Regular Paper
Legionella Survives and Elongates in Algal Consortia Containing Bacteria in Alkaline Oligotrophic Conditions
2025 年 40 巻 4 号 論文ID: ME25016
詳細
2025 年 40 巻 4 号 論文ID: ME25016
Cooling towers are a major source of Legionella, which causes Legionnaires’ disease. These bacteria grow in predatory organisms; however, the impact of non-predatory organisms in cooling towers on Legionella survival and growth remains unclear. Therefore, we investigated the effects of photosynthetic algae, the primary component of biofilms in open cooling water systems, on Legionella. We cultivated Legionella with algae collected from the towers or with pure algal strain under alkaline conditions and revealed that the Legionella 16S rRNA copy number was higher than that of Legionella alone. We also examined Legionella using an in situ hybridization chain reaction and found that some were elongated and exhibited a filamentous morphology on algal cells. Furthermore, Legionella was more active when co-cultured with pure algal strain plus Serratia spp. than when co-cultured with pure alga alone. 18S rRNA gene sequencing revealed that the algae collected had not previously been reported to coexist with Legionella. This result suggests that diverse algae in the environment support the growth of Legionella. This is the first study to experimentally demonstrate that algae promote Legionella elongation, and also that the coexistence of bacteria furthers this phenomenon. These results provide a new perspective on the ecology of Legionella and the role of non-predatory organisms.
Bacteria of the Legionella species are ubiquitously distributed in the environment. Cooling water systems are a source of Legionella (Fraser et al., 1977; Chamberlain et al., 2017). These bacteria multiply in cooling towers and are released into the environment via the mist exhaust of cooling towers, eventually infecting the human respiratory system and causing legionellosis. Other sources include humidifiers (Mahoney et al., 1992), circulating bathtubs (Joseph et al., 2005), and potting mix or compost (Whiley and Bentham, 2011).
Cooling water is generally oligotrophic and alkaline, which differs from the typical nutrient concentrations and pH of Legionella culture conditions. To survive and grow in the conditions present in cooling water, Legionella species have been proposed to coexist with or parasitize different types of organisms in biofilms or sediments. Previous studies reported that Legionella associated with various organisms in different manners, including the parasitism of predatory microbes, such as amoebae (Grimm et al., 1998; Dietersdorfer et al., 2018) and protozoans (Rasch et al., 2016; Caicedo et al., 2018), and coexistence with non-predatory microbes, such as other bacteria (Taylor et al., 2013), dead bacterial cells (Temmerman et al., 2006), algae (Pope et al., 1982), and photosynthetic microbes (Tison et al., 1980; Bohach and Snyder, 1983a). Many types of microorganisms have been suggested to contribute to the survival and growth of Legionella, which is a characteristic feature of Legionella. Amoebae are known to function as a Legionella host (Rowbotham, 1980). Amoebae feed on non-predatory organisms, such as algae and bacteria forming biofilms in cooling tower. During this process, they ingest Legionella, which survive and multiply within amoeba cells and are eventually released in cooling systems (Anand et al., 1983; Oliva et al., 2018). The parasitism of amoebae by Legionella has been extensively examined. The mechanism by which Legionella multiplies in the vacuoles of amoebae was described by Vogel et al. (1998). Many studies have reported the proliferation of Legionella in amoebae, which have been detected in most cooling water systems (Zeybek et al., 2017). Collectively, these findings suggest that Legionella multiplication in cooling towers is attributed to the presence of amoebae. However, Legionella are not always detected in water systems occupied by amoebae and, conversely, the presence of Legionella does not guarantee the coexistence of amoebae (Scheikl et al., 2016). Furthermore, the proliferation of Legionella in protozoa other than amoebae has demonstrated (Barbaree et al., 1986). Barbaree et al., (1986) reported cooling water systems in which the protozoa isolated did not exhibit Legionella proliferation as well as systems in which Legionella were detected, but protozoa were not isolated. These findings indicate that there are still some aspects to be investigated regarding whether protozoa are the sole site for Legionella proliferation in cooling water systems. Therefore, we hypothesized that organisms other than predatory protozoa coexisting with Legionella, particularly non-predatory organisms that are dominant in systems in which Legionella have been detected, may contribute to the proliferation of Legionella in this system.
To examine organisms other than protozoa that enable Legionella multiplication in cooling water systems, we selected algae as a candidate photosynthetic microbe because 1) photosynthetic microbes become dominant in open cooling systems (Di Gregorio et al., 2017) and 2) algae contribute to Legionella proliferation. However, cocultures of Legionella with algae are typically performed at pH 7.5 (Pope et al., 1982). It remains unclear whether algae contribute to the growth and survival of Legionella at the pH that generally prevails in cooling water systems (pH 8–9). In addition, we investigated whether bacteria contributed to the survival of Legionella because algae and bacteria commonly coexist in natural environments. Previous studies showed that bacteria isolated from environments supported the multiplication of Legionella. For example, Flavobacterium breve isolated from a hot water tank (Wadowsy and Yee, 1983) or Pseudomonas vesicularis (Koide et al., 1989) from a shower hose induced the satellite growth of Legionella around their colonies on an agar medium that did not support Legionella growth alone. Furthermore, Legionella were shown to persist within Pseudomonas fluorescens biofilms while maintaining its colony-forming ability (Stewart et al., 2012; Silva et al., 2024). However, it has yet to be demonstrated that bacteria from cooling water systems support the persistence of Legionella. Additionally, the effects of bacteria coexisting with algae on Legionella growth or survival have not yet been examined.
In planning this study, we considered it important to conduct morphological observations to clarify the relationships between symbiotic organisms and Legionella. The coexistence of Legionella with algae is typically observed using optical microscopy (Bohach and Snyder, 1983b). However, fluorescence in situ hybridization (FISH) has recently been used to detect Legionella coexisting with microbes in the environment (Taylor et al., 2013; Rasch et al., 2016; Caicedo et al., 2018). Since many studies have used the LEG705 probe as a Legionella genus–specific FISH probe (Manz et al., 1995), we initially applied this probe to an algal and bacterial consortium containing Legionella.
We herein investigated the effects of algae on Legionella growth in an alkaline oligotrophic environment and reported the results obtained.
Legionella pneumophila (ATCC33152) was cultured using buffered charcoal yeast extract (BCYEα) Legionella agar medium (OXOID). Vischeria stellata NIES-2148, an axenic algal strain, was purchased from the Microbial Culture Collection at the National Institute for Environmental Studies. All algal strains were grown on C medium (Microbial Culture Collection, National Institute for Environmental Studies. URL https://mcc.nies.go.jp/02medium.html#c). Serratia marcescens was isolated from biofilm samples collected from cooling water systems by single-colony isolation using R2A agar plates. The 16S rRNA gene sequence was elucidated at FASMAC using a MicroSEQTM Full Gene 16S rDNA PCR Kit with a MicroSEQ ID system (Thermo Fisher Scientific). S. marcescens was subsequently grown on R2A agar.
Biofilms were collected from two different cooling towers where Legionella had previously been detected. Specifically, biofilms were collected using a 1-mL or 10-mL syringe from the area in the tower with green biofilm growth, which was either the pit wall or the filler (Fig. S1 and S2). Each biofilm was then diluted using sterile tap water and subsequently cultured in C medium. A type of algae colony was isolated from each cooling tower, which resulted in two types of algae ultimately being isolated. The identification of the isolated algal strains by sequencing the 18S rRNA gene was performed at Techno Suruga Laboratory. Briefly, total genomic DNA was extracted using the Nucleo Spin Plant II Kit (MACHEREY-NAGEL), and was then used for PCR amplification of the 18S rRNA gene using the primers SR-1 (5′-TAC CTG GTT GAT CCT GCC AG-3′) and SR-12 (5′-CCT TCC GCA GGT TCA CCT AC-3′) (Nakayama et al., 1996). Sequencing was performed using the BigDye Terminator V3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on an ABI PRISM 3500xl Genetic Analyzer (Thermo Fisher Scientific).
Coculture tests for L. pneumophila and algaeThe two types of algae isolated from the cooling towers were used. They were named the V. stellata consortium and Klebsormidium elegans consortium. In coculture tests, 50 mL of MA medium (pH 8.6) (Microbial Culture Collection, National Institute for Environmental Studies. URL https://mcc.nies.go.jp/02medium.html#ma) was prepared in a 100-mL conical flask and then inoculated with L. pneumophila along with the V. stellata or K. elegans consortia. Inoculated organisms were prepared as follows: L. pneumophila grown on BYCEα agar was diluted with sterile tap water to attain an absorbance of 0.1 at 662 nm, the estimated Legionella CFU was approximately 106–107 CFU μL–1. This solution was added to MA medium at a diluted rate of 1/1,000 (Table 1) or 1/100,000 (Table 2). The V. stellata and K. elegans consortia grown on C medium were collected by centrifugation and washed with MA medium. One milliliter of the V. stellata consortium (OD662, 0.1) or 3 mL of the K. elegans consortium (OD662, 0.03) was added to the MA medium. Due to the lack of previous studies on the cultivation of algae, cocultures were performed under various conditions: in a growth chamber at 36°C with fluorescent light (photon flux density: 105–110 μmol m–2 S–1), at 26 or 34°C with LED light (photon flux density: 2–18 μmol m–2 S–1), or at room temperature (typically 26–28°C; minimum, 24°C; maximum, 29°C) under natural sunlight (light flux density: 8 μmol m–2 S–1) from a north-facing window without additional light. All experiments were conducted under static conditions. We conducted tests in one series because we were unable to obtain a sufficient amount of the algae used in our experiments due to the lack of previous cultivation reports.
Culture conditions and results for Legionella pneumophila cultivated with algae consortia.
| Culture No. |
Alga | Cultivation conditions | Chlorophyll concentration | Appearance of culture | ||||
|---|---|---|---|---|---|---|---|---|
| Temperature (°C) | Light source | Photon flux density (μmol m–2 s–1) |
Initial (μg mg–1) |
After cultivation (μg mg–1) |
||||
| 1 | V. stellata consortia | 26–28 (room temp.) |
Sunlight from North window | 8 | <Q.D.L. | 1.13 | green suspension | |
| 2 | K. elegans consortia | 26–28 (room temp.) |
Sunlight from North window | 8 | <Q.D.L. | 0.92 | green suspension | |
| 3 | — | 26 (room temp.) |
LED | 2 | <Q.D.L. | <Q.D.L. | transparent | |
| 4 | V. stellata consortia | 36 | Fluorescent lamp | 105–110 | <Q.D.L. | <Q.D.L. | whitening of the alga | |
| 5 | K. elegans consortia | 36 | Fluorescent lamp | 105–110 | <Q.D.L. | <Q.D.L. | whitening of the alga | |
| 6 | — | 36 | Fluorescent lamp | 105–110 | <Q.D.L. | <Q.D.L. | transparent | |
* Quantitative detection limit (Q.D.L.)
Cultivation conditions and results for Legionella pneumophila with pure cultures of Vischeria stellata NIES2148 and Serratia marcescens.
| Culture No. | Bacterium | Alga | Cultivation conditions | Chlorophyll concentration | Appearance of culture | |||
|---|---|---|---|---|---|---|---|---|
| Temperature (°C) |
Light source | Initial (μg mg–1) |
After cultivation (μg mg–1) |
|||||
| 7 | L. pneumophila | V. stellata NIES2148 | 34 | Fluorescent lamp | <Q.D.L.* | 226.8 | green suspension | |
| 8 | L. pneumophila | — | 34 | Fluorescent lamp | <Q.D.L. | <Q.D.L. | transparent | |
| 9 | — | V. stellata NIES2148 | 34 | Fluorescent lamp | <Q.D.L. | 366.3 | green suspension | |
| 10 | L. pneumophila +S. marcescens |
V. stellata NIES2148 | 34 | Fluorescent lamp | <Q.D.L. | 238.6 | green suspension | |
| 11 | L. pneumophila +S. marcescens |
— | 34 | Fluorescent lamp | <Q.D.L. | <Q.D.L. | transparent | |
* Quantitative detection limit (Q.D.L.)
The culture medium was sampled over time, and Legionella CFUs, the copy numbers of the 16S rRNA gene and 16S rRNA, and the CFUs of heterotrophic bacteria were measured. In addition, algal growth was visually observed, and the chlorophyll concentration per unit dry weight was quantified.
Legionella quantification Measurement of Legionella CFUsLegionella CFUs were quantified using glycine, vancomycin, polymyxin B, and cycloheximide (GVPC) Legionella agar medium. The GVPC agar medium was prepared using Legionella agar base (OXOID), Legionella BCYE growth supplement (OXOID), and Legionella (GVPC) selective supplement (OXOID) according to the manufacturer’s instructions. The collected culture medium was diluted with sterilized tap water; 100-μL aliquots were mixed with the same amount of KCl–HCl buffer (pH 2.2; KANTO Chemical) and plated on GVPC agar plates. The plates were incubated at 36°C for 7–10 days, the number of grey–white colonies was counted, and 5–10 were then selected for the identification of Legionella. Colonies that did not grow on BCYEα-cysteine agar (OXOID), but grew on BCYEα agar medium were identified as cysteine-requiring colonies, which were confirmed through agglutination using Legionella pneumophila sero1 serum (Denka Seiken). Agglutinated colonies were identified as L. pneumophila sero1. The detection limit was 10–2 CFU μL–1.
DNA/RNA extractionApproximately 100 μL of the sample was added to a 2-mL screw-cap tube containing 1 g of zirconia beads with a diameter of 0.1 mm and 1 mL of extraction buffer (100 mM Tris-HCl [pH 8.0], 100 mM EDTA [pH 8.0], 100 mM phosphate buffer [pH 8.0], and 1.5 M NaCl). Screw-cap tubes were loaded into a bead-beating homogenizer (Fisherbrand Bead Mill24), and bead beating was performed for 2 min. Thereafter, 110 μL of 20% sodium dodecyl sulfate (SDS) was added and the mixture was vortexed vigorously for 30 s and incubated at 60°C for 30 min. Bead beating was performed again for 2 min before centrifugation at 12,000×g at 24°C for 15 min. Seven hundred microliters of the supernatant was transferred to a 1.5-mL tube, and the same amount of chloroform: isoamyl alcohol (24:1) was added, followed by mixing and centrifugation at 7,500×g at 24°C for 5 min. A total of 630 μL of the supernatant was then transferred to a fresh 1.5-mL tube for isopropanol DNA precipitation. Precipitated DNA was dissolved in TE buffer and purified using a MonoFas DNA Purification Kit I (ANIMOS) according to the manufacturer’s instructions. This DNA was subjected to PCR for the quantification of the Legionella 16S rRNA gene copy number.
Approximately 100 μL of the sample was added to a 2-mL screw-cap tube containing 1 g of zirconia beads with a diameter of 0.1 mm, and 1 mL of ISOGEN (NIPPON GENE) was added. RNA extraction was conducted according to the manufacturer’s protocol. Total extracted RNA was used for cDNA synthesis using the PrimeScriptTM II 1st strand cDNA Synthesis Kit (Takara Bio). This cDNA was subjected to PCR for the quantification of the Legionella 16S rRNA copy number.
Quantitative PCRThe hybridization probe method was performed for qPCR. The LEG225–LEG858 primer pair (Miyamoto et al., 1997) was used for amplification with the detection probes Leg_16S1-S (5′-AGT GGC GAA GGC GGC TAG CT-3′) with a FITC label at the 3' end and Leg_16S2-S (5′-TAC TGA CAC TGA GGC ACG AAA GCG T-3′) with an LCRed640 label at the 5' end. TaKaRa Ex Taq® Hot Start Version (Takara Bio) was used for PCR amplification. Total DNA extracted from L. pneumophila grown on BYCEα agar medium was used to construct a standard curve. Light Cycler II (Roche) was used for thermal cycling under the following conditions: initial denaturation at 95°C for 10 s; 50 cycles of denaturation at 95°C for 10 s, annealing at 57°C for 15 s, and extension at 72°C for 30 s; final cooling at 40°C for 5 s. qPCR was performed in duplicate. The detection limit of DNA or RNA was 1 copy μL–1.
Quantification of heterotrophic bacteria grown on R2A agar platesHeterotrophic bacteria were counted using R2A agar plates (Gibco). The collected culture medium was diluted with sterilized tap water and inoculated onto the plates. The plates were then incubated at 30°C for 2–7 days, and the number of colonies was counted. The detection limit was 10–2 CFU μL–1.
Chlorophyll extraction and analysisOne milliliter of the culture was collected, and the medium was removed by centrifugation. Ice-cool acetone (800 μL) was added, and chlorophyll was extracted from the collected samples by incubating for 15 min in the dark with occasional mixing. After centrifugation, the supernatant was collected, the liquid volume was measured, and 2.5 mM sodium phosphate buffer (pH 7.8) was added to prepare an 80% acetone solution. Absorbance at 750, 663.6, and 646.6 nm was measured using a spectrophotometer; absorbance at 750 nm was used as the background and was subtracted from absorbance at 663.6 and 646.6 nm. These values were converted to chlorophyll concentration per unit volume using the formula of Porra et al. (1989):
chlorophyll a (μg mL–1)=12.25×A663.6–2.55×A646.6.
Dry weight measurements of algal consortiaA glass filter (Whatman) with a pore size of 1 μm and diameter of 47 mm was thoroughly washed with ultrapure water, dried at 105°C, returned to room temperature in a desiccator, and weighed. One milliliter of the culture solution was filtered through this filter, which was then incubated at 105°C for 2 h, returned to a room temperature in a desiccator, and weighed. The original weight of the glass filter was subtracted from the weight after filtration to yield the dry weight in mg. Subsequently, chlorophyll concentration per unit volume was converted on a dry weight basis.
Sample fixation for FISH and in situ Hybridization Chain Reaction (in situ HCR)Pure cultures of L. pneumophila or mixed cultures with algal consortia or pure algae were harvested by centrifugation. Samples were washed twice with phosphate-buffered saline (PBS) by centrifugation and then fixed with 4% paraformaldehyde in PBS at room temperature (24–26°C) for 30 min. After fixation, samples were washed twice with PBS and stored in 50% ethanol with PBS at –20°C.
FISHTo detect L. pneumophila, the 16S rRNA–targeting oligonucleotide probe LEG705 (5′-CTG GTG TTC CTT CCG ATC-3′), which targets sequences from the Legionellae family (Manz et al., 1995), was used. The EUB338 probe (5′-GCT GCC TCC CGT AGG AGT-3′), complementary to a region of 16S rRNA conserved in the domain Bacteria, was used as the positive control for FISH, and the non-EUB338 probe (5′-ACT CCT ACG GGA GGC AGC-3′), complementary to the EUB338 probe, was used to detect non-specific binding (Amann et al., 1990). The probes used were labeled with Alexa488 at the 5' end. Hybridization was performed at 60°C for 1.5 h or overnight in hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 0.01% SDS, and 0% formamide) containing 5 ng μL–1 of a labeled probe. To remove unhybridized probes, the glass slide was gently rinsed (0.18 M NaCl, 20 mM Tris-HCl [pH 7.2], and 0.01% SDS) and immersed in wash buffer at 46°C for 15 min. The slides were carefully rinsed with ultrapure water and air-dried; if necessary, one drop of ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific) was applied to each well. Cells were visualized using an epifluorescence microscope (BX52; Olympus) or a confocal laser scanning microscope (CLSM) (Zeiss LSM 780; Zeiss). Approximately 10 or more fields of view were observed for each sample.
In situ HCRIn situ DNA-HCR was based on the procedure described by Yamaguchi et al. (2015) with some modifications. We used LEG705, EUB338, and non-EUB338 probes, which have an initiating sequence, and the amplifying probes H1 and H2. Supplementary Table S1 lists probe sequences. Fixed cells were mounted in all wells of a 12- or 24-well glass slides. Samples were dehydrated using an ethanol series (50, 80, and 96% [v/v] ethanol for 3, 1, and 1 min, respectively). Hybridization buffer (20 mM Tris-HCl [pH 7.5], 0.9 M NaCl, 0.01% SDS, 10% dextran sulfate, 1% blocking reagent, and 35% formamide) containing initiator probes (0.5 μM) was added at 10 μL per well. Glass slides were placed in a humidified chamber and incubated at 46°C overnight. To remove the excess probe, the glass slide was gently rinsed and immersed in wash buffer (0.07 M NaCl, 20 mM Tris-HCl [pH 7.2], and 0.01% SDS) at 48°C for 30 min. The H1 and H2 amplification probes were incubated separately at 5 μM each in amplification buffer (50 mM Na2HPO4, 0.9 M NaCl, 0.01% SDS, 10% dextran sulfate, and 1% blocking reagent) at 95°C for 1.5 min and then at 25°C for 30 min; the buffers containing the two probes were then mixed. Ten microliters of the H1 and H2 amplification probe mixture was added to each well and the samples were incubated at 35°C for 1 h in a humidified chamber. To remove excess amplifier probes, slides were then immersed in 50 mL of 1× PBS containing 0.01% SDS at 4°C for 10 min. Slides were immersed in ultrapure water and 96% ethanol at room temperature for 30 s each and then stored at 4°C. If necessary, one drop of ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific) was added to each well. Cells were visualized using an epifluorescence microscope (BX52; Olympus) or CLSM (Zeiss LSM 780; Zeiss). Approximately 10 or more fields of view were observed for each sample. In the present study, filamentous Legionella were operationally defined as cells measuring 20 μm or longer. However, when cocultured with algal cell clusters, the complete visualization of Legionella cells was occasionally impeded. Under these conditions, cells measuring between 10–20 μm were also classified as filamentous.
Microscopic observations of the biofilms collected from the cooling towers revealed a single dominant algal species in each cooling tower. To isolate these algae, samples were serially diluted and cultured on C medium. Two distinct algal species were isolated, each originating from the biofilms of two separate cooling towers. A BLAST search was performed on 1,700 base pairs of the 18S rRNA gene sequences of isolated algae, which identified as V. stellata and K. elegans with homology scores of 100%. V. stellata belongs to Eustigmatophyceae and K. elegans to the algal genus Klebsormidium. Although the algae were isolated, they were in a consortium with bacteria. Therefore, they were named the V. stellata and K. elegans consortia, and we used an algal isolate containing bacteria for cocultivation with Legionella.
Culture conditions and the results obtained are shown in Table 1. All cultures, including the coculture of Legionella with algae or Legionella alone, were conducted in an oligotrophic environment using the MA medium, which does not contain L-cysteine. The pH of the MA medium was initially set at 8.6 and was maintained at pH 8.0–8.6 for the duration of the culture. Since one of the cooling towers used to obtain samples for the present study is well-maintained and cleaned monthly, the culture duration was set to approximately 1 month and was extended if necessary. An LED was used as the light source in cultures of Legionella alone (Table 1 No. 3), and light intensity was set to less than sunlight intensity (Table 1 No. 1 and 2).
Changes in Legionella numbers in the culture were monitored by counting CFUs and measuring the copy numbers of the 16S rRNA gene and 16S rRNA. The number of Legionella cells was evaluated by quantifying the 16S rRNA gene copy number, while their activity was assessed by quantifying the 16S rRNA copy number. The results obtained at 26–28°C are shown in Fig. 1A, B, C, and D. Although the MA medium for algae is an oligotrophic medium that does not contain L-cysteine, Legionella CFUs were maintained for approximately 1 month in cultures of Legionella alone or in those with algal consortia (Fig. 1A). At 0 h, the copy numbers of the 16S rRNA gene and 16S rRNA of Legionella cocultured with algal consortia were 10- to 100-fold lower than those in the pure culture of Legionella. However, CFU counts were similar between the coculture and pure culture. (Fig. 1A, B, and C). The reason for this difference was unclear; therefore, we focused on changes over time excluding 0 h. During one month of the culture, the 16S rRNA gene copy number of Legionella cocultured with algal consortia was sustained at those levels, whereas that of Legionella cultured alone slightly decreased (Fig. 1B). Furthermore, differences were observed in the 16S rRNA copy number between Legionella cultured alone and those cocultured with algal consortia (Fig. 1C). These results suggest that the algal consortia exerted a positive effect on maintaining the viability or activity of Legionella. Fig. 1D shows that the CFU count of heterotrophic bacteria on R2A agar plates changed over time. During the culture period, heterotrophic bacteria multiplied in the coculture with algal consortia, and the CFUs of heterotrophic bacteria were 10- to 100-fold higher than Legionella CFUs on R2A agar plates. CFUs were not detected in the culture of Legionella alone, indicating no bacterial contamination.
Cultivation of Legionella pneumophila with the Vischeria stellata or Klebsormidium elegans consortium at room temperature (26–28°C) under natural sunlight. (A) Count of Legionella CFUs. (B) Copy number of the 16S rRNA gene of Legionella. (C) Copy number of the 16S rRNA of Legionella. (D) Count of heterotrophic bacteria grown on R2A agar plates. The cultivation of pure cultures of L. pneumophila is shown as a control. Detailed culture conditions are shown in Table 1. Each point represents a single measurement. The detection limit of DNA or RNA was 1 copy μL–1, while that of Legionella and heterotrophic bacteria was 10–2 CFU μL–1. Data points marked with an asterisk (*) indicate values below the detection limit.
A coculture was then performed at 36°C, the optimum culture temperature for Legionella. Table 1 No. 4, 5, and 6 show culture conditions and algal growth. Under these conditions, a marked increase was not observed in algal growth and the algal cell color turned white, which were attributed to algal death. Fig. 2A shows the change in Legionella CFUs over time. Unlike the results shown in Fig. 1A, Legionella CFUs decreased in cocultures and in cultures of Legionella alone. The fastest decrease in Legionella CFUs was observed in cultures of Legionella alone, with CFUs not being detectable after 20 days. In cocultures with algal consortia, the CFU decrease was suppressed; however, after more than 20 days, an immediate reduction was noted. Fig. 2B shows changes in the 16S rRNA gene copy number over time. Although CFUs were not detectable (Fig. 2A), the 16S rRNA gene copy number did not markedly change under any culture condition. Fig. 2C shows changes in the Legionella 16S rRNA copy number over time. The 16S rRNA copy number of Legionella cultured alone slightly decreased, whereas that Legionella cocultured with algal consortia did not. The number of colonies on the R2A agar plate indicated that the algal consortia contained a high concentration of bacteria (Fig. 2D). However, CFU increases stopped after day 20 of the culture, which was attributed to algal death. According to these results, algae did not grow and Legionella CFUs decreased under all conditions, which was different from the results shown in Fig. 1. This may have been due to differences in the culture temperature or light source conditions.
Cultivation of Legionella pneumophila with the Vischeria stellata or Klebsormidium elegans consortium at 36°C under a fluorescent lamp. (A) Count of Legionella CFUs. (B) Copy number of the 16S rRNA gene of Legionella. (C) Copy number of the 16S rRNA of Legionella. (D) Count of heterotrophic bacteria grown on R2A agar plates. The cultivation of pure cultures of L. pneumophila is shown as a control. Detailed culture conditions are shown in Table 1. Each point represents a single measurement. The detection limit of DNA or RNA was 1 copy μL–1, while that of Legionella and heterotrophic bacteria was 10–2 CFU μL–1. Data points marked with an asterisk (*) indicate values below the detection limit.
To detect Legionella cells in the coculture with the V. stellata consortium (Table 1 No. 1), we initially performed conventional FISH (Supplementary Fig. S3). We observed the presence of DAPI-stained rod-like bacteria in the proximity of algae. Weak green fluorescence was detected (Fig. S3A); however, it was not possible to assess their morphology. This likely corresponded to the Legionella-targeted LEG705 probe labeled with the fluorescent dye Alexa488. Since it was challenging to detect Legionella associated with algae using conventional FISH, we performed in situ HCR, which resulted in clear fluorescence signals from the LEG705 probe that enabled Legionella detection. Legionella cells appeared as filaments extending from the algal surface and also exhibited a rod-shaped morphology (Fig. 3A and B). In contrast, Legionella cells that were cultured alone for 42 days (Table 1 No. 3) and stained with in situ HCR showed green fluorescence in the form of rods, whereas no filament-shaped cells were found (Fig. 3D). Moreover, a higher number of rods were stained with DAPI than with green fluorescence (Fig. 3E). The same sample was subjected to in situ HCR on day 34, and green fluorescence derived from LEG705 was observed even among algal aggregates (Fig. S4).
In situ HCR detection of Legionella pneumophila by the LEG705 initiator probe with Alexa488-labeled H1 and H2 amplifier probes (A), DAPI staining (B), and bright-phase imaging (C) for L. pneumophila with the Vischeria stellata consortium after 27 days of culture (Culture No. 1 in Table 1). In situ HCR detection of L. pneumophila by the LEG705 initiator probe with Alexa488-labeled H1 and H2 amplifier probes (D) and DAPI staining (E) for L. pneumophila cultured alone on day 42. Scale bars represent 20 μm. Ten or more fields of view were observed for each sample.
Cells were treated with LEG705 and stained with DAPI, and autofluorescence was imaged using CLSM (Fig. 4). Cells that reacted with the LEG705 probe were considered to be Legionella, those that stained with only DAPI were regarded as other bacteria, and autofluorescent cells were considered to be algae. Many bacilli and cocci were detected in the spaces between algal cells, and among them, filamentous or rod-shaped Legionella was noted. Filamentous Legionella cells extended along the cell surfaces of multiple algae (with gaps present within filamentous Legionella). Fig. 1A and D show that there were 100- to 1,000-fold more bacterial CFUs than Legionella CFUs, which was supported by the results of staining experiments. Therefore, the voids among algal cells appeared to be filled with bacteria, forming bacterial microcolonies, and Legionella cells were also elongated in microcolonies. These results demonstrate that algae, Legionella, and other bacteria coexisted in the consortium.
Confocal laser scanning microscopy image of in situ HCR detection of Legionella in the Vischeria stellata consortium. The bright green signal was from Legionella cells detected using the Legionella-specific LEG705 initiator probe with Alexa488-labeled H1 and H2 amplifier probes. Reddish purple indicates algae, and the blue signal is from DAPI staining. The scale bar represents 20 μm. Ten or more fields of view were observed for each sample.
On day 27, samples cocultured with the K. elegans consortium at room temperature (Table 1 No. 2) showed long filamentous bacteria stained with DAPI, and the longest bacterium size was estimated to be 50–100 μm (Fig. 5A). Green fluorescence from the LEG705 probe appeared as short filaments (Fig. 5B). This fluorescence overlapped with the ends of the filamentous bacteria stained with DAPI, which indicated that the long filaments were Legionella. The detection of green fluorescence from the LEG705 probe suggested a concentrated presence of Legionella 16S rRNA, implying that both ends of filamentous bacteria were active.
In situ HCR observation of Legionella cocultured with the Klebsormidium elegans consortium. Green fluorescence indicates Legionella detected by the LEG705 initiator probe with the Alexa488-labeled H1 and H2 amplifier probes, and orange fluorescence indicates algal autofluorescence (B), DAPI staining (A), and bright-phase imaging (C). Arrows indicate filament-like morphologies. The circle indicates the overlapping area with fluorescence from the LEG705 probe. The scale bar represents 20 μm. Ten or more fields of view were observed for each sample.
The algae isolated in the present study contained several types of bacteria; therefore, an unknown factor from coexisting bacteria may have affected the development and morphology of Legionella. We prepared a pure coculture with V. stellata strain NIES2148 and L. pneumophila to observe the morphology of Legionella. All in situ HCR images of Legionella and algae before the coculture experiment are shown in Fig. S5A, B, and C. Coculture conditions are shown in Table 2, Culture No. 7, 8, and 9.
In the present study, a fluorescent lamp was used as the light source, and light intensity was less than 20 μmol m–2 s–1. The medium containing V. stellata NIES2148 turned green and algal growth was observed. Fig. 6 shows changes in the Legionella CFU count and the copy numbers of the 16S rRNA gene and 16S rRNA over time. The Legionella CFU count and 16S rRNA gene copy number both slightly decreased, which was similar for Legionella cultured alone and in combination with V. stellata NIES2148 (Fig. 6A and B). However, changes in the Legionella 16S rRNA copy number differed between the Legionella pure culture and coculture. It decreased to undetectable levels by day 20 in the Legionella alone culture, but was maintained and then increased by day 30 in the coculture with algae (Fig. 6C).
Culture of Legionella pneumophila with pure Vischeria stellata NIES2148 and Serratia marcescens. (A) Number of Legionella CFUs. (B) Copy number of the 16S rRNA gene of Legionella. (C) Copy number of the 16S rRNA of Legionella. The incubation temperature was 34°C. Detailed cultivation conditions are shown in Table 2. Each point represents a single measurement. The detection limit of DNA or RNA was 1 copy μL–1, while that of Legionella was 10–2 CFU μ–1. Data points marked with an asterisk (*) indicate values below the detection limit.
After V. stellata and Legionella were cocultured for 30 days, in situ HCR showed faint green filamentous fluorescence derived from LEG705 along the algal surface (Fig. 7A), and a DAPI-stained filamentous form corresponding to green fluorescence was observed (Fig. 7B). Additionally, since algae cells appeared to be hollowed out, they were regarded as dead cells (Fig. 7C). On the other hand, in the culture with Legionella alone, green fluorescence was not observed, and only rod-shaped Legionella cells were detected by DAPI staining.
Coculture of Legionella pneumophila with pure Vischeria stellata. L. pneumophila was detected by in situ HCR using the LEG705 initiator probe with Alexa488-labeled H1 and H2 amplifier probes (A), DAPI staining (B), and bright-phase imaging (C). Arrows indicate filamentous morphologies of L. pneumophila. Scale bars represent 20 μm. Ten or more fields of view were observed for each sample.
Observations of cocultures of Legionella and the V. stellata consortium showed the coexistence of algae, Legionella, and other bacteria, and also the elongation of Legionella cells in this culture (Fig. 4). To clarify whether the other bacteria contributed to the development of Legionella, a coculture of Legionella, algae, and other bacteria was performed. Experimental conditions are shown in Table 2, Culture No. 10 and 11. S. marcescens isolated from a cooling system was used as the other bacterium. The LEG705 probe did not react with S. marcescens (Fig. S5D and E). Legionella CFUs were not measured because after the addition of S. marcescens, gray white colonies had increased by more than 100-fold on the GVPC agar plate, but were not identified as Legionella, while the number of S. marcescens CFUs on the R2A agar plate was 100-fold higher than that of Legionella CFU. Fig. 6B shows that the Legionella 16S rRNA gene copy number changed over time, and decreased more in the triculture system than in the culture of Legionella alone or in the coculture with V. stellata NEIS2148 or S. marcescens. The reason for this difference was unclear. The 16S rRNA copy number in the coculture was maintained, whereas that in the triculture showed an increase by an order of magnitude (Fig. 6C). Additionally, the 16S rRNA copy number in the coculture of S. marcescens and Legionella did not decrease and was maintained at the same level as that in the algal cocultures.
Fig. 8 shows in situ HCR results for the pure triculture of V. stellata, S. marcescens, and L. pneumophila. Although filamentous green fluorescence derived from the LEG705 probe was not observed until day 10, it was detected on the surface of the algae on day 21 of the culture, which was considered to indicate the presence of Legionella. These images were taken under the same conditions as those in Fig. 7; however, fluorescence intensity was markedly stronger. This result is consistent with the high RNA concentration observed. Collectively, these results indicate that Legionella collaborated with multiple species in the biofilms of non-predatory microorganisms for elongation.
Pure tri-culture of Vischeria stellata, Serratia marcescens, and Legionella pneumophila on days 1 (A), 10 (B), and 21 (C, D, E, F). L. pneumophila was detected by in situ HCR using the LEG705 initiator probe with Alexa488-labeled H1 and H2 amplifier probes (A, B, C, D), DAPI staining (E), bright-phase imaging (F). Panels D, E, and F are enlarged images of the square area in panel C. Scale bars represent 60 μm. Ten or more fields of view were observed for each sample.
In the environment, Legionella species are considered to live parasitically in predatory protozoa (Grimm et al., 1998; Rasch et al., 2016; Caicedo et al., 2018; Dietersdorfer et al., 2018) or symbiotically with non-predatory organisms, such as other bacteria (Taylor et al., 2013), photosynthetic microorganisms (Tison et al., 1980), and algae (Pope et al., 1982). Although open cooling water systems are a typical source of Legionella (Fraser et al., 1977; Chamberlain et al., 2017), algae and bacteria are also dominant in cooling towers (Di Gregorio et al., 2017). It currently remains unclear whether non-predatory organisms in these systems contribute to the growth of Legionella. Therefore, we collected algae from real cooling water systems and cocultured them with Legionella in an artificial oligotrophic medium at an alkaline pH. Quantification and morphological observations of Legionella revealed that the algae maintained the Legionella 16S rRNA copy number, and also that Legionella existed contiguously to the algae, elongating as filamentous cells on algal surfaces. These results are important not only with respect to Legionella ecology, but also public health.
Legionella behavior with or without algae under alkaline oligotrophic conditionsRepeated trial-and-error experiments were conducted to optimize culture conditions because there is currently no information on the cultivation of algae sampled from cooling towers in MA medium. Therefore, coculture conditions were not the same, whereas the microorganisms and medium used were the same in all experiments. We considered a comparison of results to help in establishing whether algae affect Legionella under alkaline oligotrophic conditions.
The 16S rRNA copy number of Legionella cultured with algae (Table 1 No. 1 and 2; Table 2 No. 7 and 10) was repeatedly found to be consistently higher than that of Legionella cultured alone (Fig. 1C, 2C, and 6C). On the other hand, in the culture of Legionella alone (Table 1 No. 3 and 6; Table 2 No. 8), the 16S rRNA copy number repeatedly decreased by an order of magnitude (Fig. 1C, 2C, and 6C). Legionella alone did not appear to be able to maintain its activity under these experimental conditions, which is possible because the Legionella culture was in a state of starvation (Li et al., 2015). Moreover, morphological observations using microscopy revealed that Legionella cells were present in these samples (Fig. 3D), suggesting that Legionella cells maintained their shape even in a state of reduced activity.
However, the CFU count showed varying changes regardless of the presence of algae: its maintenance (Fig. 1A), a decrease to an undetectable level (Fig. 2A), or a decrease (Fig. 6A), which was inconsistent. Mendis et al. (2015) previously reported that when cultured in a nutrient-poor medium, the ability of Legionella to form CFUs rapidly decreased at temperatures higher than 30°C at pH 4, 5, or 6. The present results support these findings. We attributed the inconsistency in CFUs to different incubation temperatures because higher temperatures were associated with greater reductions in CFUs. Our results also showed that this phenomenon occurred under alkaline conditions. CFUs were maintained under alkaline oligotrophic conditions at 24–34°C for approximately 1 month, mimicking cooling water environments. This is the first study to demonstrate this in a sterile medium, highlighting the necessity of Legionella suppression treatments.
As described above, CFUs decreased below the lower detection limit at 36°C; however, the Legionella 16S rRNA gene copy number remained unchanged (Fig. 2) and the cell morphology of Legionella was preserved (Fig. 3D). Some of the remaining Legionella may have been viable but nonculturable (VBNC) because previous studies demonstrated that VBNC Legionella formation was induced under oligotrophic conditions (Hwang et al., 2006; Dietersdorfer et al., 2018). These findings indicate that some VBNC Legionella strains were resuscitated in cocultures with amoebae. Therefore, it is important to establish a consensus on how to evaluate Legionella DNA or VBNC Legionella for Legionella control in cooling towers.
Although algae and Legionella are generally considered to coexist in a symbiotic relationship, only one study reported Legionella multiplication in algal biofilms (Pope et al., 1982), and the relationship between algae and Legionella in actual cooling towers has yet to be examined. This is the first study in which algae isolated from a real cooling tower were shown to contribute to the survival or growth of Legionella. Furthermore, isolated algae, which were dominant in each cooling tower, both promoted Legionella survival, and these strains had not previously been reported to coexist with Legionella. Therefore, algae that foster Legionella may be widespread in cooling water systems. However, the relationship between algae and Legionella is complex and has yet to be clarified. Bathia et al. (2022) reported that Legionella living in hydras were suppressed by Chlorella. Further studies are warranted to elucidate the relationship between algae and Legionella because this information is important for a more detailed ecological understanding and the better control of Legionella.
On the other hand, the coexistence of Legionella with non-predatory organisms other than algae and the proliferation of Legionella in these settings has been reported, including the formation of Legionella CFU under alkaline culture conditions. For example, among photosynthetic microorganisms, Legionella numbers were shown to increase in cultures with MD medium (pH 7–7.6) in which cyanobacteria proliferated (Tison et al., 1980). Tison et al. (1980) showed that Legionella numbers decreased at pH >7.5; however, they also detected Legionella CFUs at pH ≥8 (data not shown). Other studies revealed that the growth of Legionella was promoted by other organisms and that Legionella CFUs were detected even under alkaline conditions. Additionally, we focused on oligotrophic conditions at pH 8–8.6 and found that the 16S rRNA copy number of Legionella remained high if the algae consortia or pure algae grew. Since alkaline oligotrophic conditions are a common feature of cooling water, this is an important experimental result indicating the ability of Legionella to maintain a high activity level in the presence of algae in real cooling systems. Algae always coexist with bacteria in the environment. When Serratia isolated from a cooling tower was experimentally added to the coculture of algae and Legionella, a further increase was observed in the 16S rRNA copy number (Fig. 6C). Morphological observations revealed strong green fluorescence, which was considered to be derived from Legionella (Fig. 8D). Therefore, Legionella appear to coexist with two or more species and exhibit better growth under these conditions. This is the main result of this study and warrants further investigation.
Filamentous morphology of Legionella in the coculture with algaeThe 16S rRNA copy number of Legionella cultured with algae was higher than that of Legionella cultured alone. Filamentous cells considered to be Legionella were detected in these samples (Fig. 3A, S4, 4, 5B, 7A, and 8D). Even in the coculture with pure algae (Table 2 No. 7), this phenomenon was observed (Fig. 7A and B).
This is the first study in which in situ HCR was used to examine Legionella in an algal consortium. This fluorescence amplification method is crucial for observing the morphology of Legionella in the environment. The only limitations of in situ HCR are the non-specific adsorption of amplification probes and the need for the optimization of hybridization and washing conditions for each observation target.
Through the application of in situ HCR, Legionella was successfully visualized despite autofluorescence by algae, and filamentous extensions were detected on algae. To the best of our knowledge, this is the first study to report these results. In observations of Legionella cocultured with the K. elegans consortium (Table 1 Culture No. 2), a strong fluorescence signal was observed at both ends of the filament (Fig. 5), which suggested that Legionella became filamentous due to growth at both ends without division. However, it remains unclear why Legionella elongated without dividing. This coculture incubation was extended to day 54, at which point the Legionella CFU count decreased (Fig. 1A), whereas the copy numbers of the 16S rRNA gene (Fig. 1B) and 16S rRNA (Fig. 1C) did not. This contradictory behavior may be attributed to the phenomenon of Legionella elongation.
In the present study, Legionella on the surface of the algae had rod-shaped or filament-like forms. This morphology differed from that described in other studies. For example, previous studies on Legionella growth in the environment were conducted mainly with the coexistence of predatory organisms, such as amoebae and Tetrahymena (Rowbotham, 1980; Grimm et al., 1998; Caicedo et al., 2018). In these cases, Legionella appeared as a rod-shaped bacterium randomly within or around the predator cell. Additionally, FISH did not allow us to observe the Legionella cell morphology in an artificial biofilm composed of bacteria and protozoa (Taylor et al., 2013). Legionella was cocultured with photosynthetic microorganisms, and the biofilm was observed using optical microscopy. In this case, Legionella cells were scattered as rod-shaped bacteria in the slime around cyanobacteria (Bohach and Snyder, 1983b).
On the other hand, the filamentation of Legionella is a well-known phenomenon. Filamentous Legionella species are often identified in environmental water samples or clinical specimens (Blackmon et al., 1978). Paszko-Kolva et al. (1992) reported that Legionella survived for 1 year in a culture of river water from which microorganisms were removed by filtration, and the morphology of Legionella changed to a filamentous nature after approximately 1 year. The present results indicated that algae promoted the elongation of Legionella. Paszko-Kolva et al. (1992) also stated that morphological changes in Legionella were reversible; when filamentous Legionella cells were fragmented, they transformed into rods, and rod-shaped Legionella cells also transformed into filamentous cells.
Prashar et al. (2012, 2018) investigated the mechanisms underlying the infection of pulmonary epithelial cells by filamentous Legionella and showed that these cells actively attached to the epithelial cell membrane. Moreover, filamentous Legionella attached to and infiltrated cells and then divided into rods in infected cells, in which they proliferated. In the present study, Legionella CFUs did not increase; however, the possibility that filamentous Legionella infiltrate and proliferate within algae cannot be denied. Therefore, further research will be important for clarifying the mechanisms by which Legionella coexist with non-predatory microorganisms in the environment.
In the present study, Legionella cells existing closely on algae were rod-shaped or filamentous. This may be related to the culture temperature used in our study (26–28°C). The optimal culture temperature for Legionella is 36–37°C, at which Legionella become filamentous. To obtain rod-shaped Legionella, it is necessary to maintain the culture at 25–30°C. Piao et al. (2006) reported that biofilm formation by Legionella alone was affected by temperature. An incubation at 36°C produced a filamentous Legionella biofilm, while that at 30°C resulted in a biofilm of rod-shaped Legionella. Therefore, the impact of temperature on the formation of Legionella filaments warrants further research.
We propose a model for Legionella survival and growth based on the present results (Fig. 9). Even in alkaline oligotrophic water, Legionella elongate when coexisting with living algae, thereby expanding its area. The mechanisms underlying elongation and whether it occurs in cooling towers or the natural environment warrant future investigation.
A proposed model of Legionella survival and elongation. Photosynthetic microorganisms, such as algae, serve as nutrient sources for bacteria. In addition, Legionella species coexist and develop.
We investigated the effects of photosynthetic algae on Legionella survival in laboratory experiments. The present results demonstrated that algae collected from cooling water systems promoted the viability and elongation of Legionella, and also that this behavior differed from that of Legionella proliferation with predatory organisms, such as amoebae. The main results obtained herein are described below.
1) Although Legionella persisted under oligotrophic and alkaline conditions, the Legionella 16S rRNA copy number remained high due to its coexistence with live algae. Legionella cells that coexisted with live algae elongated at both ends and became filamentous. This morphology of Legionella on algal cells was observed for the first time using in situ HCR.
2) The two species of algae that were isolated from real cooling towers promoted the viability and elongation of Legionella, which suggests that diverse types of algae support Legionella in cooling systems.
3) The increase observed in the Legionella 16S rRNA copy number was greater in the presence of both algae and bacteria than in the presence of algae alone, indicating that Legionella species interact with multiple organisms to improve their survival.
In the present study, non-predatory organisms that are dominant in biofilms promoted Legionella elongation under alkaline and oligotrophic conditions. Since Legionella species coexist with diverse types of eukaryotic and prokaryotic cells, each symbiotic mechanism needs to be clarified. If these mechanisms are elucidated, it may become possible to control Legionella in artificial environments more safely and efficiently.
Satou, W., Nagai, N., and Hatamoto, M. (2025) Legionella Survives and Elongates in Algal Consortia Containing Bacteria in Alkaline Oligotrophic Conditions. Microbes Environ 40: ME25016.
https://doi.org/10.1264/jsme2.ME25016
We thank Ms. Oomori and Ms. Matsuzaki for their technical assistance, Mr. Kato for his instructions on microscopy observations, Ms. Tanaka for her help with the confocal laser microscope, and Mr. Kakimoto for his literature review.
