Development of a Multiplex-PCR Serotyping Assay for Characterizing Legionella pneumophila Serogroups Based on the Diversity of Lipopolysaccharide Biosynthetic Loci.

Legionella pneumophila, which is the main cause of Legionnaires' disease, comprises at least 15 serogroups (SGs). We show here the diversity of lipopolysaccharide biosynthetic loci among serogroups and describe the development of a PCR serotyping assay for 15 SGs based on the sequences of LPS biosynthetic loci. Using this multiplex-PCR (M-PCR) system, serogroups were detected using primers that specifically amplify the sequences of SG1, SG2, SG5, SG7, SG8, SG9, SG11, SG13, SG3/15, and SG6/12. When PCR products of the expected sizes were not detected, we used primers that identified SG4/10/14. The PCR serotyping system specifically amplified the sequences corresponding to SGs of 238 L. pneumophila strains. This method will be very useful for conducting epidemiological studies and investigating outbreak of Legionnaires' disease.

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.

Legionella pneumophila and Other Legionella Species Isolated from Legionellosis Patients in Japan between 2008 and 2016.

The Legionella Reference Center in Japan collected 427 Legionella clinical isolates between 2008 and 2016, including 7 representative isolates from corresponding outbreaks. The collection included 419 Legionella pneumophila isolates, of which 372 belonged to serogroup 1 (SG1) (87%) and the others belonged to SG2 to SG15 except for SG7 and SG11, and 8 isolates of other Legionella species (Legionella bozemanae, Legionella dumoffii, Legionella feeleii, Legionella longbeachae, Legionella londiniensis, and Legionella rubrilucens). L. pneumophila isolates were genotyped by sequence-based typing (SBT) and represented 187 sequence types (STs), of which 126 occurred in a single isolate (index of discrimination of 0.984). These STs were analyzed using minimum spanning tree analysis, resulting in the formation of 18 groups. The pattern of overall ST distribution among L. pneumophila isolates was diverse. In particular, some STs were frequently isolated and were suggested to be related to the infection sources. The major STs were ST23 (35 isolates), ST120 (20 isolates), and ST138 (16 isolates). ST23 was the most prevalent and most causative ST for outbreaks in Japan and Europe. ST138 has been observed only in Japan, where it has caused small-scale outbreaks; 81% of those strains (13 isolates) were suspected or confirmed to infect humans through bath water sources. On the other hand, 11 ST23 strains (31%) and 5 ST120 strains (25%) were suspected or confirmed to infect humans through bath water. These findings suggest that some ST strains frequently cause legionellosis in Japan and are found under different environmental conditions.IMPORTANCELegionella pneumophila serogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.

Outbreak of Legionnaire's Disease Caused by Legionella pneumophila Serogroups 1 and 13.

In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires' disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

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.

Variation and association of fibronectin-binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates.

Fibronectin-binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N-terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67-B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA-fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67-B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2-N3 three-dimensional structural models indicated that not only the variable amino acid residues, but also well-conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.

A case of pneumonia caused by Legionella pneumophila serogroup 12 and treated successfully with imipenem.

The patient was an 83-year-old man hospitalized for Haemophilus influenzae pneumonia, who developed recurrent pneumonia after improvement of the initial episode. Legionella pneumophila serogroup 12 was isolated from the sputum, accompanied by increased serum antibody titers to L. pneumophila serogroup 12. Therefore, the patient was diagnosed as having Legionella pneumonia caused by L. pneumophila serogroup 12. Case reports of pneumonia caused by L. pneumophila serogroup 12 are rare, and the case described herein is the first report of clinical isolation of this organism in Japan. When the genotype was determined by the protocol of The European Working Group for Legionella Infections (Sequence-Based Typing [SBT] for epidemiological typing of L. pneumophila, Version 3.1), the sequence type was ST68. Imipenem/cilastatin therapy was found to be effective for the treatment of Legionella pneumonia in this patient.

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.

Distribution of monoclonal antibody subgroups and sequence-based types among Legionella pneumophila serogroup 1 isolates derived from cooling tower water, bathwater, and soil in Japan.

Legionella pneumophila serogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates of L. pneumophila SG 1 in Japan using monoclonal antibody (MAb) typing and sequence-based typing (SBT). Samples were analyzed from bathwater (BW; n = 50), cooling tower water (CT; n = 50), and soil (SO; n = 35). The distribution of MAb types varied by source, with the most prevalent types being Bellingham (42%), Oxford (72%), and OLDA (51%) in BW, CT, and SO, respectively. The ratios of MAb 3/1 positive isolates were 26, 2, and 14% from BW, CT, and SO, respectively. The environmental isolates from BW, CT, and SO were divided into 34 sequence types (STs; index of discrimination [IOD] = 0.973), 8 STs (IOD = 0.448), and 11 STs (IOD = 0.879), respectively. Genetic variation among CT isolates was smaller than seen in BW and SO. ST1 accounted for 74% of the CT isolates. The only common STs between (i) BW and CT, (ii) BW and SO, and (iii) CT and SO were ST1, ST129, and ST48, respectively, suggesting that each environment constitutes an independent habitat.

[Four cases of respiratory infections caused by Legionella pneumophila serogroup 3].

Legionella pneumonia tends to be severe and is known to be fatal. Introduction of the urinary Legionella antigen test and changes in the Infectious Disease Law have led to increased numbers of reports, and milder cases are now occasionally seen. We experienced three cases demonstrating mild respiratory infections and one case demonstrating nosocomial pneumonia associated by Legionella pneumophila serogroup 3. Case 1 showed multiple ground-glass opacities on HRCT and productive cough. Cases 2 and 3 showed abnormal findings on chest X-ray, and chest CT findings in both cases suggested chronic respiratory infection. Case 4 experienced fever and hypoxia, and pulmonary edema was noted on X-ray. All of them four cases were diagnosed with respiratory infections isolated L. pneumophila serogroup 3 by culture results, and three of them cases were treated in the outpatient clinic. Thus, milder cases of Legionella pneumonia may be encountered during routine care, and tests for Legionella should be performed in such cases.

Complete Genomic Sequence of the Clinical Isolate Legionella pneumophila Serogroup 1 Strain 80-045 from Japan.

We report the complete genomic sequence of Legionella pneumophila serogroup 1 strain 80-045, isolated from autopsy lung tissue of the first patient diagnosed with Legionnaires' disease in Japan.

Persistent Legionella contamination of water faucets in a tertiary hospital in Japan.

OBJECTIVE: The feasibility of the decontamination procedure for Legionella pneumophila of water systems in healthcare facilities varies by water purification and disinfection methods in each country. We evaluated the efficacy of feasible decontamination strategies in Japan. METHODS: This study was conducted at Tokyo Medical University Hospital (1015 beds) between 2015 and 2018. Samples from the water system and cooling tower were cultured periodically. Hyper-chlorination of cool tap water (>0.2 ppm), increases in the temperature of hot water (>55 °C), and flushing were used as decontamination strategies. The case of healthcare-associated legionellosis was surveyed. Environmental and clinical isolates were genotyped. RESULTS: 1439 environmental samples were collected; 19 (1.3%) samples tested positive for L. pneumophila from water faucets of patient rooms, toilets, waste rooms, and water sourced from wells. Genotyping of 12 isolates confirmed that the same strains were present in eight environmental isolates and two isolates from patients over three years. Although the environmental contamination of the water system was persistent, the number of positive locations of hospital environments gradually decreased; eight in 2015, four in 2016, three in 2017, and four in 2018, respectively. CONCLUSIONS: Monitoring contamination, hyper-chlorination, controlling temperature, and flushing were effective as a Legionella decontamination strategy.

Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR.

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates.

Pulsed-field gel electrophoresis (PFGE), which determines the genomic relatedness of isolates, is currently used for the epidemiological investigation of infectious agents such as bacteria. In particular, this method has been used for the epidemiological investigation of Legionella outbreaks. However, it takes 4 days to complete a Legionella-PFGE analysis. Due to partial digestion and DNA damage, the reproducibility of the obtained fragment digestion patterns is poor for this pathogen. In this study, we report an improved protocol that takes only 2 days to complete and that allows clear discrimination of the restriction profile with higher reproducibility than that previously achieved.

[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.

Distribution of lag-1 Alleles, ORF7, and ORF8 Genes of Lipopolysaccharide and Sequence-Based Types Among Legionella pneumophila Serogroup 1 Isolates in Japan and China.

Approximately 85% of cases of Legionnaires' disease are caused by Legionella pneumophila serogroup 1. In this study, we analyzed the distribution of lag-1 alleles, ORF 7 and ORF 8 genes of lipopolysaccharide (LPS) and sequence-based types of 616 L. pneumophila serogroup 1 strains isolated in Japan (206 clinical, 225 environmental) and China (13 clinical and 172 environmental). The lag-1 gene was harbored by significantly more of the clinical isolates compared with the environmental isolates (90.3 vs. 19.1% and 61.6 vs. 3.0%, respectively; both P < 0.001). ORF 7 genes were detected in 51.0% of Japanese clinical and 36.0% of Japanese environmental (P = 0.001) isolates, as well as 15.3% of Chinese clinical and 9.9% of Chinese environmental isolates (P = 0.544). ORF 8 genes were detected in 12.1% of Japanese clinical and 5.8% of Japanese environmental (P = 0.017) isolates, as well as 7.7% of Chinese clinical and 3.4% of Chinese environmental isolates (P = 0.388). The Japanese and Chinese isolates were assigned to 203 and 36 different sequence-types (ST), respectively. ST1 was predominant. Most isolates with the same ST also had the same lag-1, ORF 7, and ORF 8 gene subgroups. In conclusion, the lag-1 was present in most of the clinical isolates, but was absent from most of the environmental isolates from both China and Japan, regardless of the water source and SBT type. PCR-based serotyping and subgrouping methods can be used to define a hierarchy of virulence genotypes that require stringent surveillance to prevent human disease.

[Largest outbreak of legionellosis associated with spa baths: comparison of diagnostic tests].

In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. Among the visitors, 295, including suspected cases had pneumonia and/or symptoms of fever, coughing, etc. Of these, 37% were hospitalized and 7 died. Clinical samples from 95 mainly inpatients were collected and microbiologically tested at our laboratory. Legionella pneumophila serogroup (SG) 1 was isolated from 3 of 24 in sputum culture, and none of the 3 had been treated effectively with antibiotics at sputa collection. L. pneumophila antigen in urine was detected by using enzyme immunoassay and/or immunochromatographic kits in 23 of 75 patients. Serum antibodies to L. pneumophila SG1 and Legionella dumoffii were detected in 5 each of 66 patients--9 cases including a case at mixed infection-by microplate agglutination test and/or indirect immunofluorescence assay. At our laboratory, 32 were diagnosed with legionellosis. In this outbreak, 14 were diagnosed at other laboratories, resulting in 46 confirmed cases. Urine antigen was detected more frequently by Binax NOW immunochromatographic assay than by Biotest EIA-31% versus 16% of cases tested. Both assays detected urine antigen only in samples collected within 4 weeks after onset. Antigen concentration in urine enhanced sensitivity-58% and 51%-and extended the period of antigen detection beyond 5 weeks. Both antibody titers to L. pneumophila SG1 and L. dumoffii in more than 90% of sera collected within 3 weeks after onset were < 1:16. The rate of serum antibody titer to > or = 1:128 within 3 weeks was 1.6%, during 4 to 6 weeks less than 10%, and after 7 weeks or more 8 to 25%. After an administrative report was published, L. pneumophila DNA in sputa was detected in 5 of 17 patients by nested PCR, resulting in extra 3 cases. Altogether, urinary antigen detection and PCR were more effective in laboratory diagnostic tests than culture and serology. Culture combined with molecular epidemiology is critical, however, for confirming the source of infection.

A Case of Community-Acquired Pneumonia Due to Legionella pneumophila Serogroup 9 Wherein Initial Treatment with Single-Dose Oral Azithromycin Appeared Useful.

Legionella species are important causative pathogens for severe community-acquired pneumonia (CAP). Most cases of Legionella pneumonia are due to Legionella pneumophila serogroup 1, and CAP due to L. pneumophila serogroup 9 is rare. A fourth case of CAP due to L. pneumophila serogroup 9 has been reported, and initial treatment using single-dose oral azithromycin appeared useful. Azithromycin or fluoroquinolone injection is usually recommended for the treatment of Legionella pneumonia, and no previous reports have shown the effectiveness of single-dose oral azithromycin. This case report is therefore valuable from the perspective of possible treatment for mild to moderate Legionella pneumonia using single-dose oral azithromycin.