Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are a major source of Legionella infections in Japan. In this study, we performed 16S rRNA gene amplicon sequencing to characterize the bacterial community in bath and shower water from public bath facilities, along with chemical parameters, and investigated the effect of the bacterial microbiome on the presence of Legionella species. Although no significant difference in bacterial community richness was observed between bath and shower water samples, there was a remarkable difference in the bacterial community structure between them. Distance-based redundancy analysis revealed that several factors (free residual chlorine, pH, and conductivity) were correlated with the bacterial community in bath water. The most abundant bacterial genera in the samples were Pseudomonas (13.7%) in bath water and Phreatobacter (13.6%) in shower water, as indicated by the taxonomic composition, and the dominant bacteria differed between these environmental samples. Legionella pneumophila was the most frequently detected Legionella species, with additional 15 other Legionella species detected in water samples. In Legionella-positive water samples, several unassigned and uncultured bacteria were enriched together. In addition, the co-occurrence network showed that Legionella was strongly interconnected with two uncultured bacteria. Corynebacterium and Sphingomonas negatively correlated with Legionella species. The present study reveals the ecology of Legionella species, especially their interactions with other bacteria that are poorly understood to date. IMPORTANCE: Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

Gene expression of axenically-isolated clinical Entamoeba histolytica strains and its impact on disease severity of amebiasis.

The severity of Entamoeba histolytica infection is determined by host immunology, pathogen virulence, and the intestinal environment. Conventional research for assessing pathogen virulence has been mainly performed using laboratory strains, such as a virulent HM-1: IMSS (HM-1) and an avirulent Rahman, under various artificial environmental conditions because of the difficulties of axenic isolation of the clinical strains. However, it is still unclear whether scientific knowledge based on laboratory strains are universally applicable to the true pathogenesis. Hereby, we performed transcriptomic analysis of clinical strains from patients with different degrees of disease severity, as well as HM-1 under different conditions. Even after several months of axenization, Clinical strains show the distinct profile in gene expression during in vitro passage, moreover, difference between any 2 of these strains was much greater than the changes on the liver challenge. Interestingly, 26 DEGs, which were closely related to the biological functions, were oppositely up- or down regulated between virulent Ax 19 (liver abscess) and avirulent Ax 11 (asymptomatic carrier). Additionally, RNAseq using laboratory strain (HM1) showed more than half of genes were differently expressed between continuously in vitro passaged HM1 (in vitro HM1) and periodically liver passaged HM1 (virulent HM1), which was much greater than the changes on the liver passage of virulent HM1. Also, transcriptomic analysis of a laboratory strain revealed that continuous environmental stress enhances its virulence via a shift in its gene expression profile. Changes in gene expression patterns on liver abscess formation were not consistent between clinical and laboratory strains.

Microbial Contamination of Rubber Ducks Floating in Bathtubs of Bathing Facilities, and an Evaluation of Their Washing Methods.

Microbiological contamination inside rubber ducks floating in the bathtub at a "duck bath" of a bathing facility was analyzed by examining bacterial and amoebic counts. The results of microbial tests, such as standard plate count, heterotrophic plate count and Legionella-LAMP (Loopmediated isothermal amplification) , showed that microbial contamination increased in the rubber ducks over time. When the ducks were washed with sodium hypochlorite, those microbial contaminations were not detected; but when the ducks were washed with an electrolyzed water, the standard plate counts and the heterotrophic plate counts were detected in the amount of 103 per duck in the wipe samples. Without proper washing of rubber ducks, bacteria and free-living amoeba can grow and colonize biofilms, and can thereby cause infection in humans. Also, microbial contamination inside ducks may reduce chlorination of the entire bathtub and cause bacterial infection such as Legionellosis from the bathtub water.

Near-chromosome level genome assembly reveals ploidy diversity and plasticity in the intestinal protozoan parasite Entamoeba histolytica.

BACKGROUND: Amoebozoa is a eukaryotic supergroup composed of unicellular and multicellular amoebic protozoa (e.g. Acanthamoeba, Dictyostelium, and Entamoeba). They are model organisms for studies in cellular and evolutionary biology and are of medical and veterinary importance. Despite their importance, Amoebozoan genome organization and genetic diversity remain poorly studied due to a lack of high-quality reference genomes. The slime mold Dictyostelium discoideum is the only Amoebozoan species whose genome is available at the chromosome-level. RESULTS: Here, we provide a near-chromosome-level assembly of the Entamoeba histolytica genome, the second semi-completed Amoebozoan genome. The availability of this improved genome allowed us to discover inter-strain heterogeneity in ploidy at the near-chromosome or sub-chromosome level among 11 clinical isolates and the reference strain. Furthermore, we observed ploidy-independent regulation of gene expression, contrary to what is observed in other organisms, where RNA levels are affected by ploidy. CONCLUSIONS: Our findings offer new insights into Entamoeba chromosome organization, ploidy, transcriptional regulation, and inter-strain variation, which will help to further decipher observed spectrums of virulence, disease symptoms, and drug sensitivity of E. histolytica isolates.

Hydroxyapatite powder cake filtration reduces false positives associated with halophilic bacteria when evaluating Escherichia coli in seawater using Colilert-18.

Escherichia coli is an important fecal indicator bacterium that is used to evaluate the microbiological quality of water. The Colilert-18 (Quanti-Tray/2000) is a widely used, rapid, and simple quantitative method for detecting E. coli in drinking water, bathing water, and wastewater. However, Colilert-18 method is less reliable for seawater; false positives are often caused by halophilic bacteria such as Vibrio. While false positives can be avoided by diluting the sample by 10 times or more, the resulting decrease in detection limit makes it difficult to quantify E. coli in seawater. In this study, we combined cake filtration, using hydroxyapatite powder, with the Colilert-18 method to remove salinity without diluting the water sample. When quantifying E. coli in river water, the E. coli concentration obtained from the cake filtration/Colilert-18 method showed a high quantitative value of 90% or more, on average, compared to the concentration obtained with the original Colilert-18 method. The E. coli concentrations in seawater determined using the developed method were similar to those determined using the modified m-TEC method, with no false positives. Highly reliable quantitative values can be obtained using the proposed method because it is possible to measure 100 times as much sample compared to the dilution method. Thus, the developed method is expected to be a powerful tool that can eliminate the problem of false positives.

The Molecular Detection of Cryptosporidium and Giardia in Sika Deer (Cervus Nippon Centralis) in Japan.

Fecal specimens (271 samples) from wild deer, Cervus nippon centralis, were collected from nine different areas in Japan; these samples were subjected to a real-time reverse transcription PCR for Cryptosporidium-and Giardia-specific 18S ribosomal RNA to investigate the prevalence of Cryptosporidium and Giardia infection. The incidence of Cryptosporidium and Giardia in the nine areas ranged from 0% to 20.0% and 0% to 3.4%, respectively. The prevalence of Cryptosporidium among male and female deer was 8.1% and 3.9%, respectively, while that of Giardia was 0.7% and 0.8%. Sequence analysis identified the Cryptosporidium deer genotype, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium meleagridis from the sequence of Cryptosporidium-specific partial 18S ribosomal RNA and Giardia intestinalis assemblage A from the partial sequence of Giardia-specific 18S rRNA. The variation in regional prevalence indicates that Cryptosporidium infection depends on environmental factors, and that bovine Cryptosporidium was detected more frequently than cervine Cryptosporidium. These data suggest that wild deer might be a healthy carrier of bovine Cryptosporidium.

Draft Genome Sequence of Mycobacterium sp. Strain shizuoka-1, a Novel Mycobacterium Isolated from Groundwater of a Bathing Facility in Shizuoka, Japan.

Mycobacterium sp. strain shizuoka-1 is a rapidly growing scotochromogenic mycobacterium and was isolated from well water for a bathing facility in Shizuoka Prefecture in Japan. Here, we report the draft sequence of its genome, comprising a 6.5-Mb chromosome. This mycobacterium has 83.1% identity with Mycobacterium rhodesiae, a human pathogen.

Characterization of Sarcocystis fayeri's actin-depolymerizing factor as a toxin that causes diarrhea.

Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15-kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea-inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15-kDa protein. The deduced amino acid sequence showed homology to actin-depolymerizing factor (ADF). A recombinant 15-kDa protein depolymerized prepolymerized actins in a test tube. The 15-kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15-kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15-kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15-kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor-α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeri's ADF, our findings may offer new insight into research on parasites and parasite-instigated food poisoning.

Complete sequence and characterization of the mitochondrial genome of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium (Cestoda: Diphyllobothriidae), using next generation sequencing.

We first constructed and characterized the complete mitochondrial genome (mitogenome) sequence of Diphyllobothrium stemmacephalum, the type species of genus Diphyllobothrium, using next generation sequencing (NGS). The mitogenome of D. stemmacephalum was 13,716bp, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 longer intergenic non-coding regions, and has features common to mitogenomes of other cestodes. Although it has been accepted that tRNA for serine (trnS2(UCN)) in Platyhelminthes lacks a D arm, the trnS2(UCN) of D. stemmacephalum was predicted to have a paired D arm as in Diplogonoporus balaenopterae. The non-coding region 2 contained eight tandem repeat units (34nucleotides/unit). This study also corroborated that D. stemmacephalum is phylogenetically more closely related to Dip. balaenopterae than to Diphyllobothrium latum and Diphyllobothrium nihonkaiense. As demonstrated here, mitogenome sequence data obtained using NGS is useful for gaining a better understanding of the systematics, phylogeny and taxonomic revisions involving valuable specimens preserved in museums, universities or research institutes for which sequence data are not yet available, and also for making diagnoses based on clinical samples.

The Development of a Novel, Validated, Rapid and Simple Method for the Detection of Sarcocystis fayeri in Horse Meat in the Sanitary Control Setting.

Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10(4) copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure.

Depletion of Cryptosporidium parvum oocysts from contaminated sewage by using freshwater benthic pearl clams (Hyriopsis schlegeli).

The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.

Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).

We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water.

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0×10(3)(3.48log)counts mL(-1). We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥3.48 or <3.48log total bacterial counts mL(-1) were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe(2+), Mn(2+), NH(4)(+), skin debris, and/or biofilms in the water.

Plasmodium falciparum: differing effects of non-esterified fatty acids and phospholipids on intraerythrocytic growth in serum-free medium.

Different combinations of non-esterified fatty acids (NEFA) had variable effects on intraerythrocytic growth of Plasmodium falciparum. All stages of the parasite cultured in medium supplemented with cis-9-octadecenoic acid (C18:1-cis-9), hexadecanoic acid (C16:0), phospholipids (Pld) and bovine albumin free of NEFA were similar to those grown in complete growth medium. Three typical growth patterns indicating suppressed schizogony (SS), suppressed formation of merozoites (SMF), and inhibited invasion of merozoites (IMI) resulted from culture in other combinations of lipids. Unsaturated or saturated NEFA with longer or shorter carbon chains than C18:1-cis-9 or C16:0, higher degree of unsaturation, and trans-forms mainly resulted in SS and SMF effects. However, IMI or partial IMI was observed with tetradecanoic acid or octadecanoic acid enriched with C18:1-cis-9, and cis-9-hexadecenoic acid plus C16:0. Isoforms of C18:1-cis-9 also mainly resulted in partial IMI. SMF also occurred with C18:1-cis-9 plus C16:0 in the absence of Pld. Thus different NEFA exerted distinct roles in erythrocytic growth of the parasite by sustaining development at different stages.

[Legionella contamination risk factors in non-circulating hot spring water].

We examined water from 182 non-circulating hot spring bathing facilities in Japan for possible Legionella occurrence from June 2005 to December 2006, finding Legionella-positive cultures in 119 (29.5%) of 403 samples. Legionellae occurrence was most prevalent in bathtub water (39.4%), followed by storage tank water (23.8%), water from faucets at the bathtub edge (22.3%), and source-spring water (8.3%), indicating no statistically significant difference, in the number of legionellae, having an overall mean of 66 CFU/100mL. The maximum number of legionellae in water increased as water was sampled downstream:180 CFU/100 mL from source spring, 670 from storage tanks, 4,000 from inlet faucets, and 6,800 from bathtubs. The majority--85.7%--of isolated species were identified as L. pneumophila : L. pneumophila serogroup (SG) 1 in 22%, SG 5 in 21%, and SG 6 in 22% of positive samples. Multivariate logistic regression models used to determine the characteristics of facilities and sanitary management associated with Legionella contamination indicated that legionellae was prevalent in bathtub water under conditions where it was isolated from inlet faucet/pouring gate water (odds ratio [OR] = 6.98, 95% confidence interval [CI] = 2.14 to 22.8). Risk of occurrence was also high when the bathtub volume exceeded 5 m3 (OR = 2.74, 95% CI = 1.28 to 5.89). Legionellae occurrence was significantly reduced when the bathing water pH was lower than 6.0 (OR = 0.12, 95% CI = 0.02 to 0.63). Similarly, occurrence was rare in inlet faucet water or the upper part of the plumbing system for which pH was lower than 6.0 (OR = 0.06, 95% CI = 0.01 to 0.48), and when the water temperature was maintained at 55 degrees C or more (OR = 0.10, 95% CI = 0.01 to 0.77). We also examined the occurrence of amoeba, Mycobacterium spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus in water samples.

Plasmodium falciparum: development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer.

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.

Occurrence of Cryptosporidium sp. in snakes in Japan.

The aim of this study was to determine the prevalence of Cryptosporidium in snakes in Japan. Fecal samples or intestinal contents of 469 snakes, consisting of five species, were analyzed and Cryptosporidium oocysts were detected only from the Japanese grass snake Rhabdophis tigrinus. The mean prevalence of Cryptosporidium sp. in Japanese grass snakes was approximately 26% in the region studied. Histopathological observations revealed that the organism caused proliferative enteritis in the small intestine. Sequence analysis of a fragment of the small subunit rRNA gene has shown that the partial sequence of Cryptosporidium sp. isolated from the snakes was identical to that of the Cryptosporidium snake genotype W11 from New Guinea viper boa.

Expression of stromelysin-3 (matrix metalloproteinase-11) in macrophages of murine thymus following thymocyte apoptosis.

High expression of stromelysin-3 (ST-3), also known as matrix metalloproteinase-11, has been implicated in tumor progression and intense tissue remodeling. Nonetheless, details of the cell type(s) expressing ST-3 are less well defined. Here, we report that ST-3 expression was elevated in mouse thymus following thymocyte apoptosis after administration of anti-CD3 Ab. TUNEL analysis revealed that many thymocytes in the cortical region were induced to apoptotic cell death 14 h after the injection. After an additional 2-6 h, ST-3 expression in the thymus was more apparent. Co-staining analysis by anti-ST-3 and F4/80 Abs showed that most F4/80-positive macrophages were also positive for ST-3. Murine peritoneal macrophages were found to constitutively express ST-3, and exposure to apoptotic cells hardly affected ST-3 expression in the macrophages. Taken together, our results indicate that ST-3 is not involved in the execution process of thymocyte apoptosis, and the increased levels of ST-3 in the thymus may be due to the presence of macrophages responsible for clearance of apoptotic cells.