Legionella is an important waterborne pathogen that causes Legionnaires' disease (LD). Several outbreaks associated with wastewater treatment plants (WWTPs) have been reported in recent years; however, the prevalence of Legionella in WWTPs in Japan has rarely been investigated. In this study, we investigated the distribution of Legionella in influent wastewater collected from two WWTPs in Kobe, Japan between April 2023 and March 2024. The concentrations for Legionella in all seasons varied between 104 and 106 copies/100 mL in all seasons. Among the 10 Legionella species detected in the influent wastewater, Legionella pneumophila was the most commonly isolated. Genotyping revealed that pathogenic L. pneumophila strains were widely distributed in the influent wastewater in Japan with genetic diversity. LD is one of the most important infectious diseases during natural disasters. This study highlights the importance of influent wastewater as a potential source of LD in Japan, where natural disasters occur frequently.

  • Legionella, a waterborne pathogen, causes Legionnaires' disease.

  • Outbreaks linked to wastewater treatment plants (WWTPs) have been reported.

  • A study in Kobe, Japan, investigated Legionella distribution in influent wastewater.

  • Legionella concentrations varied between 104 and 106 copies/100 mL in all seasons.

  • L. pneumophila was the most common species in influent wastewater of Japan, with diverse genotypes.

Legionnaires' disease (LD) is caused by the inhalation of aerosols contaminated with Legionella. Of the 66 Legionella species identified to date, at least 30 are known to cause disease in humans. Legionella pneumophila is responsible for most LD cases. These bacteria are ubiquitous in freshwater and soil at low concentrations. However, when contaminated at high concentrations in man-made water installations, such as building water systems, cooling towers, and bath facilities, they are a major source of LD. In Japan, public bath facilities are the main source of Legionella infection; however, in many cases, the source of infection remains unknown (Amemura-Maekawa et al. 2018).

In recent years, several cases of LD associated with wastewater treatment plants (WWTPs) have been reported (Kusnetsov et al. 2010; Nogueira et al. 2016). Legionella has been detected in Dutch wastewater at concentrations of up to 108 CFU/L (Loenenbach et al. 2018). Aerosols formed during wastewater treatment processes contain Legionella derived from WWTPs, which can be dispersed over long distances and reach the local population as aerosols of inhalable size (Vermeulen et al. 2021). A risk assessment for WWTPs for Legionella growth and emissions has also been reported (van den Berg et al. 2023). On the other hand, the risk of aerosol generation during the treatment process of WWTPs is considered to be very low in general in Japan, because treatment facilities in WWTPs are covered, and influent wastewater is treated immediately and decomposed. However, there is a potential risk to WWTPs during the frequent natural disasters that occur in Japan. In fact, many WWTPs and pumping stations stopped functioning after the Great East Japan Earthquake and tsunami of March 11, 2011 (Matsuhashi et al. 2014). In addition, recent guerrilla downpours have caused untreated wastewater to overflow from manholes. In view of the increased occurrence of large-scale earthquakes and guerrilla downpours in future (Japan Meteorological Agency 2023), the investigation of Legionella in wastewater is warranted. However, the prevalence of Legionella in wastewater in Japan has rarely been investigated. The genetic diversity of L. pneumophila isolates from wastewater has not yet been analyzed using molecular typing techniques. In this study, we investigated the prevalence and genetic characteristics of Legionella isolates from influent wastewater in Kobe, Japan to determine whether it is a reservoir of Legionella.

Sample collection and isolation of Legionella species

Influent wastewater samples, which comprised wastewater before treatment, were collected from two WWTPs (A and B) in Kobe. A total of 48 composite samples corresponding to 24 h were collected twice a month from April 2023 to March 2024. The wastewater samples (400 mL) were concentrated to 4 mL by cooling and centrifuging at 10,000 × g for 10 min. Amoebic co-culture was performed using Acanthameba castelanii ATCC30010 for 7 days (Schalk et al. 2012). The concentrated samples and the amoeba co-culture solutions were subjected to one or more of the following pretreatment methods: 1) heating at 50 °C for 30 min, 2) mixing with an equal volume of 0.2 M KCl–HCl buffer (pH 2.2) at room temperature for 30 min, and 3) heating at 50 °C for 30 min and mixing with an equal volume of 0.2 M KCl–HCl buffer (pH 2.2) at room temperature for 30 min. Additionally, we performed the pretreatment method of heating at 50 °C for 30 min and mixing with an equal volume of 0.1 M citric acid/citrate buffer (pH 2.2) at room temperature for 30 min to inhibit the growth of contaminating bacteria (Kasuga et al. 2002). The treated sample solutions were diluted with sterile water and grown on GVPC (glycine–vancomycin–polymyxin–cycloheximide), MWY (Modified Wadowsky-Yee) (Kanto Chemical, Tokyo, Japan), and WYOα (Wadowsky-Yee-Okuda Potassium α-ketoglutarate) agar plates (Eiken Chemical, Tokyo, Japan). The agar plates were incubated at 36 °C for 7 days in a moist chamber. The mosaic-cut, glass-like, cysteine-requiring colonies were presumed to be Legionella. Species of Legionella were determined using MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany) and sequencing of mip genes as previously described (Ratcliff et al. 1998). Serogroups (SGs) of L. pneumophila were determined using the slide agglutination test with commercial antisera (DENKA Corporation, Tokyo, Japan).

Quantitative polymerase chain reaction (qPCR)

For the quantitative polymerase chain reaction (qPCR), DNA was extracted from 1 mL of the concentrated solution using a QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany). The 100-fold concentrated 1 mL samples were further concentrated to a final volume of 100 μL. The real-time qPCR was performed using the Cycleave PCR Legionella (16S rRNA) Detection Kit (Takara Bio, Shiga, Japan). Briefly, DNA template solutions (5 μL) were added to the reaction mixtures (20 μL) and were subjected to PCR amplification in the Thermal Cycler Dice Real Time System IV (Takara Bio). The amplified 16S rRNA gene of Legionella and the internal control gene were detected by FAM and ROX, respectively. According to the manufacturer's instructions, the copies of the 16S rRNA gene in each sample were automatically calculated by comparing the threshold cycle values to the standard curve we constructed and then were multiplied 20-fold to obtain the number of copies in 100 mL influent wastewater.

Genotyping and detection of lag-1

Genotyping was performed by sequence-based typing (SBT) following the protocol of the European Working Group for Legionella infections (EWGLI) as previously described (Gaia et al. 2005; Ratzow et al. 2007). Briefly, genomic DNA was extracted from isolates and then amplified using primers targeting seven specific genes (flaA, pilE, asd, mip, mompS, proA, and neuA). Amplicons were sequenced with specific primers, and the resulting consensus sequences were trimmed and compared to previously assigned allele numbers. The combination of alleles was defined as seven allelic profiles and a sequence type (ST) represented by a number. The PCR was performed to detect lag-1 using the primers lag-F (5′-CTCACAACAAGTCAAGCAAC-3′) and lag-R (5′-AAACCATACCAAAGCAACAT-3′) as previously described (Kozak et al. 2009).

Prevalence and distribution of Legionella isolates

Legionella DNA was detected at concentrations between 104 and 106 copies/100 mL in all samples collected monthly from WWTP-A and WWTP-B (Figure 1). Legionella concentrations tended to be lower in summer and higher in winter at both WWTPs; however, there was no clear seasonal variation.
Figure 1

Legionella distribution in influent wastewater in Kobe City, Japan. The number of isolates in each Legionella species is the sum of two samples for each month, and the Legionella gene concentration is the average of two samples for each month.

Figure 1

Legionella distribution in influent wastewater in Kobe City, Japan. The number of isolates in each Legionella species is the sum of two samples for each month, and the Legionella gene concentration is the average of two samples for each month.

Close modal

Ten Legionella spp. were detected in 14 of 48 influent wastewater samples (29.2%). The isolation rate at WWTP-B was 37.5% (9/24), which was higher than that of 20.8% (5/24) in WWTP-A. Eight strains comprising three species were isolated from WWTP-A, and 19 strains comprising nine Legionella species were isolated from WWTP-B (Figure 1). L. pneumophila (12/27, 44.4%) was the most frequently isolated species, accounting for 75.0% (6/8) and 31.6% (6/19) of isolates from WWTP-A and WWTP-B, respectively. In contrast, L. birminghamensis (n = 3), L. moravica (n = 2), and L. septentrionalis (n = 1), which are not often found in bathwater or other sources, were isolated from WWTPs.

Genotypes in L. pneumophila isolates

The genetic characteristics of the 12 L. pneumophila isolates obtained from the two WWTPs are shown in Table 1. KL2670, KL2685, KL2514/KL2533, and KL2491 were identified as novel STs. The genotypes of the isolates from the two WWTPs differed. Two L. pneumophila SG1 strains, K2495 and KL2685, were included in the soil group (S3) and bath group (B2), respectively, according to previously reported grouping (Amemura-Maekawa et al. 2018). KL2685, which was designated as the novel ST ST-KW2, harbored lag-1. Four STs reported in Japanese patients with LD (ST68, ST537, ST1032, and ST1427) were present in the influent wastewater samples. These results indicate that pathogenic L. pneumophila was distributed throughout the influent wastewater.

Table 1

STs in 12 L. pneumophila isolates

WWTPsStrain no.Month of isolationSGSTsaSBT profile
lag-1Amemura-Maekawa et al.’s group
flaApilEasdmipmompSproAneuA
KL2495 Apr 260 12 11 23 29 26 – S3 
KL2494, KL2669 Apr, Oct 1,424 23 12 31 48 31 220 NT  
KL2679 Sep 1,032 13 14 38 NT  
KL2677 Dec 1,427 12 14 220 NT  
KL2670 Oct ST-KW1 13 28 NT  
KL2685 May ST-KW2 17 21 35 31 B2 
KL2514, KL2533 Mar, May ST-KW3 10 28 14 NT  
KL2491 Apr ST-KW4 10 19 28 14 11 NT  
KL2518 Apr 68 13 28 14 NT  
KL2686 Feb 537 13 28 12 NT  
WWTPsStrain no.Month of isolationSGSTsaSBT profile
lag-1Amemura-Maekawa et al.’s group
flaApilEasdmipmompSproAneuA
KL2495 Apr 260 12 11 23 29 26 – S3 
KL2494, KL2669 Apr, Oct 1,424 23 12 31 48 31 220 NT  
KL2679 Sep 1,032 13 14 38 NT  
KL2677 Dec 1,427 12 14 220 NT  
KL2670 Oct ST-KW1 13 28 NT  
KL2685 May ST-KW2 17 21 35 31 B2 
KL2514, KL2533 Mar, May ST-KW3 10 28 14 NT  
KL2491 Apr ST-KW4 10 19 28 14 11 NT  
KL2518 Apr 68 13 28 14 NT  
KL2686 Feb 537 13 28 12 NT  

aST-KW1 to ST-KW4 are novel STs.

NT, not tested.

In this study, we investigated the prevalence of Legionella isolated from influent wastewater in Japan. Legionella spp., particularly L. pneumophila, are widely distributed in influent wastewater. The concentration of Legionella in all seasons varied between 104 and 106 copies/100 mL, which is comparable with the results of previous studies (Lund et al. 2014; Bonetta et al. 2022). The concentrations detected in WWTPs tended to be slightly higher, although Legionella was also detected in bathwater samples (Inoue et al. 2015). DNA from Legionella species detected in WWTPs is derived from viable Legionella cells but also from viable but non-culturable (VBNC) cells and dead cells. While previous studies have demonstrated that VBNC Legionella in water samples can regain culturability in amoebic co-culture (Steinert et al. 1997; García et al. 2007), our investigation did not observe an increase in Legionella DNA following amoebic co-culture over a 1-week period in samples from WWTPs (data not shown). Enhancements to the amoebic co-culture method, such as optimizing the selection of amoebal strains and adjusting incubation periods, may facilitate the isolation of Legionella from WWTPs. Additionally, further investigations employing techniques such as the use of ethidium monoazide and qPCR in combination are necessary. Such approaches selectively quantify viable Legionella cells by preventing the amplification of DNA from membrane-damaged dead cells, providing a more comprehensive understanding of the state of Legionella within WWTP environments.

WWTP-B had a higher isolation rate and more Legionella species than WWTP-A. The genotypes of L. pneumophila differed between WWTP-A and WWTP-B. WWTP-B is a combined system in which wastewater and rainwater are conveyed through the same pipe, whereas WWTP-A is a separate system in which the wastewater and rainwater are treated separately. This difference in the wastewater systems may be correlated with the prevalence of Legionella species. Legionella species detected at WWTP-B have been isolated from environmental water and soil (Casati et al. 2009; Zhan et al. 2022), suggesting that an influx from the natural environment may increase Legionella concentrations in WWTP-B.

Legionella species, L. gormanii, L. dumoffii, L. bozemanae, and L. birminghamensis, which were not isolated from the bath water, were detected in the WWTPs (Komatsu et al. 2023). These species have been associated with several clinical cases (McDade 2008; Amemura-Maekawa et al. 2018).

Of the two L. pneumophila SG1 strains, KL2685 was assigned to a novel ST (allelic profile: 7, 6, 17, 21, 35, 31, and 9), harboring lag-1, and was included in the B2 group, according to Amemura-Maekawa et al. ST68, ST537, and ST1032 have been detected in shower and bath waters in Japan (Kanatani et al. 2017). In our previously reported sample set, ST68 and ST1424 were detected in the bath water of L. pneumophila SG6 and SG5 (data not shown) (Komatsu et al. 2023), suggesting that the STs of L. pneumophila detected in bath water are discharged as domestic wastewater and are present in WWTPs. Additionally, four STs (ST68, ST537, ST1032, and ST1427) have been isolated from patients with LD (Kozak et al. 2009). The source of infection in the LD cases caused by ST1032 is unknown. Furthermore, Amemura-Maekawa et al. reported a case of LD caused by L. pneumophila ST68, which occurred following drowning in mud from the tsunami during the Great East Japan Earthquake on March 11, 2011.

To date, no cases of LD associated with WWTPs have been reported in Japan. This may be attributed to the typically low likelihood of such occurrences under normal operational conditions, owing to the structured and efficient processing within these facilities. However, the potential risk becomes evident during natural disasters when the functioning of WWTPs is disrupted. For instance, the Great East Japan Earthquake in 2011 severely impacted 120 WWTPs, with many facilities and pumping stations along the coastal areas of affected prefectures ceasing operations (Matsuhashi et al. 2014). Urban areas experienced untreated wastewater overflow from manholes as a consequence (Division of Sewerage, Department of Civil Engineering, Miyagi Prefecture 2013). Furthermore, the frequency of sudden heavy rainfall events, known in Japan as guerrilla downpours, has been increasing (Japan Meteorological Agency 2023). Several recent reports have revealed that puddles on asphalt roads serve as potential environmental reservoirs for L. pneumophila (Sakamoto et al. 2009; Kanatani et al. 2013, 2021). These results suggest that the presence of Legionella in puddles on roads could be spread by moving cars, resulting in the aerosolization of puddle water, especially on rainy days. Given Japan's susceptibility to natural disasters, influent wastewater containing Legionella species and potentially pathogenic genotypes poses a significant concern for the transmission of LD.

To conclude, pathogenic L. pneumophila strains were widely distributed in influent wastewater in Japan with genetic diversity. Despite no reported cases of LD associated with WWTPs in Japan, the potential for exposure to untreated influent wastewater during natural disasters is a matter of concern. This study highlights the need to consider influent wastewater as a source of LD in Japan during frequent natural disasters.

The Japan Society supported this research for the Promotion of Science, KAKENHI (Grant No. 23K11469). We thank Kanna Kodama, Taketoshi Shimizu, Hironobu Ueshiro, Michiko Ogawa, Hiroshi Teraoka, Shigeki Nada, Futoshi Ihama, and Michiko Hashikawa at Public Construction Projects Bureau, Kobe City, for their cooperation in wastewater sampling. The authors also thank Shinobu Tanaka at the Kobe Institute of Health for technical support.

S.K. and N.N. designed the study methods and wrote the first draft of the manuscript. N.N., S.K., and C.F. performed bacterial isolation, identification typing, and PCR. S.K. and N.N. analyzed the data. All the authors have read and agreed to the published version of this manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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