acanthamoeba S13WT required the Neochlamydia endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid agar.

Long-term survival of Naegleria polaris from Antarctica after 10 years of storage at 4 °C.

A free-living amoeba, Naegleria is ubiquitously distributed in various natural environments. Since some Naegleria spp. are exclusively distributed in the Arctic and sub-Antarctic regions, we hypothesized that the amoeba may be useful to determine long-term survival of Naegleria in laboratory conditions at 4 °C. The main objective of the study is to determine that a species of an environmental amoebal isolated can live at low temperatures after a long time. Here, we therefore show long-term survival of an amoeba, Naegleria polaris isolated from a sediment sample, which was collected from Antarctica 10 years ago, and since stored at 4 °C. The sample was put on non-nutrient agar plates with heat-killed Escherichia coli, and then the plate was incubated at 4, 15, or 30 °C. Motile amoebae were seen only when the plate was incubated at 15 °C. The sequencing of ribosomal DNA including internal transcribed spacers (ITS) 1, 5.8S rDNA, and ITS2 region revealed the amoebae to be N. polaris, which is exclusively distributed in the Arctic and sub-Antarctic regions. Scanning electron microscopic observation showed that no typical sucker-like structure was seen on the surface of N. polaris, but the cysts were similar to those of Naegleria fowleri. Thus, our result shows, for the first time, that N. polaris can survive after 10 years of storage at 4 °C. This finding may help us understand the still undescribed effects of environmental samples on viability of amoebae.

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry.

Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells.

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXRα inhibitor; and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism.

Walker occupancy has an impact on changing airborne bacterial communities in an underground pedestrian space, as small-dust particles increased with raising both temperature and humidity.

Although human occupancy is a source of airborne bacteria, the role of walkers on bacterial communities in built environments is poorly understood. Therefore, we visualized the impact of walker occupancy combined with other factors (temperature, humidity, atmospheric pressure, dust particles) on airborne bacterial features in the Sapporo underground pedestrian space in Sapporo, Japan. Air samples (n = 18; 4,800L/each sample) were collected at 8:00 h to 20:00 h on 3 days (regular sampling) and at early morning / late night (5:50 h to 7:50 h / 22:15 h to 24:45 h) on a day (baseline sampling), and the number of CFUs (colony forming units) OTUs (operational taxonomic units) and other factors were determined. The results revealed that temperature, humidity, and atmospheric pressure changed with weather. The number of walkers increased greatly in the morning and evening on each regular sampling day, although total walker numbers did not differ significantly among regular sampling days. A slight increase in small dust particles (0.3-0.5µm) was observed on the days with higher temperature regardless of regular or baseline sampling. At the period on regular sampling, CFU levels varied irregularly among days, and the OTUs of 22-phylum types were observed, with the majority being from Firmicutes or Proteobacteria (γ-), including Staphylococcus sp. derived from human individuals. The data obtained from regular samplings reveled that although no direct interaction of walker occupancy and airborne CFU and OTU features was observed upon Pearson's correlation analysis, cluster analysis indicated an obvious lineage consisting of walker occupancy, CFU numbers, OTU types, small dust particles, and seasonal factors (including temperature and humidity). Meanwhile, at the period on baseline sampling both walker and CFU numbers were similarly minimal. Taken together, the results revealed a positive correlation of walker occupancy with airborne bacteria that increased with increases in temperature and humidity in the presence of airborne small particles. Moreover, the results indicated that small dust particles at high temperature and humidity may be a crucial factor responsible for stabilizing the bacteria released from walkers in built environments. The findings presented herein advance our knowledge and understanding of the relationship between humans and bacterial communities in built environments, and will help improve public health in urban communities.

Measurement of NET formation in vitro and in vivo by flow cytometry.

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane-impermeable DNA-binding dye, SYTOX Green. SYTOX Green-positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Green-positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co-localization of MPO and plasma membrane-appendant DNA of SYTOX Green-positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green-positive cells induced by PMA. The collective evidence suggests that SYTOX Green-positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well-used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Diversity changes of microbial communities into hospital surface environments.

Previous works have demonstrated considerable variability in hospital cleanliness in Japan, suggesting that contamination is driven by factors that are currently poorly controlled. We undertook 16S rRNA sequence analysis to study population structures of hospital environmental microbiomes to see which factor(s) impacted contamination. One hundred forty-four samples were collected from surfaces of three hospitals with distinct sizes ("A": >500 beds, "B": 100-500 beds, "C": <100 beds). Sample locations of two ward types (Surgical and Internal) included patient room bed table (multiple) (4BT), patient overbed table (multiple) (4OT), patient room sink (multiple) (4S), patient room bed table (single) (SBT), patient overbed table (single) (SOT), patient room sink (single) (SS), nurse desk (ND), and nurse wagon (NW). Total DNA was extracted from each sample, and the 50 samples that yielded sufficient DNA were used for further 16S rRNA sequencing of hospital microbiome populations with cluster analysis. The number of assigned bacterial OTU populations was significantly decreased in hospital "C" compared to the other hospitals. Cluster analysis of sampling locations revealed that the population structure in almost all locations of hospital "C" and some locations in the other hospitals was very similar and unusually skewed with a family, Enterobacteriaceae. Interestingly, locations included patient area (4OT, 4BT, SBT) and nurse area (ND), with a device (NW) bridging the two and a place (4S and SS) shared between patients or visitors. We demonstrated diversity changes of hospital environmental microbiomes with a skewed population, presumably by medical staff pushing NWs or sinks shared by patients or visitors.

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation.

BACKGROUND: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. RESULTS: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heat-killed Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified. CONCLUSION: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.