Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants

International Journal of Hygiene and Environmental Health 213 (2010) 158–166

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International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.de/ijheh

Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants Daniele Corsaro a , Gemma Saucedo Pages b , Vicente Catalan c , Jean-Franc¸ois Loret d , Gilbert Greub a,∗ a

Center for Research on Intracellular Bacteria, Institute of Microbiology, Faculty of Biology and Medecine, University of Lausanne, Bugnon 46, 1011 Lausanne, Switzerland Aigues de Barcelona, General Batet 1-7, 08028 Barcelona, Spain Labaqua, Dracma 16-18, Pol. Ind. Las Atalayas, 03114 Alicante, Spain d SUEZ Environment, CIRSEE, 38 rue du Président Wilson, 78230 Le Pecq, France b c

a r t i c l e

i n f o

Article history: Received 5 August 2009 Received in revised form 18 February 2010 Accepted 19 March 2010 Keywords: Amoebae Chlamydiae Legionellae Mycobacteria Water treatment plant

a b s t r a c t In this study, we enlarged our previous investigation focusing on the biodiversity of chlamydiae and amoebae in a drinking water treatment plant, by the inclusion of two additional plants and by searching also for the presence of legionellae and mycobacteria. Autochthonous amoebae were recovered onto non-nutritive agar, identified by 18S rRNA gene sequencing, and screened for the presence of bacterial endosymbionts. Bacteria were also searched for by Acanthamoeba co-culture. From a total of 125 samples, we recovered 38 amoebae, among which six harboured endosymbionts (three chlamydiae and three legionellae). In addition, we recovered by amoebal co-culture 11 chlamydiae, 36 legionellae (no L. pneumophila), and 24 mycobacteria (all rapid-growers). Two plants presented a similar percentage of samples positive for chlamydiae (11%), mycobacteria (20%) and amoebae (27%), whereas in the third plant the number of recovered bacteria was almost twice higher. Each plant exhibited a relatively high specific microbiota. Amoebae were mainly represented by various Naegleria species, Acanthamoeba species and Hartmannella vermiformis. Parachlamydiaceae were the most abundant chlamydiae (8 strains in total), and in this study we recovered a new genus-level strain, along with new chlamydiae previously reported. Similarly, about 66% of the recovered legionellae and 47% of the isolated mycobacteria could represent new species. Our work highlighted a high species diversity among legionellae and mycobacteria, dominated by putative new species, and it confirmed the presence of chlamydiae in these artificial water systems. © 2010 Elsevier GmbH. All rights reserved.

Introduction Within the water distribution systems, a peculiar microbial ecology is to be defined, taking into account the interplay of different physical, chemical and biotic variables, like temperature, type of pipe surface, nutrient levels, presence of disinfectant chemicals, biofilms, predatory protists and their endosymbionts (Berry et al., 2006). Among the protists, free-living amoebae have gained growing interest, as it has been shown that they are widespread in aquatic habitats, and successfully colonize many man-made water systems, like cooling towers, humidifiers, hospital water networks, or drinking water production plants (Corsaro et al., 2009; Hoffmann and Michel, 2001; Thomas et al., 2006b, 2008). Amoebal species

∗ Corresponding author at: Center for Research on Intracellular Bacteria (CRIB), Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011 Lausanne, Switzerland. Tel.: +41 21 314 49 79; fax: +41 21 314 40 60. E-mail address: [email protected] (G. Greub). 1438-4639/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2010.03.002

developing cysts are generally resistant to treatment processes (Gerba et al., 2003; Hijnen et al., 2006; Loret et al., 2008a). Some species and strains of amoebae are opportunistic parasites (amphizoic amoebae) of vertebrates, including humans, in which they cause mainly keratitis and meningo-encephalitis (Visvesvara et al., 2007). In addition, several microorganisms, including established pathogens, are able to infect and survive within different amoebae, and thus may by-pass disinfection treatment (Greub and Raoult, 2004; Loret et al., 2008b). Due to this peculiar lifestyle, laboratory strains of amoebae, mainly Acanthamoeba sp., have been used as host cells to isolate amoeba-resisting bacteria from either environmental (Collingro et al., 2005a; Pagnier et al., 2008; Thomas et al., 2006b, 2008) and clinical (Rowbotham, 1998; Greub et al., 2004) samples. Legionellae and mycobacteria are well known inhabitants of aquatic biofilms. Various species within both groups are recognized pathogens (Legionella pneumophila, Mycobacterium avium complex) or highly suspected pathogens (Fields et al., 2002; Greub and Raoult, 2004; Primm et al., 2004), and the elimination of pathogenic microorganisms should be performed prior to the entry in the final

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distribution systems (Council Directive 98/83/EC). Our interest for the chlamydiae, comes from the evidences of their huge and unexplored diversity in the environment (Corsaro et al., 2003, 2009), and from the possibility that novel chlamydial pathogens may occur in the environment (Corsaro and Venditti, 2004; Corsaro and Greub, 2006). In this study we used Acanthamoeba co-culture to isolate three main groups of amoeba-associated bacteria, i.e. Chlamydiales, Legionella spp., and Mycobacterium spp., from three water treatment plants producing drinking water in Spain. This work extended our recent investigation focusing on the biodiversity of chlamydiae and amoebae in one of these three plants (plant C) (Corsaro et al., 2009).

Recovery of bacteria by amoeba co-culture

Materials and methods

Recovery of autochthonous amoebae and screening for bacterial endosymbionts

Chlamydiae, legionellae and mycobacteria potentially present in the samples were recovered by amoeba co-culture in 24-well microplates (Costar, Corning, NY), using Acanthamoeba sp. (strain ATCC 30010), as described previously (Corsaro et al., 2009). Briefly, 100 ␮l of each sample were inoculated in serial dilution onto Acanthamoeba monolayers, centrifuged (1500 × g for 30 min), and incubated at 32 ◦ C in a humidified atmosphere in the dark. At 6 days post-inoculation, subcultures were performed on fresh Acanthamoeba (second co-culture). Along the first and the second co-culture, amoebae were screened for by using specific PCR (see below), and bacterial strains were identified by sequencing a portion of the 16S rRNA gene (see below).

Samples Autochthonous amoebae from each sample were isolated onto bacterized non-nutritive agar (NNA) at 32 ◦ C in the dark, as described previously (Page, 1967; Rowbotham, 1980; Corsaro et al., 2009). Distinct morphotypes were subcultured onto NNA to obtain clonal amoebae, which were identified by sequencing portion of the 18S rRNA gene (see below). Naturally harboured bacteria were searched for by applying the specific 16S rRNA gene PCR.

A total of 125 samples was collected from August, 2006 to July, 2007, from three treatment plants (A, B and C) producing drinking water, located in Spain. Samples originated from different points upstream and downstream the major steps of the industrial processes (Table 1). Samples of 1 litre were filtered through a 0.2-␮m polycarbonate membrane, and the filter was resuspended in 50 ml of sterile distilled water in Falcon tubes, and sent to the laboratory at room temperature. Prior to any inoculation assay onto agar plates or amoeba co-culture, tubes were vortexed for 15–30 s in order to resuspend the microorganisms present on the filters. Temperature, turbidity (Nephelometric Formazine Units, NFU) and quantitative microbiological analyses per litre for total amoebae (most probable number, MPN), and aerobic bacteria (colony-forming unit, CFU) were performed, as described previously (Corsaro et al., 2009).

DNA extraction, gene amplifications and sequencing Total DNA was extracted from infected Acanthamoeba cocultures, and from clonal amoebae recovered directly from the NNA, with the AquaPure Genomic DNA extraction kit (Bio-Rad). PCR were performed in 50 ␮l reaction tubes containing specific primer sets. To detect chlamydiae, the almost complete 16S rRNA gene was amplified with the primers 16SIGF (5 -CGGCGTGGATGAGGCAT-

Table 1 Types of samples and summary of results. Samples

n

Samples positive fora Chlamydiae

Plant A

Plant B

Plant C

a b c d

Raw surface water Settled water Sand-filtered water Finished water Sludge from clarifier Biofilm from filter Total (%)

2 4 8 2 2 3 21

Raw surface water Settled water Sand-filtered water Finished water Sludge from clarifier Biofilm from clarifier Total (%)

2 5 9 2 5 2 25

Raw groundwater Raw surface water Sand-filtered water Ozonated water GAC-filtered water Finished water Biofilm from distribution system Sediment from distribution system Water from dead leg Water from distribution system Total (%)

8 8 8 1 8 8 11 12 5 10 79

Legionellae 1

1

Mycobacteria 1

1

1 1 1

2 1 (4.7)

Amoebae

4 (19.0)

1 2 (9.5)

1b

3 (14.2) 2

1 3 1 1 3 (12) 4

1 2 2c

9 (11.4)

1

1

1 (4)

5 (20)

4 1 7 (28)

4 1

7 2

3 5d 5 1 6 d

2 5 3 1 33 (41.7)

Bacteria were recovered by Acanthamoeba co-culture, autochthonous amoebae onto bacterized NNA. A Protochlamydia strain was recovered within its natural host, an Acanthamoeba T4. A Neochlamydia strain was recovered within its natural host, an Acanthamoeba T4. Two Legionella were recovered within their natural hosts, a Naegleria australiensis (raw water), and an Acanthamoeba T4 (biofilm).

3 6 1 2 17 (21.5)

1 1 2 7 2 22 (27.8)

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3 ) and rP2chlam (5 -CTACCTTGTTACGACTTCAT-3 ) (Thomas et al., 2006a), under reaction conditions of 5 min at 94 ◦ C, followed by 40 cycles at 94 ◦ C for 30 s, 54 ◦ C for 30 s, 72 ◦ C for 1 min 30 s, and final extension for 10 min at 72 ◦ C. To detect legionellae, a 660-bp portion of the 16S rRNA gene was amplified using the primers Leg225 (5 -AAGATTAGCCTGCGTCCGAT-3 ) and Leg858 (5 -GTCAACTTATCGCGTTTGCT-3 ) (Miyamoto et al., 1997). Reaction conditions were: 5 min at 94 ◦ C, followed by 40 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 1 min, and a final extension for 10 min at 72 ◦ C. To detect mycobacteria, a 1020-bp portion of the 16S rRNA gene was amplified using the primers 285b (5 -GAGAGTTTGATCCTGGCTCAG-3 ) and 264 (5 -TGCACACAGGCCACAAGGGA-3 ) (Kirschner et al., 1993). Reaction conditions were: 5 min at 94 ◦ C, followed by 40 cycles at 94 ◦ C for 30 s, 64 ◦ C for 30 s, 72 ◦ C for 1 min 30 s, and a final extension for 10 min at 72 ◦ C. Autochthonous amoebae were identified by amplifying and sequencing a portion of the 18S rRNA gene (ranging from 600 to 1000 bp), with the primers Ami6F1 (5 -CCAGCTCCAATAGCGTATATT-3 ), Ami6F2 (5 -CCAGCTCCAAGAGCGTATATT-3 ) and Ami9R (5 -GTTGAGTCGAATTAAGCCGC-3 ) specific for the eukaryotic 18S rRNA gene (Thomas et al., 2006b). Reaction conditions were: 5 min at 94 ◦ C, followed by 40 cycles at 94 ◦ C for 1 min, 55 ◦ c for 30 s, 72 ◦ C for 2 min, and a final extension for 10 min at 72 ◦ C. PCR products from at least three independent reactions were purified using the QIAquick PCR purification kit (Qiagen, France). Sequencing was performed using a series of inner primers for the chlamydial 16S rDNA, and the same PCR primers for the legionella and mycobacterial 16S and the amoebal 18S rDNA, in an automated sequencer (ABI 3130XL, Applied Biosystem) by the BigDye Terminator Cycle kit. Sequence data were edited with Chromas Lite (Technelysium, Australia) and contigs were constructed with the Vector NTI software (Informax, Invitrogen). Genetic and phylogenetic analyses The small subunit rDNA sequences obtained were compared with sequences available in GenBank via the BLAST server (www.ncbi.nlm.nih.gov), aligned with selected sequences retrieved from GenBank, and the alignments were adjusted by removing the 5 and 3 ends of the longer sequences. Phylogenetic analyses were performed using MEGA3 (Kumar et al., 2004) by applying distance matrix [neighbour-joining (NJ) and minimum evolution (ME)] and maximum parsimony (MP) approaches, with 1000 (NJ and ME) and 100 (MP) bootstrap re-sampling. Biodiversity analysis Operational taxonomic units (OTU) were defined on the basis of sequence similarity values >97% for Legionella sp., or >98% for Mycobacterium sp., and on the congruence with phylogenetic analysis. The ˛-diversity was estimated by measuring the following indices: Shannon [H = −pi log(pi)], Eq (Eq = H /H max ), Simpson [D = 1 − (pi2 )], and Margalef [M = (S − 1)/ln N]. The Shannon index H varies from 0 (communities with only a single species) to high values (communities with n species; the higher is H , the lower is the number of individuals in each species). The Simpson index D varies from 0 (only few species predominate in the sample) to 1 (presence of many different species). The evenness given by Eq varies from 0 (predominance of one species) to 1 (many species occur in the sample at similar levels). The Margalef index M indicates the species richness. The ˇ-diversity was estimated by measuring the following indices: Sorensen [ˇS = 2c/S1 + S2 ], Jaccard [ˇJ = c/r], and Mountford [ˇM = 2c/2(S1 S2 ) − (S1 + S2 )c], where S1 and S2 represent the total number of species in community 1 and 2, respectively, c repre-

sent the number of species common to both communities, and r represent the number of species found only in community 1 or 2. Values vary from 0 (no species overlap between communities) to 1 (complete species overlap). Results The type of water samples analysed from the three water treatment plants (n = 125), and the number of positive samples for the three bacterial taxa and amoebae, are summarized in Table 1. The graphic representations of the ˛- and ˇ-diversities are presented in Fig. 1. Chlamydial and amoebal diversity from plant C was discussed in a previous study (Corsaro et al., 2009). Physical data Temperature values for plant A and B were constant, ranging from 18.5 to 21.5 ◦ C and from 24.0 to 25.2 ◦ C, respectively, while more important changes were observed for plant C, from 9.5 to 28.5 ◦ C (Corsaro et al., 2009). Turbidity in plants A and B showed values <7 and <50 NFU, respectively, in raw surface waters, then decreased below 0.4 in treated waters (0.11–0.39). By contrast, values in plant C were higher for raw surface waters (24–230 NFU), and they varied from 0.3 to 0.8 NFU in treated waters. Quantitative microbiological data Plants A and B resulted poor in total microbiota, as compared to plant C. Values for raw surface waters were as follows: amoebae 93–1100 MPN l−1 (plant A), 15–43 MPN l−1 (plant B), 210–4600 MPN l−1 (plant C); aerobic bacteria 6.3 × 106 to 11.5 × 106 CFU l−1 (plant A), 500 to 28 × 104 CFU l−1 (plant B), 11 × 106 to 284 × 106 CFU l−1 (plant C). The filtration steps reduced greatly the microbial charge: mean values were of 0.6–1.7 MPN l−1 for amoebae, and between 4400 and up to 70 × 106 CFU l−1 for bacteria. As expected, the highest numbers of amoebae were found in sludges and sediments. In these samples, amoebae were regularly >1100 MPN l−1 , and reached values up to 12 × 106 MPN l−1 . Bacteria were more abundant in sludges (values range from 21 × 107 to 310 × 107 CFU l−1 ) than in biofilms (values range: 28 × 104 to 22 × 106 CFU l−1 ). With the exception of a single sample from a dead leg, the microbial charge in the distribution system was below 3 MPN l−1 for amoebae and 22 000 CFU l−1 for aerobic bacteria. Autochthonous amoebae Three samples out of 21 (14.2%), 7 out of 25 (28%), and 22 samples out of 79 (27.8%), were positive for amoebae onto non-nutritive agar (NNA), from the plants A, B and C, respectively. Amoebae of plant C have previously been analysed (Corsaro et al., 2009). They showed high biodiversity (H = 1.81, D = 0.9), with six genera (Acanthamoeba, Echinamoeba, Hartmannella, Stenamoeba, Vannella, and Naegleria) and 14 amoebal species recognized from a total of 28 isolates (M = 3.9). Dominant amoebae were Naegleria (12 isolates), with N. clarki, N. australiensis and two unnamed species. Five putative species of Acanthamoeba genotype T4 (7 isolates) were also recovered from plant C. From plant A, two isolates identified as Hartmannella vermiformis were recovered from raw surface water and from finished water, respectively, whereas one Naegleria fultoni isolate was recovered from the sludge of a clarifier. Among the seven amoebae isolated from plant B, we identified three different species: N. australiensis (three isolates, one from raw surface water, one from the sludge and one from the biofilm of a clarifier), Acanthamoeba sp. genotype T4, from raw surface water, and H. vermiformis, from the sludge of a clarifier. Two amoebae, also recovered from the

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Fig. 1. ˛-Biodiversity (A) and ˇ-biodiversity (B) analysis. Data for the autochthonous amoebae, recovered onto NNA, and the three bacterial taxa, all identified by small subunit ribosomal gene sequencing, are analysed by means of commonly used diversity indices, and presented graphically.

same sludge, remained uncharacterized due to failure of 18S rDNA amplification and sequencing. Biodiversity was lower for plant A (H = 0.27, D = 0.47, M = 0.91) with respect to plant B (H = 0.41, D = 0.56, M = 1.24), and both plants showed low amoebal biodiversity with respect to plant C (Fig. 1A). H. vermiformis was the only species isolated from all three plants, and ˇ values were very low (0.013–0.105). N. australiensis was found in plants B and C, while the various Acanthamoeba strains, also found in plants B and C, belonged to distinct lineages within the genotype T4, considered as separate OTU. Plants A and B showed a higher proportion of species in common (ˇ ranged from 0.28 to 0.4). In contrast, plants A and C presented nearly plant-specific amoebal population (ˇ ranged from 0.05 to 0.125). Plants B and C exhibited intermediate ˇ values that ranged from 0.08 to 0.235 (Fig. 2B). All the amoebal strains (n = 38) were screened for the presence of endosymbionts by specific PCR, and six (15.7%) resulted positive. Three were chlamydiae, detected within three distinct Acanthamoeba T4 strains, and isolated each from raw surface water (plant B), biofilm and sediment samples from the distribution system (plant C, strains CRIB53 and CRIB59). The remaining three were legionellae, detected within N. australiensis (strain CRIB44) from raw surface water, and two distinct Acanthamoeba T4 strains, from raw surface water (strain CRIB45), and from the biofilm of a dis-

tribution system (strain CRIB54), all originating from the plant C. Mycobacteria were not detected within recovered amoebae. Chlamydiae Chlamydiae of plant C were described in a previous study (Corsaro et al., 2009). Parachlamydiaceae (four isolates) were the dominant clade, but we also recovered members of Criblamydiaceae, of the Rhabdochlamydia cluster and of a novel lineage, indicative for a relative high biodiversity (H = 0.82, D = 0.84, M = 2.73). From plant A, we recovered one strain by amoebal co-culture from sand-filtered water (strain CRIB39). From plant B, two strains were recovered by amoebal co-culture respectively from the biofilm and from the sludge of a clarifier (strains CRIB40 and CRIB41). An additional strain was a natural Acanthamoeba endosymbiont (strain CRIB44). All the strains from plants A and B belonged to the Protochlamydia clade (family Parachlamydiaceae) (Fig. 2). The strains CRIB40 and CRIB44 emerged very robustly as the sister-group of Protochlamydia amoebophila under various treeing methods, and showed 98 and 98.1% 16S rDNA sequence similarity with the type strain UWE25. The strain CRIB41 belonged clearly to Protochlamydia naegleriophila (99.5% sequence similarity with the reference strain KNic). The strain CRIB39 showed sequence simi-

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Fig. 2. Neighbour-joining tree based on a 1200-bp portion of the 16S rRNA gene of the Chlamydiae, with particular emphasis on the parachlamydial strains recovered in this study (in bold, GenBank accession numbers given in parenthesis). Origin from the three plants is marked by different symbols. Numbers at nodes indicates percentage bootstrap values for distance and maximum parsimony methods (1000 and 100 replications, respectively). Verrucomicrobium spinosum (Verrucomicrobia) is used as outgroup.

larity values <95% with both P. amoebophila and P. naegleriophila, and thus could represent a new species. The ˛-diversity values could be estimated only for the plant B (H = 0.27, D = 0.44, M = 0.05) (Fig. 1A). P. naegleriophila was found in both plants B and C (ˇ values ranged from 0.105 to 0.222) (Fig. 1B). Legionellae Four samples out 21 (19%) from plant A, two out 25 (8%) from plant B, and 31 out 79 (39.2%) from plant C, resulted positive for legionellae in amoebal co-culture. Two additional legionellae were detected within two autochthonous amoebae isolated from raw surface water (Naegleria australiensis strain CRIB44) upstream of plant C and from the biofilm of a distribution system (Acanthamoeba T4 strain CRIB54) downstream of plant C. One isolate from plant B and 8 from plant C, including both intra-amoebal bacteria remained undetermined, as we failed to subculture them on BCYE agar and we did not obtain definite 16S rDNA sequences. These samples were excluded from the subsequent analyses. The four isolates from plant A were clustered into three OTU (OTU-7, -15, -21); the unique isolate of plant B formed the OTU-1; and the 25 isolates from the plants C were clustered into 20 OTU (OTU-2 to OTU-20, and OTU-22) (Fig. 3). Overall, legionellae were recovered mainly from GAC-filtered waters (20%, 6/30 isolates, all from plant C), raw surface and sand-filtered waters (16.6%, 5 iso-

lates from each type of sample) and sediments (13.3%, 4 isolates). Legionellae were less frequently recovered in plant A as compared to plant C (H = 0.45 vs 1.23, D = 0.62 vs 0.93, M = 1.4 vs 5.6) (Fig. 1A). Only two OTU (OTU-7 and OTU-15) were found in two plants, A and C (ˇ values ranged from 0.057 to 0.181) (Fig. 1B), while the unique strain from plant B showed no close relationship with any other strains originating from the other plants. Five OTU out 20 (25%) from plant C could be assigned to known species: L. lytica (OTU-2), L. shakespearei (OTU-6), L. dumoffii (OTU-12), L. nautarum (OTU-17), and L. drozanskii (OTU-18), while all the OTU from the plants A and B seemed to represent new species. L. pneumophila was not recovered. The only isolate showing significant sequence similarity with L. pneumophila (97.5%), leg-S025, from plant A, emerged as an independent lineage with two other isolates. They formed a relatively robust clade with both distance and parsimony methods, and were considered as unique OTU (OTU-8 to OTU-10). Mycobacteria Two samples out 21 (9.5%) from plant A, five out 25 (20%) from plant B, and 17 out 79 (21.5%) from plant C, resulted positive for mycobacteria in amoeba co-culture. We failed to obtain definite 16S rDNA sequences for three isolates from plant C, which were excluded from the successive analysis. Autochthonous amoebae were found free of mycobacteria.

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Fig. 3. Neighbour-joining tree based on a 650-bp portion of the 16S rRNA gene of the legionellae. Strains recovered in this study are in bold, GenBank accession numbers in parenthesis. Symbols indicated the plant of origin. At the right, strains were clustered into putative 22 OTU, and assigned to known species or labelled as Legionella sp. Numbers at nodes indicates percentage bootstrap values for distance and maximum parsimony methods (1000 and 100 replications, respectively). Piscirickettsia salmonis and Coxiella burnetii were used as outgroups.

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Fig. 4. Neighbour-joining tree based on a 1050-bp portion of the 16S rRNA gene of the mycobacteria. Strains recovered in this study are in bold, GenBank accession numbers in parenthesis. Symbols indicated the plant of origin. At the right, strains were clustered into 12 putative OTU, and assigned to known species or labelled as Mycobacterium sp. Numbers at nodes indicates percentage bootstrap values for distance and maximum parsimony methods (1000 and 100 replications, respectively). Since all recovered strains were rapid-growers, Mycobacterium tuberculosis was used as outgroup.

The two strains from plant A were assigned to OTU-3 and OTU-6; the five strains from plant B were clustered into four OTU (OTU-6 to OTU-8 and OTU-11); and the 14 strains from plant C were clustered into ten OTU (OTU-1 to OTU-7, OTU-9, OTU-10 and OTU-12) (Fig. 4). All isolates belonged to the rapid-grower group of mycobacteria. On the whole, mycobacteria were recovered mainly from raw surface waters (23.8%, 5/21 isolates), sand-filtered waters and biofilms (19%, 4 isolates for each type of sample), and sediments (14.2%, 3 isolates). Biodiversity for mycobacteria was the highest in plant C (H = 0.95, D = 0.88, M = 3.79), and the lowest in plant A (H = 0.301, D = 0.5, M = 0.3), plant B showing intermediate values (H = 0.57, D = 0.72, M = 1.4) (Fig. 1A). Different strains of a single OTU (OTU6) were present in all three plants (ˇ ranged from 0.012 to 0.117). Strains assigned to two additional OTUs were present in plants A and C (OTU-3) or plants B and C (OTU-7). Plants A and B, as well as plants A and C, showed a higher proportion of mycobacterial taxa in common (ˇ ranged from 0.2 to 0.33 for both pairs of plants), compared to plants B and C (ˇ ranged from 0.068 to 0.26) (Fig. 1B). Four OTU could be assigned to known species: M. chlorophenolicum (OTU-1), M. frederiksbergense (OTU-6), M. rhodeasiae (OTU-7), and M. neglectum (OTU-11) (Fig. 4). The other isolates exhibited relatively low (<97%) 16S rRNA sequence similarity values. One of these isolates, myc-S088 (OTU-2), showed a sequence similarity value of only 95% with its closest relative, M. mageritense.

Overall biodiversity index Plant C was the richest for all bacterial and amoeba taxa, with a Shannon index of 1.63 and a Simpson index (1 − D) of 0.97. Species richness was estimated at 11.6. These values were higher as compared to values of plants A and B, which presented Shannon values of 0.87 and 0.99, Simpson values of 0.86 each, and species richness of only 3.04 and 3.4, respectively (Fig. 1A). The evenness was always >0.85, indicating an almost equal repartition of strains among the taxa.

Discussion In this study, we analysed samples originating from three drinking water treatment plants for the presence of autochthonous amoebae, and of three groups of amoeba-associated bacteria, namely chlamydiae, legionellae and mycobacteria. Bacteria were recovered by Acanthamoeba co-culture directly from the samples. Bacteria were also searched for in recovered autochthonous amoebae. As shown in Table 1, plant C was more heavily colonized with legionellae than plants A or B. Plants B and C presented a similar percentage of samples positive for chlamydiae, mycobacteria and amoebae. This rate was twice lower in plant A.

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Biodiversity was analysed for each plant (˛-diversity) and between plants (ˇ-diversity) (Fig. 1). The latter analysis indicated that each plant exhibited a relative specific microbiota, mainly composed of rare new species. The plants A and B exhibited a more common microbiota (Sorensen and Jaccard indices of 0.23 and 0.2), but the Mountford index, less dependent of sampling bias, was of only 0.036 (Fig. 1B). Samples positive for amoebae were mainly raw surface waters, biofilms and sediments from reservoirs or clarifiers, and sludge from clarifiers (Corsaro et al., 2009, this study). Overall, Naegleria spp. (Heterolobosea) were the most common amoebae, with 16 (44.4%) of a total of 36 strains. Acanthamoeba was the second most abundant genus, with nine strains (25%), followed by Hartmannella vermiformis, with five strains (13.8%). All the other amoebae were found in plant C (Corsaro et al., 2009). Noteworthy, Acanthamoeba, a relatively widespread amoeba was not isolated from plant A and was very rare in plant B (a single isolate was recovered). Six amoebae (15.7%) were found positive for endosymbionts: five Acanthamoeba (three for chlamydiae and two for legionellae) and one N. australiensis (for legionella). A Naegleria harbouring legionellae was previously reported (Thomas et al., 2008); unfortunately, the endosymbiont was lost during the axenization of the amoeba. The chlamydiae found as natural endosymbionts of Acanthamoeba were identified as new strains of Neochlamydia (strain CRIB37, see Corsaro et al., 2009) and Protochlamydia (strain CRIB44, this study). Mycobacteria were absent from our collection of autochthonous amoebae, but previous studies reported the natural occurrence of mycobacteria within amoebae (Thomas et al., 2008; Yu et al., 2007). One and two species of chlamydiae were recovered from plants A and B, respectively. This low species richness contrasted with the high species richness previously reported in plant C (Corsaro et al., 2009). Three strains isolated from plant B belonged to the Protochlamydia genus, which was expected to be richer in their representatives (Corsaro and Venditti, 2006). P. naegleriophila, initially discovered as a natural symbiont of Naegleria (Michel et al., 2000), may also grow within Acanthamoeba and might be an emerging pathogen (Casson et al., 2008). We recovered this species in plant B (one strain, this study) and C (two strains) by Acanthamoeba co-culture. Amoebae isolated from the same sample from which we recovered P. naegleriophila were naegleriae (N. australiensis), but they were free of symbionts. The other two Protochlamydia strains (CRIB40 and CRIB44) were closely related to P. amoebophila (Collingro et al., 2005b). The very few strains assigned to this species are endosymbionts of Acanthamoeba recovered from soils (Fritsche et al., 2000; Schmitz-Esser et al., 2008), and a human respiratory sample (Haider et al., 2008). To our knowledge, we recovered for the first time a P. amoebophila strain from an aquatic biotope. In our study, we recovered by amoeba co-culture, from a total of 125 samples, 37 legionellae (29.6%) and 24 mycobacteria (19.2%). Among them, we identified by 16S rDNA sequencing and phylogenetic analyses, 30 strains of legionellae, clustered into 22 OTU, and 21 strains of mycobacteria, clustered into 12 OTU. Noteworthy, only five and four OTU, comprising 10 legionellae (33%) and 11 mycobacteria (52%) strains, respectively, could be assigned to known species. This means that about 66% of the legionellae and 48% of the mycobacteria that were recovered could represent new species. Legionella spp. are commonly found in natural freshwater and artificial water systems, where they seem to alternate between a free-living planktonic stage and an intracellular stage within protists. Various species have been associated to respiratory diseases. Amoebae play a role in the virulence and the ecology of these intracellular bacteria as reservoir and vector (Greub and Raoult, 2004). Indeed, infection by legionellae mainly occurs by aerosolization and not by person-to-person transmission. L. pneumophila is the

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primary pathogen, responsible for about 90% of cases of legionellosis, but other species may cause diseases, like L. longbeachae (4%), L. bozemannii (2.5%) and L. micdadei (0.6%) (Fields et al., 2002; Muder and Yu, 2002; Lamoth and Greub, 2010). Among the species recovered in this study, three are known to be potential agents of respiratory diseases in humans: L. dumoffii, L. lytica (LLAP3) and L. drozanskii (LLAP1). For the putative new species, further studies will be necessary to evaluate their clinical relevance. The high proportion of putative new species of Legionella recovered in this study by amoeba co-culture is congruent with previous reports. Thus, using a culture-independent, PCR-based approach, Wullings and van der Kooij (2006) found that about 65% (132 out 202 clones) of legionellae identified were not assignable to known species, and L. pneumophila represented only a minor part, with five clones (2.5%). Similarly, several new legionella species were recovered from cooling tower by Wéry et al. (2008), although L. pneumophila predominated. Mycobacteria also are widely present in the environment. In vitro studies have shown their ability to infect, survive and/or multiply within amoebae and amoebal cysts, and even ciliates (Primm et al., 2004; Thomas and McDonnell, 2007). Yu et al. (2007) described a strain of the M. avium complex as natural endosymbiont of Acanthamoeba, and recently a strain of M. mucogenicum was isolated in amoeba co-culture from a lysate of Echinamoeba (Thomas et al., 2008). In addition, some rapid-grower mycobacteria seem to be recoverable only by Acanthamoeba co-culture (Wang et al., 2006). Noteworthy, all the identified strains isolated in amoeba co-culture in this study were rapid-growers, despite extension of amoeba co-culture to up to 3 weeks. Our samples were untreated, in contrast to our previous methodology (Thomas et al., 2008), in which various slow-grower strains were recovered with a similar protocol after treatment by NaOH, heat and/or acid. Some slow-growers such as M. avium are able to grow in amoebae and may be present in similar biotopes (Adékambi et al., 2006; Hilborn et al., 2006). Although, coinfection of amoebae by more than one bacterial species may occur, rapid-grower mycobacteria may impair recovery of slow-growers. Samples for the recovery of mycobacteria are generally chemically treated to eliminate other microbiota. Then, slow-grower strains may be selected by pre-treatment whereas untreated samples will favour recovery of rapidly growing mycobacteria (Hilborn et al., 2006; Wang et al., 2006). Moreover, amoebae could represent a widespread reservoir for mycobacteria, as confirmed by the association between the presence of amoebae and mycobacteria in man-made aquatic environments (Thomas et al., 2006b, 2008). In our study, we recovered mycobacteria in 50% (12/24) of samples positive for amoebae and in 12% (12/101) of samples negative for amoebae (p < 0.005), confirming the association observed by Thomas et al. (2006b). However, in a recent study conducted in natural environments, mycobacteria were recovered in water positive or negative for amoebae without significative difference (Eddyani et al., 2008). In another study, Berk et al. (2006) compared the rate of amoebae infected by bacteria recovered from natural waters and cooling towers, finding a significant difference of 7.5% vs 55%. Several factors could explain such difference, including the presence of biofilms in some parts of the artificial water systems, where the amoeba-bacteria association would be favoured. In contrast, in natural water, density population is expected to be diluted. Moreover, ecosystems are more complex, with a larger variety of potential hosts, including other protists, invertebrates and small vertebrates. In addition, in the absence of biocide treatment (largely used in water treatment plant), a free-living lifestyle could predominate for facultative intracellular bacteria such as legionellae and mycobacteria. In addition, a sampling bias should always be considered, as not all the amoebae could be recovered onto NNA, and the laboratory strain of amoeba used for co-culture could not be susceptible to all the

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bacterial strains searched for. In this respect, it is to note that all the samples from which autochthonous amoebae with endosymbionts were recovered, resulted negative by Acanthamoeba co-culture. For all six amoebae with natural endosymbionts, we failed to transfer the symbionts to our Acanthamoeba lab strain. The Legionella endosymbiont of Naegleria was lost during the axenization of the natural host, and it seemed unable to grow onto agar. Similarly, Thomas et al. (2008) failed to transfer legionella symbionts of Naegleria in the same Acanthamoeba strain. Also, Neochlamydia CRIB37 and Protochlamydia CRIB44 recovered in these plants, could only be propagated into their natural Acanthamoeba hosts. Overall, this work showed (i) the occurrence of a significant diversity of chlamydiae, legionellae and mycobacteria in the water plants studied, which probably inhabit protists, since they promptly grow in Acanthamoeba co-culture and (ii) a limited recovery of amoebae exhibiting symbionts. Acknowledgements We thank S. Aeby for the excellent technical assistance. This work was supported by SUEZ ENVIRONNEMENT (Paris). G. Greub is supported by the Leenards Foundation through a career award entitled ‘Bourse Leenards pour la relève académique en médecine clinique à Lausanne’. References Adékambi, T., Salah, S.B., Khlif, M., Raoult, D., Drancourt, M., 2006. Survival of environmental mycobacteria in Acanthamoeba polyphaga. Appl. Environ. Microbiol. 72, 5974–5981. Berk, S.G., Gunderson, J.H., Newsome, A.L., Farone, A.L., Hayes, B.J., Redding, K.S., Uddin, N., Williams, E.L., Johnson, R.A., Farsian, M., Reid, A., Skimmyhorn, J., Farone, M.B., 2006. Occurrence of infected amoebae in cooling towers compared with natural aquatic environments: implications for emerging pathogens. Environ. Sci. Technol. 40, 7440–7444. Berry, D., Xi, C., Raskin, L., 2006. Microbial ecology of drinking water distribution systems. Curr. Opin. Biotechnol. 17, 297–302. Casson, N., Michel, R., Müller, K.-D., Aubert, J.D., Greub, G., 2008. Protochlamydia naegleriophila as etiologic agent of pneumonia. Emerg. Infect. Dis. 14, 168–172. Collingro, A., Poppert, S., Heinz, S., Schmitz-Esser, S., Essig, A., Schweikert, M., Wagner, M., Horn, M., 2005a. Recovery of an environmental chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp. Microbiology 151, 301–309. Collingro, A., Toenshoff, E.R., Taylor, M.W., Fritsche, T.R., Wagner, M., Horn, M., 2005b. ‘Candidatus Protochlamydia amoebophila’, an endosymbiont of Acanthamoeba spp. Int. J. Syst. Evol. Microbiol. 55, 1863–1866. Corsaro, D., Greub, G., 2006. Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria. Clin. Microbiol. Rev. 19, 283–297. Corsaro, D., Venditti, D., 2004. Emerging chlamydial infections. Crit. Rev. Microbiol. 30, 75–106. Corsaro, D., Venditti, D., 2006. Diversity of the Parachlamydiae in the environment. Crit. Rev. Microbiol. 32, 185–199. Corsaro, D., Valassina, M., Venditti, D., 2003. Increasing diversity within Chlamydiae. Crit. Rev. Microbiol. 29, 37–78. Corsaro, D., Feroldi, V., Saucedo, G., Ribas, F., Loret, J.F., Greub, G., 2009. Novel Chlamydiales strains isolated from a water treatment plant. Environ. Microbiol. 11, 188–200. Council Directive 98/83/EC, 1998. Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. Off. J. Eur. Commun. L 330, 32–54. Eddyani, M., De Jonckheere, J.F., Durnez, L., Suykerbuyk, P., Leirs, H., Portaels, F., 2008. Occurrence of free-living amoebae in Southern Benin in communities of low and high endemicity of Buruli ulcer. Appl. Environ. Microbiol. 74, 6547–6553. Fields, B.S., Benson, R.F., Besser, R.E., 2002. Legionella and Legionnaires’ disease: 25 years of investigation. Clin. Microbiol. Rev. 15, 506–526. Fritsche, T.R., Horn, M., Wagner, M., Herwig, R.P., Schleifer, K.H., Gautom, R.K., 2000. Phylogenetic diversity among geographically dispersed Chlamydiales endosymbionts recovered from clinical and environmental isolates of Acanthamoeba spp. Appl. Environ. Microbiol. 66, 2613–2619. Gerba, C.P., Nwachuku, N., Riley, K.R., 2003. Disinfection resistance of waterborne pathogens on the United States Environmental Protection Agency’s Contamination Candidate List (CCL). J. Water Supply Res. Technol. AQUA 52, 81–94.

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