Biotic interactions between the human pathogen Legionella pneumophila and nematode grazers in cooling tower biofilms

Christin Ortlieb; Aurélie Labrosse; Liliane Ruess; Michael Steinert

doi:10.1371/journal.pone.0309820

Abstract

Biofilms in cooling towers represent a common habitat for the human pathogen Legionella pneumophila. Within the biofilm consortium, frequent interactions with protozoa, i.e. amoebae and ciliates, were reported, while nematodes have only recently been considered as potential environmental reservoir for the pathogenic bacteria. This study is the first approach to investigate the biotic interactions between L. pneumophila and bacterial-feeding nematodes in a semi-natural biofilm model. The species were Diploscapter coronatus, Diploscapter pachys, Plectus similis and Plectus sp., which all co-occur with L. pneumophila in the environment. Biofilms derived from cooling towers were either inoculated with mCherry-labeled L. pneumophila solely or in combination with GFP-labeled Escherichia coli. All experiments were conducted in single-species set-ups and multi-species (D. coronatus and P. similis) set-ups, to account for interspecific competition. Bacterial ingestion was assessed after 24 and 96 h as fluorescence patterns in the digestive tract of the nematodes using confocal laser scanning microscopy. L. pneumophila cells were ingested by all nematode species, with D. coronatus having the highest pathogen load. The fluorescence intensity (i.e. bacterial load) varied between compartments within the digestive tract and was independent of incubation time. Bacterial cells accumulated mostly around the cardia and in the intestine, while less cells were found within stoma and pharynx. Interspecific competition changed the pattern, i.e. with incubation of D. coronatus and P. similis in the same biofilm a significantly higher pathogen load occurred in the intestine of D. coronatus than P. similis after 24 h and 96 h. Remarkably, when given a choice between L. pneumophila and E. coli, P. similis was the only nematode species containing both bacteria after incubation for 24 h. None of the other nematode species contained E. coli after 24 h and 96 h incubation, while L. pneumophila was present. This study thus provides the first evidence, that under environmental conditions L. pneumophila is a frequent diet of bacterial-feeding nematodes, highlighting their potential as pathogen vectors or even host in cooling tower habitats.

Citation: Ortlieb C, Labrosse A, Ruess L, Steinert M (2024) Biotic interactions between the human pathogen Legionella pneumophila and nematode grazers in cooling tower biofilms. PLoS ONE 19(10): e0309820. https://doi.org/10.1371/journal.pone.0309820

Editor: Marta Palusinska-Szysz, Maria Curie-Sklodowska University, POLAND

Received: May 2, 2024; Accepted: August 19, 2024; Published: October 25, 2024

Copyright: © 2024 Ortlieb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was funded by the German Science Foundation (https://www.dfg.de/de) with the grant numbers RU 780/19-1 to LR, to fund and support the work of Christin Ortlieb, and STE 838/12-1 to MS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

In the aquatic environment, prokaryotic and eukaryotic cells often join together to form complex communities adhering to inorganic or organic surfaces, known as biofilms [1]. Here, cells are embedded in a matrix of extracellular polymeric substances (EPS), which protects from a wide range of environmental challenges, e.g. predation, UV exposure, metal toxicity, dehydration and salinity, phagocytosis as well as antimicrobial agents [2, 3]. Especially in man-made water systems (e.g. dental units, drinking water systems, showerheads, air conditioning units, cooling towers), the enhanced resistance of biofilms to antibiotics is a serious concern for human health [4–7]. One bacterial pathogen that naturally grows and persists in such technical water systems is Legionella pneumophila, which is also classified as one of the highest-priority infectious disease pathogens of general public health concerns in Germany [8, 9].

L. pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease, with a case fatality rate of 9% in Europe [10]. The inhalation of Legionella-contaminated aerosols can result in a bacterial infection of the human lungs, leading to alveolar damage, cellular infiltrations of neutrophils and macrophages, and eventual dispersal of extracellular bacteria to other organs [11]. Large community-associated outbreaks of Legionnaire’s disease are most frequently associated with cooling towers, which can disperse Legionella-contaminated aerosols over long distances of up to 10–15 km [12, 13]. In Germany, at least four cooling tower-related outbreaks of Legionnaires’ disease were documented since 2009 [14]. The largest outbreak to date with 159 suspected cases occurred in 2013 in Warstein [15].

Cooling towers provide an excellent environment for the growth and proliferation of diverse microorganisms due to elevated operation temperatures, a neutral pH and continuous aeration [16]. Within cooling towers, the availability of surfaces in the evaporative fill material, heat exchangers, water reservoir and cooling water pipelines allow for the extensive growth of biofilms [17]. Particularly the water temperatures between 25°C and 35°C support the growth of the thermophilic L. pneumophila [18]. The majority of cooling towers apparently contain a core Legionella community, that quickly recovers after chemical disinfection [19]. Especially the floating biofilms at the water-air interface in the basin represent a common bacterial habitat [20].

As an intracellular parasite L. pneumophila is regularly found in microbial grazers, predominantly amoebae (e.g. Acanthamoeba, Vermamoeba, Naegleria) and ciliates (Tetrahymena) [21]. Amoebae isolated from cooling towers are 16 times more likely to be infected (albeit not necessarily with Legionella) than amoebae isolated from natural fresh waters [22], making cooling towers a hot spot for Legionella-amoebae interactions [23]. However, virtually nothing is known on the interactions with other major bacterial grazers, the nematodes, in the biofilms of cooling towers.

Among metazoa, nematodes are the most abundant and diverse group in both natural biofilms and granular or biological filters in technical waters [24, 25]. As filter-feeders, nematodes ingest bacteria suspended in liquid. Food particles are transported from the stoma to the intestine via the pharynx, a neuromuscular organ functioning as pump [26]. A grinder located in a bulb at the end of the pharynx crushes the bacteria before they are pushed through a valve and enter the intestine [27]. By grazing on bacteria nematodes influence key biofilm processes such as detachment, oxygen turnover, carbon mineralization and release of secondary metabolites [28, 29]. Nematode feces and mucus excreta may further enhance microbial growth, while bioturbation increases biofilm permeability and provides new niches for bacteria [30, 31].

Generally, L. pneumophila cells can survive the passage through the nematode grinder and colonize the intestinal tract [32]. Here, the pathogen replicates in the intestinal lumen but also invades intestinal cells establishing Legionella-containing vacuoles (LCVs) [32, 33]. Colonization and persistence of the pathogen within the intestine leads to a shortened lifespan compared to individuals fed with E. coli OP50, as shown for C. elegans [32]. Next to this parasitic relationship, host-bacteria interactions can be commensal, i.e. bacteria obtain nutrients off its host without introducing disease [34].

By harboring and excreting bacteria in viable conditions, nematodes contribute to their dissemination in the environment, which makes them a potential health risk as vectors of human pathogens including Legionella. However, infection assays with nematodes are mainly restricted to the model host Caenorhabditis elegans [32, 33, 35], which is typically not present in aquatic environments. Recently, a survey screening the biofilms of natural (26 swimming lakes) and technical (7 cooling towers) water habitats, revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers [36]. Seven nematode taxa that thrive in Legionella-positive cooling tower biofilms were isolated and cultured, including Plectus and Diploscapter. This provides an ideal basis for studying the interactions between nematodes and Legionella with biota that co-occur in nature, to address the role of nematodes as environmental reservoirs. In a previous study, we were able to show that the L. pneumophila cooling tower isolate KV02 impairs the feeding activity of the environmental biofilm isolates P. similis and Plectus sp. [36]. Examination of the pharyngeal pumping activity showed, that L. pneumophila decreased the pumping rate of both nematode species in comparison to the standard lab food E. coli OP50 by 70% [36]. This suggests that the pathogen, at least in an artificial set-up on an agar plate, is not a common food source for nematodes.

In order to further decipher the Legionella-nematode interaction, the current study investigates the relationship between L. pneumophila and bacterial-feeding nematodes in an ecological set-up, i.e. a semi-natural biofilm model. To achieve the most realistic picture of this interspecific interaction, the tested L. pneumophila strain KV02, the nematode P. similis, and the biofilm material were all isolated from the same cooling tower. Additionally, Plectus sp., isolated from a natural Legionella-positive spa bath [37], was used. Further, the thermophilic Diploscapter coronatus and Diploscapter pachys were tested. Diploscapter is a common taxon in cooling tower biofilms and known vector for enteric pathogens such as Salmonella and Listeria [36, 38]. The taxa Plectus and Diploscapter have different ecological life-strategies. Diploscapter is an enrichment opportunist, continuously pumping with its pharynx at high microbial densities, while Plectus is a generalist opportunist, pumping less frequently and also surviving on poor food conditions [39]. These different feeding strategies of the two tested nematode taxa may affect the grazing activity on L. pneumophila. Test assays were performed with Legionella only and Legionella with the common food bacterium Escherichia coli in combination to investigate the food choice of nematodes. L. pneumophila was labeled with mCherry, and E. coli with GFP, to allow tracing of cells in biofilm and nematodes. The following hypotheses were investigated (1) Ingestion of L. pneumophila by nematodes varies between species, with enrichment opportunists showing the highest uptake, (2) the distribution pattern of bacterial cells within the digestive tract of nematode grazers differs due to interspecific interactions (e.g. commensalism or parasitism), and (3) nematodes prefer E. coli over the pathogen L. pneumophila when given a choice.

2. Materials and methods

2.1 Cultivation of bacterial strains

The Escherichia coli strain DH10β harboring the GFP-expressing plasmid pXDC31 was cultivated on lysogeny broth (LB) agar supplemented with 12.5 μl ml^-1 chloramphenicol. The E. coli strain OP50 was grown in LB medium. Both E. coli strains were long-term laboratory cultures and were incubated on the respective media overnight at 37°C. The L. pneumophila strain KV02 was isolated from biofilms of a cooling tower [36]. The bacteria were labeled with mCherry (plasmid pXDC50) and grown on buffered charcoal-yeast extract (BCYE) agar, supplemented with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 5 μl ml^-1 chloramphenicol at 37°C for 2 days. Prior to the experiments, bacteria were carefully removed from the agar plate and resuspended in sterile-filtered cooling tower water. Then, bacterial density, expressed as colony forming units (CFU ml^-1), was measured at OD600nm.

2.2 Cultivation of nematodes

All assays were performed with free-living, bacterial-feeding nematodes, reported as common fauna in cooling tower biofilms [36]. This comprised the generalist opportunists Plectus similis (0.53 ± 0.10 mm; Zell 1993) and Plectus sp. (0.44 ± 0.80 mm), and the enrichment opportunists Diploscapter coronatus (0.45 ± 0.63 mm; Cobb 1893) and Diploscapter pachys (0.38 ± 0.61 mm; Steiner 1942). The taxa Diploscapter (c-p 1) and Plectus (c-p 2) are grouped along a colonizer-persister continuum ranging from one to five on the basis of several character sets, including the reaction to resource pulses [40].

P. similis was isolated from a cooling tower biofilm in Lower Saxony, Germany [36] and Plectus sp. from a biofilm in a thermal spa bath in Aix-les Bains, France [37]. Both Plectus species were grown on Page’s Amoeba saline (PAS) agar on their natural bacterial microbiome, without additional food supply. D. coronatus was kindly provided by Dr. Philipp Schiffer and the D. pachys strain PF1309 by Hélène Fradin [41]. Stock cultures of Diploscapter were grown on nematode growth medium (NGM) agar plates inoculated with an E. coli OP50 lawn at 20°C. Since nematodes do not fall under the animal welfare regulations in Germany (i.e. laboratory animal reporting regulations), no Ethics statement is required. The same applies to the flora and fauna originating from the biofilm material.

2.3 Biofilm assay

Natural biofilms were obtained from a cooling tower with proven occurrence of L. pneumophila located in the Lower Saxony region in Germany [36]. Floating biofilms from the air-water interface were collected in glass bottles and stored at 4°C until further processing. The cooling tower biofilm was mainly composed of benthic cyanobacteria (predominantly Lyngbya, Oscillatoria, Phormidium), benthic Charophyta (predominantly Klebsormidium) and epiphytic Bacillariophyta (predominantly Amphora, Navicula, Nitzchia).

To achieve nematode-free biofilms, 20 ml subsamples were screened for biofilm-dwelling nematodes using an inverse microscope (CKX31, Olympus, Tokyo, Japan). To avoid food competition between the autochthone grazers and the four test nematode species, the natural nematode fauna was removed using a micropipette. The removal of nematode eggs was not possible, yet the feeding pressure of newly hatched nematode larvae can be considered as minor. Then, a volume of each 250 μl nematode-free biofilm were transferred into chamber slides (each chamber with a total volume of 300 μl; ibidi, Gräfelfing, Germany) as experimental microcosms (S1 Fig).

For the inoculation of natural biofilms with bacteria and nematodes we adapted a method described by Rasch et al. [37]. In the L. pneumophila-only assay, the feeding of nematodes on the pathogenic bacteria inhabiting the biofilm was tested. For this, 50 μl of mCherry-labeled L. pneumophila KV02 (final concentration 2 x 10⁸ CFU ml^-1), resuspended in sterile-filtered cooling tower water, were inoculated and left overnight at 25°C to adapt. As next step, hand-picked test nematodes (30 individuals per microcosm) were inoculated per microcosm (n = 2). Nematodes were age-synchronized by selecting only young females (visual inspection using a binocular) in order to exclude age-dependent differences in ingestion rates. Two scenarios were investigated: i) single set-ups with one of the four test species each, and ii) mixed set-ups with a combination of P. similis and D. coronatus with each 15 individuals per microcosm (n = 2). These two species were chosen because they are most likely to share technical biofilms as habitat: P. similis occurred in cooling tower biofilms together with L. pneumophila [36] and D. coronatus was abundant in biofilter reactors [42].

For the second test series, the L. pneumophila versus E. coli assay, 50 μl of GFP-labeled E. coli DH10β were inoculated together with 50 μl of mCherry-labeled L. pneumophila KV02 (final concentration 6 x 10⁸ CFU ml^-1) prior to the addition of nematodes. The same two scenarios as for the L. pneumophila-only assay were tested, either single set-ups (one of the four test species with 30 individuals per microcosm) and mixed set ups (combination of P. similis and D. coronatus with 15 individuals each) per microcosm (n = 2). All microcosms were incubated at 25°C for either 24 h or 96 h. This time span covers the initial ingestion of bacteria until reaching a plateau of the intestinal bacterial load [32, 43]. Prior to killing the nematodes with 15% paraformaldehyde for microscopic analysis, 50% of the individuals were checked for viability using a binocular microscope.

The palatability of GFP-labeled E. coli DH10β for all nematode species was verified in preliminary choice tests after Abada et al. [44] against the isogenic strain DH10β not expressing GFP and against E. coli OP50 (S2 Fig). Both, E. coli DH10β and E. coli OP50 are common lab foods of nematodes [45, 46]. Yet, as D. coronatus and D. pachys were grown on E. coli OP50 the strain E. coli DH10β was used for the biofilm assays to minimize potential effects of food familiarity. Additionally, ingestion of GFP-labeled E. coli DH10β in sterile-filtered cooling tower water was confirmed microscopically (S3 Fig).

2.4 Fluorescence microscopy and data analysis

The biofilm microcosms were screened for nematode colonization and ingestion of L. pneumophila KV02 and E. coli DH10β by confocal laser scanning microscopy (CLSM; Leica TCS SP8, Wetzlar, Germany). The excitation wavelengths 488 nm and 552 nm were used to excite GFP and mCherry, respectively. Emission spectra were collected between 492 and 544 nm (GFP) and 564 and 724 nm (mCherry). In some cases, full z-series images were obtained for 3D animations to verify the rod shape of single bacterial cells and to verify that the bacterial cells were located within the nematode body and not outside on the cuticula. To account for nematode autofluorescence, potentially interpreted as bacteria-derived fluorescence, nematodes from microcosms without labeled bacteria served as control.

The mean fluorescence intensity (MFI) of mCherry-labeled L. pneumophila KV02 and GFP-labeled E. coli DH10β was quantified in the nematode digestive system. The software Fiji was used, an open-source platform focused on biological-image analysis [47]. The final MFI (bacterial signal) was calculated by subtracting the MFI of the non-fluorescent image background from that of the body tissue MFI. The MFI data are given as arbitrary unit (a.u.) ± standard error (SEM). Graphs are expressed as mean per nematode, with nematode individuals as biological repeats composed of 2 technical replicates (i.e. 2 microcosms).

The presence of bacteria was assigned in six parts of the nematode digestive tract: mouth cavity (stoma), the oesophagus (pharynx, terminal bulb, cardia), and the gut (intestine, rectum) (Fig 1 and S2 Fig). Each confocal image was scanned for the presence of fluorescent bacteria within nematodes. Then, the entire body part (region of interest, ROI) was circled with a drawing pen and the MFI was measured for the defined area by the software. This procedure was repeated for all nematode individuals with proven ingestion of bacteria and set-up. For each nematode examined the MFI was measured for all six body parts. To ensure that the original image data from the microscope software were comparable, the underlying image properties (e.g. contrast, saturation) were not changed between recordings. Nematodes in which no bacteria were found were not considered for the calculation of the MFI.

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Fig 1. Representative confocal laser scanning microscopy images of nematodes harboring Legionella pneumophila KV02.

(A) Diploscapter coronatus. (B) Diploscapter pachys. (C) Plectus similis. (D) Plectus sp. Nematodes harbor L. pneumophila KV02 (mCherry, red) in the stoma (Plectus sp.), terminal bulb (D. coronatus), cardia (D. coronatus), intestine (D. coronatus, P. similis), and rectum (D. coronatus, D. pachys). Number of investigated individuals (n), number of individuals that harbored L. pneumophila KV02 (KV02) and number of individuals without L. pneumophila KV02 (none) after incubation for 24 h and 96 h are shown for each nematode species.

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The data are analyzed using R version 4. 2. 3 “Shortstop Beagle” [48]. Prior to statistical tests all data were checked for normal distribution and homogeneity of variances. Then, the data were subjected to the Mann-Whitney U test or the Kruskal-Wallis rank sum test with Dunn’s post hoc test (with Bonferroni correction). A significance level of p < 0.05 was applied to all statistical analyses.

3. Results

3.1 No-choice assays—L. pneumophila-only

Single-species set-up.

With L. pneumophila KV02 and nematodes in single test assays, bacterial cells were ingested by all nematode species, yet detected at different compartments within the digestive system (Figs 1 and 2). After 24 h incubation all individuals of D. coronatus harbored bacteria (Fig 1A), with the bacterial load (expressed as MFI) highest around the cardia, followed by the intestine and the terminal bulb, with 102, 86, and 49 a.u. Ind^-1; respectively (Fig 2). On the other hand, stoma and rectum harbored only few bacteria, which was significant for the stoma compared to the intestine (p < 0.028). After 96 h incubation, the overall MFI decreased by about half, while the fluorescence location pattern remained similar: The bacterial load was again highest in the intestine (56 a.u. Ind^-1) and around the cardia (48 a.u. Ind^-1), followed by the terminal bulb (28 a.u. Ind^-1). The lowest MFI was emitted by the pharynx and rectum (p < 0.001) and no bacteria were detected in the stoma.

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Fig 2. Bacterial load of the digestive system of nematodes after incubation with Legionella pneumophila KV02.

Bacterial load as measured by mean fluorescence intensity (MFI: mean ± SEM Ind.^-1). Nematodes were incubated with L. pneumophila KV02 (mCherry, red) for 24 h and 96 h. Compared were the mouth cavity (stoma), the oesophagus (pharynx, terminal bulb, cardia), and the gut (intestine, rectum). Bars with no or the same letters are not statistically different according to Dunn’s post hoc test (p < 0.05).

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After 24 h incubation, L. pneumophila KV02 was present in 50% of D. pachys specimen (Fig 1B). The MFI was highest around cardia (60 a.u. Ind^-1) and terminal bulb (50 a.u. Ind^-1; Fig 2), and lowest in the intestine (28 a.u. Ind^-1). As for D. coronatus, no fluorescence was detected in the pharynx. After 96 h incubation, only 25% of D. pachys individuals harbored L. pneumophila KV02. The bacterial load was comparable in terminal bulb, cardia, intestine and rectum, with a mean MFI of 74 a.u. Ind^-1, but much lower in stoma and pharynx (Fig 2).

After 24 h incubation, P. similis comprised most bacteria in the intestine and terminal bulb with 45 and 33 a.u. Ind^-1; respectively (Fig 2). No fluorescence signal was detected around the cardia, while here the MFI was 51 a.u. Ind^-1 after 96 h. Generally, L. pneumophila was only found in 26% of individuals after 96 h incubation. The bacterial load was significantly higher in the intestine than in the pharynx (p < 0.049), whereas no bacteria were present in the stoma, terminal bulb and rectum.

After 24 h incubation, Plectus sp. harbored L. pneumophila in 40% of individuals (Figs 1D and 2). The bacterial load was highest in the intestine (62 a.u. Ind^-1). Otherwise, L. pneumophila KV02 was distributed evenly between stoma, cardia and rectum, with an MFI of about 37 a.u. Ind^-1. After 96 h incubation, L. pneumophila KV02 was only present in 9% of nematodes. The bacteria homogenously spread in the pharynx, cardia and intestine, with an MFI of around 31 a.u. Ind^-1. Comparable to P. similis, no fluorescence was detected in the stoma, terminal bulb and rectum.

For each of the four nematode species, the MFI of L. pneumophila KV02 in the different parts of the digestive system was independent of incubation time (p > 0.057). Moreover, the bacterial load of the investigated compartments did not differ between species, except for a significantly higher bacterial load in the rectum of D. coronatus compared to D. pachys after 96 h incubation (p < 0.002).

Mixed-species set-up.

To test whether interspecific competition of bacterial feeders affects the uptake of L. pneumophila KV02, the nematodes D. coronatus and P. similis were incubated in the same microcosms. After 24 h incubation, the bacterial load of the intestine was significantly higher in D. coronatus (116 a.u. Ind^-1) than in P. similis (18 a.u. Ind^-1; p < 0.015) (Fig 3). A similar species-specific bacterial ingestion rate was observed after 96 h, with 91 and 13 a.u. Ind^-1 for D. coronatus and P. similis, respectively (p < 0.021). After 24 h, the bacterial load of the intestine did not differ to the respective single-species set-ups. On the other hand, after 96 h in the mixed-species set-up, D. coronatus and P. similis harbored significantly more bacteria than in the respective single-species set-ups (D. coronatus = p < 0.021; P. similis = p < 0.012). Further, for D. coronatus, also the bacterial load of the pharynx with 22 a.u. Ind^-1 (p < 0.001) and cardia with 104 a.u. Ind^-1 (p < 0.044) was higher in the mixed compared to the single set-up.

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Fig 3. Presence of mCherry-labeled Legionella pneumophila KV02 in the digestive system of nematodes.

Diploscapter coronatus (plain) and Plectus similis (dots) were incubated together in the same biofilm microcosm for 24 h and 96 h. Number of investigated individuals (n), number of individuals that harbored L. pneumophila KV02 (KV02) and number of individuals without bacteria are shown for each nematode species. The bacterial load is measured as mean fluorescence intensity (MFI: mean ± SEM Ind.^-1). Statistical differences according to the Mann-Whitney U test with * and ** at p < 0.05 and p < 0.01, respectively.

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Regarding the other body compartments of nematodes, the overall fluorescence pattern in D. coronatus in mixed- and single-species set-up did not differ. The terminal bulb, cardia and intestine had the highest bacteria load after 24 h (87% of individuals, n = 16), as well as after 96 h incubation (80% of individuals, n = 10). Similarly, for P. similis, the overall fluorescence pattern in mixed- and single-species set-up did not differ. After 96 h incubation in the mixed-species set-up, bacteria were distributed between pharynx, intestine and rectum with a mean of 67% of nematodes observed (n = 12).

3.2 Bacterial choice assays—L. pneumophila versus E. coli

Single-species set-up.

To investigate potential food preference when given a choice between the human pathogen and a standard bacterial diet, nematodes were incubated with biofilm inoculated simultaneously with L. pneumophila KV02 and E. coli DH10β. After 24 h, P. similis was the only nematode species feeding on both bacteria (Fig 4). In 17% of the 36 specimens investigated high abundance occurred in the intestine with 35 and 44 a.u. Ind^-1 for E. coli DH10β and L. pneumophila KV02, respectively (Fig 5). Fluorescence signals were also detected in the stoma and pharynx with MFIs more than twice as high for E. coli DH10β. On the other hand, solely L. pneumophila KV02 was present around the cardia (2 a.u. Ind^-1) and in the rectum (33 a.u. Ind^-1) of P. similis, where E. coli DH10β was completely lacking.

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Fig 4. Legionella pneumophila KV02 and Escherichia coli DH10β in the digestive system of Plectus similis.

L. pneumophila KV02 (mCherry, red), E. coli DH10β (GFP, green) and P. similis were incubated together for 24 h in a biofilm model. (A) Stoma. (B) Pharynx. (C) Intestine. Confocal laser scanning microscopy images show the distribution of L. pneumophila KV02 (left) and E. coli DH10β (middle) separately and in merged images (right).

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Fig 5. Presence of mCherry-labeled Legionella pneumophila KV02 and GFP-labeled Escherichia coli DH10β in nematodes.

L. pneumophila KV02 (red), E. coli DH10β (gray) and nematodes were incubated together for 24 h in a biofilm model. Number of investigated individuals (n), number of individuals that harbored L. pneumophila KV02 (KV02) or E. coli DH10β (DH10β) and number of individuals without bacteria are shown for each nematode species. The bacterial load is measured as mean fluorescence intensity (MFI: mean ± SEM Ind^.-1). Bars with no or the same letters are not statistically different according to Dunn’s post hoc test (p < 0.05).

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The other three nematode species tested did only feed on L. pneumophila KV02, and no cells of E. coli DH10β were detected after 24 h. This was surprising, as general palatability and ingestion of E. coli DH10β were verified in a control set-up (S3 Fig). The species exhibited different fluorescence patterns (Fig 5): The overall MFI of L. pneumophila KV02 was significantly higher in D. coronatus compared to Plectus sp. and D. pachys (p < 0.001). Fluorescence was detected in more than 90% of the D. coronatus specimen, but only in 43 and 26% of the observed Plectus sp. and D. pachys, respectively. In detail, for D. coronatus the highest bacterial load occurred around the cardia (105 a.u. Ind^-1), followed by the intestine (78 a.u. Ind^-1), while less bacteria were detected in the stoma, pharynx and rectum (p < 0.001). In D. pachys, the bacterial load increased from 14 a.u. Ind^-1in the terminal bulb over 32 a.u. Ind^-1 in the intestine to 36 a.u. Ind^-1 in the rectum. No fluorescence was detected in the stoma and pharynx. In Plectus sp., the bacterial load was highest in the intestine (39 a.u. Ind^-1) and significantly lower in all other body compartments (p < 0.001).

After 96 h incubation none of the four nematode species contained E. coli DH10β (S4 Fig), while cells of L. pneumophila KV02 were still evident. The overall fluorescence pattern of the bacteria in the nematodes were not affected by time, with signals comparable to 24 h, i.e. again highest around the cardia (D. coronatus) or intestine (D. pachys, P. similis, Plectus sp.).

Mixed-species set-up.

Incubation of different nematode species in the same biofilm had variable effects on the ingestion and distribution of L. pneumophila KV02 and E. coli DH10β in their digestive system (Fig 6). Generally, the bacterial load of L. pneumophila KV02 was always higher in D. coronatus than P. similis (except in the rectum after 24 h incubation), which was significant for the cardia (p < 0.012) and intestine (p < 0.002) after 24 h incubation. Moreover, irrespective of the presence of another bacterial feeder, in D. coronatus the cardia and intestine showed the highest bacterial loads, and P. similis harbored most L. pneumophila KV02 in its intestine.

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Fig 6. Presence of mCherry-labeled Legionella pneumophila KV02 and GFP-labeled E. coli DH10β in nematodes.

L. pneumophila KV02 (red), E. coli DH10β (gray) and the nematode species Diploscapter coronatus (plain) and Plectus similis (dots) were incubated together in the same biofilm microcosm for 24 h and 96 h. Number of investigated individuals (n), number of individuals that harbored L. pneumophila KV02 (KV02) or E. coli DH10β (DH10β) and number of individuals without bacteria are shown for each nematode species. The bacterial load is measured as mean fluorescence intensity (MFI: mean ± SEM Ind.^-1). Statistical differences according to the Mann-Whitney U test with * and ** at p < 0.05 and p < 0.01, respectively.

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Comparable to the single-species set-ups, E. coli DH10β scarcely was detected in D. coronatus, with only one individual exhibiting a fluorescence signal in the pharynx after 24 h incubation (24 a.u. Ind^-1). In contrast, the bacterial location in P. similis varied. While in single species set-ups, nematodes harbored E. coli DH10β in stoma, pharynx and intestine, in the presence of D. coronatus bacteria were only detected in the intestine. Here, 19 and 42 a.u.

Ind^-1 were detected after 24 and 96 h, respectively. This corresponds to a reduction in fluorescence by 46% after 24 h compared to the single-species set-up, yet this effect was not significant.

4. Discussion

4.1 Nematode traits affecting pathogen load

The co-occurrence of the human pathogen L. pneumophila and nematodes in cooling tower biofilms suggests nematodes as potential reservoir for the human pathogen. This is supported by the observation, that each tested nematode species readily fed on L. pneumophila KV02 in a semi-natural biofilm model. However, the bacterial load varied with genus as well as with species, as indicated by the fluorescence intensity of the ingested bacterial cells. In general, the number of ingested particles is dependent on feeding type (i.e. size and morphology of the stoma) and feeding behavior of nematodes [49, 50]. Diploscapter, as an enrichment opportunist (c-p 1) ingests food at high pumping rates of the pharynx, while Plectus (c-p 2) pumps more irregularly with active sweeping motions during feeding [51]. In line with this, Diploscapter had a higher mean load of L. pneumophila KV02 cells than Plectus in the single set-ups of both, the no-choice (L. pneumophila-only) and choice (L. pneumophila vs. E. coli) assays. Additionally, the wider stoma of Diploscapter (3.1 ± 0.5 μm) compared to Plectus (2.1 ± 0.3 μm) likely also contributed to its higher bacterial ingestion rates.

Differences in pharyngeal muscle contraction patterns between Diploscapter and Plectus may have also determined the bacterial loads as pumping behavior strongly affects the efficiency of pharyngeal food transport [52]. Further, Plectus has a less complex pharynx than Diploscapter, lacking a muscular median bulb, probably resulting in less efficient bacterial ingestion. For example, compared to Poikilolaimus oxycercus with median and terminal bulb, Panagrolaimus lacking the median bulb needs two more pumping cycles to transport particles towards the terminal bulb [53]. A morphological peculiarity of Diploscapter is the existence of two cuticularized chambers posterior the grinder. This so-called double haustrulum allows for a “rumination” of particles, as they can re-enter the grinder from the intestine. This increases the grinding efficiency as not all bacteria are crushed by the grinder in the first passage [53]. Grinding efficiency may therefore be a further factor influencing the bacterial load in the intestine of the tested genera.

Apart from uptake, the bacterial load in the intestine is determined by its length and volume, the absorption rate of digested products, and the defecation rate [54]. Likely, differences in the intestinal volume of Diploscapter and Plectus determined the number of bacterial cells entering the intestine, while the microvilli frequency and density of necessary transporters affected bacterial digestion [54], resulting in the observed differences in fluorescence intensities. The attachment of L. pneumophila to intestinal cells may be facilitated by the surface-associated heat shock protein Hsp60 [33]. While speculative, it may be possible that taxon-specific variations in membrane receptors affect the binding of Hsp60 to the intestinal cells of Diploscapter and Plectus.

Finally, defecation rates vary with nematode species [55], resulting in different bacterial loads. Schiemer et al. [56] reported that the assimilation efficiency, i.e. the ratio between ingestion and egestion (defecation) rate, in Plectus palustris decreases with increasing food availability as food particles pass the gut too quickly for effective digestion.

Overall, single intact L. pneumophila KV02 cells could be detected past the grinder in all tested nematode species. Subsequent defecation disperses undigested bacterial cells into the environment. Thus, in addition to C. elegans, the two investigated nematode taxa fulfill a major prerequisite to act as a reservoir and transmission vehicle for L. pneumophila.

4.2 Distribution pattern of L. pneumophila in the digestive tract of nematodes

The distribution pattern of L. pneumophila KV02 varied in the digestive tract of biofilm-dwelling nematodes. A key process of nematode feeding is the quick transport of food particles through the oesophagus into the intestine, which is realized by a continuous pumping motion of the pharynx. With a duration of ca. 300 ms per contraction-relaxation cycle [27, 57], pumping is too fast to effectively accumulate bacteria in the pharyngeal lumen. Instead, food particles are trapped in the terminal bulb of the oesophagus, before being transported into the intestine via the cardia. A corresponding fluorescence pattern, i.e. high fluorescence intensities around the cardia and further on in the intestine, but low intensities in stoma and pharynx, was best observable in D. coronatus (Fig 2). In contrast, in D. pachys, P. similis and Plectus sp. bacteria were quite evenly distributed across body regions.

In the current study, the bacterial load of the gut did not decrease with observation time, rather bacteria accumulated around the cardia and along the intestine at least for 96 h. As bacterial residence time in the intestine is regarded as short with 2 to 10 minutes [58], this indicates successful pathogen persistence despite the hosts immune response. Especially in D. pachys the evenly distributed L. pneumophila KV02 cells after 96 h incubation (Fig 2) may point to a Legionella parasitism. Yet, also commensalism might be a possible interaction, as no typical symptoms of Legionella infection, i.e. extrusion of viscera through the vulva and intestinal and anal distension [32] were detectable after 96 h of incubation. The bacterial load neither decreased nor increased with time, thus it cannot be concluded whether nematodes just harbored L. pneumophila KV02 or if bacterial multiplication took place. Yet, the presence of L. pneumophila KV02 in the nematode rectum at both observation times suggests that a fraction of cells is constantly released into the environment after the passage through the gut. As nematodes defecate 30 to 60% of ingested bacteria in viable conditions [59], this makes them potential transport vehicles of Legionella in cooling towers.

Overall, bacterial cells were not transported along the intestine immediately after leaving the oesophagus. This lag time in the cardia region as well as the intestine may offer the potential as replicative niche for human pathogens.

4.3 Nematode food choice and competition

To account for differences in nematode feeding traits as well as for interspecific competition multi-species set-ups with D. coronatus and P. similis in the same biofilm were conducted. Like in the single set-ups, the L. pneumophila KV02 load was clearly higher in the intestine of D. coronatus than P. similis at both observation times. Interestingly, the L. pneumophila KV02 fluorescence intensity in the P. similis intestine was significantly higher in single species set-ups than in the multi-species set-ups. This was the case for both the no-choice assay (after 96 h) and choice assay (after 24 h). In addition to species-specific morphological and metabolic traits (see above) this could be due to exploitation competition, i.e. D. coronatus and P. similis interacted indirectly as they competed for food. D. coronatus, as superior competitor, ingested L. pneumophila KV02 more rapidly thereby decreasing the amount of available food for P. similis.

Interestingly, in D. coronatus, D. pachys and Plectus sp. only the presence of L. pneumophila KV02 but not the presence of E. coli DH10β could be detected in the digestive tract when bacteria were offered simultaneously (Fig 5 and S4 Fig), which points to a food preference for the pathogen. P. similis was the only species feeding on both, L. pneumophila KV02 and E. coli DH10β after co-inoculation for 24 h (Figs 4 and 5). Perhaps, this result is related to a greater food requirement of P. similis, since it was the largest of the four tested species. For example, the species P. palustris consumes 7.2 × 10⁶ bacterial cells daily [60], while the smaller Cephalobus persegnis has a consumption rate of 6.6 × 10⁵ cells per day [61]. Thus, it may be possible that when the density of the preferred diet L. pneumophila KV02 was declining, P. similis started feeding on the less preferred E. coli DH10β, too.

In contrast to the biofilm model, a previous study showed that, compared to E. coli OP50, L. pneumophila KV02 significantly impairs the pharyngeal pumping activity, i.e. pumping rates were reduced by 70% in P. similis and even by 300% in Plectus sp. on agar plate [36]. Apparently, the semi-natural test environment of a biofilm results in a distinctly different nematode food choice compared to agar plate assays. This fact emphases the need for experiments that reflect the habitat conditions in order to achieve realistic interactions between bacteria and nematodes.

Several reasons may explain the differences detected between agar plate and biofilm assays. Firstly, the co-ingestion of the natural biofilm microbiota could have altered the pathogenicity of L. pneumophila. Indeed, feeding co-cultures of beneficial and detrimental bacteria to C. elegans resulted in the active suppression of the pathogenicity of certain detrimental strains [62]. Moreover, Bacillus megaterium and Pseudomonas mendocina increased resistance to infection with P. aeruginosa in C. elegans, compared to individuals fed with E. coli OP50 [63]. Also, nematode exposure to pathogenic agents varies between agar plate and natural habitats. While the aquatic biofilm environment likely has attenuated the impact of effector proteins secreted by Legionella, these were not diluted on agar plate, thereby downregulating the grazing activity of nematodes. Secondly, nematodes can develop strategies to avoid potential pathogens, called aversive learning, i.e. nematodes modify their food preference after pathogen infection and cellular damage [64]. For instance, the initial preference of C. elegans for P. aeruginosa PA14 over E. coli OP50 was reversed after feeding on the pathogen, ultimately resulting in a preference for E. coli [65]. However, no diet switch from L. pneumophila to E. coli was observed over the entire experimental period, suggesting aversive learning as unlikely. Thirdly, a crucial process for bacterial biofilm formation is known as quorum sensing mediated by extracellular signal molecules (autoinducers—AIs) for cell-cell communication [66]. Several AIs are recognized by nematodes and stimulate chemotaxis and feeding, including the acylated homoserine lactone (aHSL) AIs of Gram-negative bacteria [67]. Thus, Legionella AIs functioning as chemoattractant for nematodes might explain the preference for L. pneumophila KV02 over E. coli DH10β in the biofilm assays.

Shaheen & Ashbolt [68] investigated the feeding preferences of free-living amoebae regarding L. pneumophila and two E. coli strains in amoeba-bacteria co-cultures. Using fluorescence microscopy, they observed a “forced-feeding” situation, where amoebae mostly fed on L. pneumophila, when the preferred E. coli sources were depleted [68]. However, no remarkable decrease of E. coli DH10β cells could be determined in the biofilm microcosms after incubation with nematodes for 24 h. Also, the existence of the natural biofilm microbial community as an alternative food source to L. pneumophila KV02 speaks against such a "forced-feeding" condition.

Another aspect is competition for colonization niches in the nematode gut between L. pneumophila KV02 and E. coli DH10β. The higher Legionella load may indicate that the pathogen has a competitive advantage against non-pathogenic strains, i.e. E. coli DH10β has difficulties surviving in an environment in which an active host response, triggered by Legionella, is present. This immune system response includes pathways involving e.g. the DAF-2 (insulin/IGF-I like) receptor, p38 MAP kinase and the transforming growth factor β (TGF-β) [43]. For instance, C. elegans p38 MAP kinase mutants were more, while daf-2 insulin signaling pathways mutants were less susceptible to Legionella infection [32]. Indeed, Portal-Celhay & Blaser [69] reported that in C. elegans grown on mixed lawns of bacteria, S. enterica is able to establish persistent colonization in the gut, while completely outcompeting E. coli OP50. Yet, S. enterica also outcompeted E. coli OP50 in daf-2 mutants, which indicates that gut colonization resilience is more bacterial strain specific and less dependent on innate immunity [69].

In sum, all tested nematode species exhibited a high load of L. pneumophila KV02 but a low presence of E. coli DH10β in their gut (except P. similis), when feeding in the semi-natural biofilm environment. This indicates a preference for the pathogen over the non-fluorescent autochthonous microflora and GFP-labeled E. coli DH10β. Competition between the bacterial strains within the nematode gut may have reduced the presence of E. coli DH10β additionally. The underlying mechanisms of Legionella-nematode interactions have to be further explored in test systems reflecting natural habitat conditions.

5. Conclusion

This study using a semi-natural biofilm model provides first evidence that bacterial-feeding nematodes co-occurring with L. pneumophila in cooling towers preferentially ingest the human pathogen over the standard laboratory diet E. coli. The comparison between bacterial load patterns in Diploscapter and Plectus revealed species-specific regions for bacterial accumulation, which may have implications for their potential as a replicative niche for L. pneumophila. A high load of L. pneumophila compared to common bacteria such as E. coli may be a sign of food preference, yet bacterial competition within the nematode gut cannot be excluded. The presence of the pathogen after 96 h underlines the potential of biofilm-dwelling nematodes to serve as reservoir and transmission vehicle of L. pneumophila in cooling towers. Future environmental studies on nematode-Legionella interactions should consider a wider time span to reveal possible long-term effects of Legionella pathogenicity on nematode grazing. Additionally, investigation of different experimental temperatures is desirable to mimic alterations in the thermal regime of cooling towers.

Supporting information

S1 Fig. Illustration of the experimental set-up.

(A) Subsample of the cooling tower biofilm material. (B) Illustration of a chamber slide. 250 μl of nematode-free biofilm within separate chambers as experimental microcosms. Depending on the assay, either 50 μl of mCherry-labeled L. pneumophila KV02 (2 x 10⁸ CFU ml^-1), or 50 μl of GFP-labeled E. coli DH10β together with 50 μl of mCherry-labeled L. pneumophila KV02 (6 x 10⁸ CFU ml^-1), resuspended in sterile-filtered cooling tower water, were inoculated and left overnight at 25°C to adapt. (C) Nematode inoculation. Hand-picked nematodes (30 individuals per microcosm) were inoculated. Two scenarios were investigated: single set-ups each comprised one of the four test species (D. coronatus, D. pachys, P. similis, Plectus sp.), while mixed set-ups were a combination of P. similis and D. coronatus with each 15 individuals, respectively, per microcosm (n = 2). All microcosms were incubated at 25°C for either 24 h or 96 h.

https://doi.org/10.1371/journal.pone.0309820.s001

(TIF)

S2 Fig. Choice index (CI) after Abada et al. [44].

The GFP-labeled strain E. coli DH10β was tested against the isogenic strain DH10β not expressing GFP, as well as against E. coli OP50. A CI of -1.0 represents a total preference for the GFP-expressing strain, a CI of 1.0 represents a total preference for the isogenic strains DH10β or OP50, while a CI of 0 represents an equal distribution of nematode individuals between both bacterial diets. Food choice assays were performed on 9 cm NGM agar plates at 20°C. For this, 30 μl of freshly prepared bacterial suspensions adjusted to 10⁶ CFU ml^-1 were seeded at opposite sides of the assay plate at a distance of 1.5 cm from the plates’ edge. Then, 100 young adult nematodes suspended in sterile mineral water (Volvic, Danone Deutschland GmbH, Germany) were placed at the center of each plate. Each trial was replicated 5 times with 3 repeats per replicate. Bars with no or the same letters are not statistically different according to the Tukey HSD test (p < 0.05).

https://doi.org/10.1371/journal.pone.0309820.s002

(TIF)

S3 Fig. Representative confocal laser scanning microscopy images of nematodes harboring E. coli DH10β.

Nematodes harbor E. coli DH10β (GFP, green) in the pharynx (Diploscapter coronatus, Plectus sp.), terminal bulb (Plectus similis), and rectum (Diploscapter pachys). Bacteria and nematodes were incubated in sterile-filtered cooling tower water for 24 h at 25°C.

https://doi.org/10.1371/journal.pone.0309820.s003

(TIF)

S4 Fig. Presence of mCherry-labeled Legionella pneumophila KV02 and GFP-labeled Escherichia coli DH10β in nematodes.

L. pneumophila KV02 (red), E. coli DH10β (gray) and nematodes were incubated together for 96 h in a biofilm model. Number of investigated individuals (n), number of individuals that harbored L. pneumophila KV02 (KV02) and number of individuals without bacteria are shown for each nematode species. The bacterial load is measured as mean fluorescence intensity (MFI: mean ± SEM Ind^.-1). Bars with no or the same letters are not statistically different according to Dunn’s post hoc test (p < 0.05).

https://doi.org/10.1371/journal.pone.0309820.s004

(TIF)

S1 Dataset. Choice index of nematodes.

https://doi.org/10.1371/journal.pone.0309820.s005

(XLSX)

S2 Dataset. Mean fluorescence intensity (MFI) of Legionella pneumophila (no-choice assay).

https://doi.org/10.1371/journal.pone.0309820.s006

(XLSX)

S3 Dataset. Mean fluorescence intensity (MFI) of Legionella pneumophila and Escherichia coli DH10β (choice assay).

https://doi.org/10.1371/journal.pone.0309820.s007

(XLSX)

Acknowledgments

We gratefully acknowledge the help of Dr. Lothar Täuscher, who identified the algal composition of the biofilm material.

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Figures

Abstract

1. Introduction

2. Materials and methods

2.1 Cultivation of bacterial strains

2.2 Cultivation of nematodes

2.3 Biofilm assay

2.4 Fluorescence microscopy and data analysis

3. Results

3.1 No-choice assays—L. pneumophila-only

Single-species set-up.

Mixed-species set-up.

3.2 Bacterial choice assays—L. pneumophila versus E. coli

Single-species set-up.

Mixed-species set-up.

4. Discussion

4.1 Nematode traits affecting pathogen load

4.2 Distribution pattern of L. pneumophila in the digestive tract of nematodes

4.3 Nematode food choice and competition

5. Conclusion

Supporting information

S1 Fig. Illustration of the experimental set-up.

S2 Fig. Choice index (CI) after Abada et al. [44].

S3 Fig. Representative confocal laser scanning microscopy images of nematodes harboring E. coli DH10β.

S4 Fig. Presence of mCherry-labeled Legionella pneumophila KV02 and GFP-labeled Escherichia coli DH10β in nematodes.

S1 Dataset. Choice index of nematodes.

S2 Dataset. Mean fluorescence intensity (MFI) of Legionella pneumophila (no-choice assay).

S3 Dataset. Mean fluorescence intensity (MFI) of Legionella pneumophila and Escherichia coli DH10β (choice assay).

Acknowledgments

References