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.

Detection of Legionella species, the influence of precipitation on the amount of Legionella DNA, and bacterial microbiome in aerosols from outdoor sites near asphalt roads in Toyama Prefecture, Japan.

BACKGROUND: Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites, such as buildings and private homes, using amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing. RESULTS: Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = - 0.29, P < 0.01) before the sampling day, respectively. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features. CONCLUSIONS: DNA from Legionella species was widely present in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Size resolved characteristics of urban and suburban bacterial bioaerosols in Japan as assessed by 16S rRNA amplicon sequencing.

To study the size-resolved characteristics of airborne bacterial community composition, diversity, and abundance, outdoor aerosol samples were analysed by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing with Illumina MiSeq. The samples were collected using size-resolved samplers between August and October 2016, at a suburban site in Toyama City and an urban site in Yokohama City, Japan. The bacterial communities were found to be dominated by Actinobacteria, Firmicutes, and Proteobacteria. At the genus level, we found a high abundance of human skin-associated bacteria, such as Propionibacterium, Staphylococcus, and Corynebacterium, in the urban site. Whereas, a high abundance of bacteria associated with soil and plants, such as Methylobacterium and Sphingomonas, was observed in the suburban site. Furthermore, our data revealed a shift in the bacterial community structure, diversity, and abundance of total bacteria at a threshold of 1.1-µm diameter. Interestingly, we observed that Legionella spp., the causal agents of legionellosis in humans, were mainly detected in > 2.1 µm coarse particles. Our data indicate that local environmental factors including built environments could influence the outdoor airborne bacterial community at each site. These results provide a basis for understanding the size-resolved properties of bacterial community composition, diversity, and abundance in outdoor aerosol samples and their potential influence on human health.

Global distribution of epidemic-related Shiga toxin 2 encoding phages among enteroaggregative Escherichia coli.

Since the Shiga toxin-producing enteroaggregative Escherichia coli (Stx-EAEC) O104:H4 strain caused a massive outbreak across Europe in 2011, the importance of Stx-EAEC has attracted attention from a public health perspective. Two Stx-EAEC O86 isolates were obtained from patients with severe symptoms in Japan in 1999 and 2015. To characterize the phylogeny and pathogenic potential of these Stx-EAEC O86 isolates, whole-genome sequence analyses were performed by short-and long-read sequencing. Among genetically diverse E. coli O86, the Stx-EAEC O86 isolates were clustered with the EAEC O86:H27 ST3570 subgroup. Strikingly, there were only two loci with single nucleotide polymorphisms (SNPs) between the Stx2a phage of a Japanese O86:H27 isolate and that of the European epidemic-related Stx-EAEC O104:H4 isolate. These results provide evidence of global distribution of epidemic-related Stx2a phages among various lineages of E. coli with few mutations.

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase.

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.

Complete Genome Sequence of an Enterohemorrhagic Escherichia coli O111:H8 Strain Recovered from a Large Outbreak in Japan Associated with Consumption of Raw Beef.

We present the complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain. This strain was isolated from a hemolytic-uremic syndrome patient and was responsible for a large outbreak associated with the consumption of raw beef in 2011.

Distribution and Molecular Characterization of Acinetobacter baumannii International Clone II Lineage in Japan.

Multidrug-resistant (MDR) Acinetobacter spp. have been globally disseminated in association with the successful clonal lineage Acinetobacter baumannii international clone II (IC II). Because the prevalence of MDR Acinetobacter spp. in Japan remains very low, we characterized all Acinetobacter spp. (n = 866) from 76 hospitals between October 2012 and March 2013 to describe the entire molecular epidemiology of Acinetobacter spp. The most prevalent species was A. baumannii (n = 645; 74.5%), with A. baumannii IC II (n = 245) accounting for 28.3% of the total. Meropenem-resistant isolates accounted for 2.0% (n = 17) and carried ISAba1-blaOXA-23-like (n = 10), blaIMP (n = 4), or ISAba1-blaOXA-51-like (n = 3). Multilocus sequence typing of 110 representative A. baumannii isolates revealed the considerable prevalence of domestic sequence types (STs). A. baumannii IC II isolates were divided into the domestic sequence type 469 (ST469) (n = 18) and the globally disseminated STs ST208 (n = 14) and ST219 (n = 4). ST469 isolates were susceptible to more antimicrobial agents, while ST208 and ST219 overproduced the intrinsic AmpC ß-lactamase. A. baumannii IC II and some A. baumannii non-IC II STs (e.g., ST149 and ST246) were associated with fluoroquinolone resistance. This study revealed that carbapenem-susceptible A. baumannii IC II was moderately disseminated in Japan. The low prevalence of acquired carbapenemase genes and presence of domestic STs could contribute to the low prevalence of MDR A. baumannii A similar epidemiology might have appeared before the global dissemination of MDR epidemic lineages. In addition, fluoroquinolone resistance associated with A. baumannii IC II may provide insight into the significance of A. baumannii epidemic clones.

Rapid and Accurate Diagnosis Based on Real-Time PCR Cycle Threshold Value for the Identification of Campylobacter jejuni, astA Gene-Positive Escherichia coli, and eae Gene-Positive E. coli.

We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, [RFBS24 ver.5]) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the results from RFBS24 ver.5 and bacterial culture methods (BC), we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks using RFBS24 ver.5 and evaluated the correlation between the cycle threshold (CT) value of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae, with positive predictive values (PPV) of 45.5-87.0% and a kappa coefficient (KC) of 0.60-0.92, respectively. The CTs were significantly different between BC-positive and -negative samples (p ＜ 0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower confidence interval (CI) limit of 95% or 99% for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) to discriminate for assured BC-positive results (d-CTs: 27.42-30.86), and subsequently the PPVs (94.7%-100.0%) and KCs (0.89-0.95) of the 3 targets were increased. Together, we concluded that the implication of a d-CT-based approach would be a valuable tool for rapid and accurate diagnoses using the RFBS24 ver.5 system.

Prevalence of Legionella species isolated from shower water in public bath facilities in Toyama Prefecture, Japan.

AIMS: We investigated the prevalence of Legionella spp. isolated from shower water in public bath facilities in Toyama Prefecture, Japan. In addition, we analyzed the genetic diversity among Legionella pneumophila isolates from shower water as well as the genetic relationship between isolates from shower water and from stock strains previously analyzed from sputum specimens. METHODS: The isolates were characterized using serogrouping, 16S rRNA gene sequencing, and sequence-based typing. RESULTS: Legionella spp. were isolated from 31/91 (34.1%) samples derived from 17/37 (45.9%) bath facilities. Isolates from shower water and bath water in each public bath facility were serologically or genetically different, indicating that we need to isolate several L. pneumophila colonies from both bath and shower water to identify public bath facilities as sources of legionellosis. The 61 L. pneumophila isolates from shower water were classified into 39 sequence types (STs) (index of discrimination = 0.974), including 19 new STs. Among the 39 STs, 12 STs match clinical isolates in the European Working Group for Legionella Infections database. Notably, ST505 L. pneumophila SG 1, a strain frequently isolated from patients with legionellosis and from bath water in this area, was isolated from shower water. CONCLUSIONS: Pathogenic L. pneumophila strains including ST505 strain were widely distributed in shower water in public bath facilities, with genetic diversity showing several different origins. This study highlights the need to isolate several L. pneumophila colonies from both bath water and shower water to identify public bath facilities as infection sources in legionellosis cases.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan--Quantitative Shiga toxin detection from stool during EHEC outbreak.

Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection.

[Bacterial infections, what we can learn from each outbreak].

Studies on outbreaks or incidences of infectious diseases and food poisonings are the starting points in research. Analyses of the outbreaks will provide the mechanism by which the illnesses occur and the establishment of countermeasure. We report here some sensational outbreaks which recently occurred in Japan: 1) enterohemorrhagic Escherichia coli (EHEC) O157 outbreaks by pickled Chinese cabbage in Hokkaido, 2) EHEC O157/O111 outbreaks by raw beef in Toyama Prefecture, 3) parasitic food poisoning due to raw olive flounder consumption in the western Japan, and 4) botulism due to the consumption of vacuum packed food in Tottori Prefecture.

Serodiagnosis using microagglutination assay during the food-poisoning outbreak in Japan caused by consumption of raw beef contaminated with enterohemorrhagic Escherichia coli O111 and O157.

A microagglutination (MA) assay to identify antibodies to Escherichia coli O111 and O157 was conducted in sera collected from 60 patients during a food-poisoning outbreak affecting 181 patients in Japan which was caused by the consumption of contaminated raw beef. Enterohemorrhagic E. coli (EHEC) O111:H8 and/or O157:H7 was isolated from the stools of some of the patients, but the total rate of positivity for antibodies to O111 (45/60, 75.0%) was significantly higher than that for antibodies to O157 (10/60, 16.7%). The MA titers of antibodies to O111 measured in patients with hemolytic-uremic syndrome and bloody diarrhea were higher than those measured in patients with only diarrhea. In patients from whose stool no isolates of E. coli O111 and O157 were obtained, the positive antibody detection rates were 12/19 (63.2%) for O111 and 2/19 (10.5%) for O157, and the MA titers of antibodies to O111 measured were higher than those to O157. Similarly, the MA titers of antibodies to O111 were significantly higher than those to O157, regardless of the other groups, including groups O111, O111 and O157, and O157. These serodiagnosis results suggest that EHEC O111:H8 stx2 played a primary role in the pathogenesis of this outbreak. Furthermore, our findings suggest that the isolates from the patients' stool specimens were not always the major causative pathogen in patients with multiple EHEC infections, because the sera from patients from whose stools only O157 was isolated were positive for antibodies to O111. Measuring antibodies to E. coli O antigen is helpful especially in cases with multiple EHEC infections, even with a non-O157 serotype.

[Water-borne outbreak of Yersinia enterocolitica O8 due to a small scale water system].

A water-borne outbreak of Yersinia enterocolitica O:8 associated with a small-scale water system occurred during July-August 2011 in Toyama Prefecture, Japan. Escherichia coli was not detected in tap water from the small-scale water system. However, the maximum concentration of viable bacteria in the tap water was 700CFU/mL, which exceeds the legal standard for purity of tap water (100CFU/mL). Furthermore, Y. enterocolitica O8 was isolated from the tap water with the use of immunomagnetic beads prepared with anti-Y. enterocolitica O8 antibodies. Pulsed-field gel electrophoresis analysis identified 3 isolates from tap water and 5 isolates from 4 patient stool specimens as belonging to the outbreak strain. An epidemiological investigation revealed improper management of the residual chlorine concentration in the tap water. This is the first report of an outbreak of Y. enterocolitica due to tap water from a small-scale water system in Japan.

Close genetic relationship between Legionella pneumophila serogroup 1 isolates from sputum specimens and puddles on roads, as determined by sequence-based typing.

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.