First Evidence of AmoebaeâMycobacteria Association in Drinking

Article pubs.acs.org/est

First Evidence of Amoebae−Mycobacteria Association in Drinking Water Network Vincent Delafont,†,‡ Faïza Mougari,§ Emmanuelle Cambau,§ Michel Joyeux,‡ Didier Bouchon,† Yann Héchard,† and Laurent Moulin*,‡ †

Université de Poitiers, Laboratoire Ecologie et Biologie des Interactions, UMR CNRS 7267, Equipes Microbiologie de l’Eau & Ecologie, Evolution, Symbiose, Poitiers 86000, France ‡ Eau de Paris, Direction de la Recherche et du Développement pour la Qualité de l’Eau, R&D Biologie. 33, avenue Jean Jaurès, 94200 Ivry sur Seine, France § APHP Groupe Hospitalier Saint-Louis Lariboisière-Fernand-Widal, Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux, F-75010 Paris, France S Supporting Information *

ABSTRACT: Free-living amoebae are protozoa ubiquitously found in water systems. They mainly feed on bacteria by phagocytosis, but some bacterial species are able to resist or even escape this lethal process. Among these amoeba resistant bacteria are numerous members of the genus Mycobacterium. Nontuberculous Mycobacteria (NTM) are opportunistic pathogens that share the same ecological niches as amoebae. While several studies have demonstrated the ability of these bacteria to colonise and persist within drinking water networks, there is also strong suspicion that mycobacteria could use amoebae as a vehicle for protection and even replication. We investigated here the presence of NTM and FLA on a drinking water network during an all year round sampling campaign. We observed that 87.6% of recovered amoebal cultures carried high numbers of NTM. Identification of these amoeba and mycobacteria strains indicated that the main genera found in drinking water networks, that is, Acanthamoeba, Vermamoeba, Echinamoeba, and Protacanthamoeba are able to carry and likely to allow replication of several environmental and potentially pathogenic mycobacteria including M. llatzerense and M. chelonae. Direct Sanger sequencing as well as pyrosequencing of environmental isolates demonstrated the frequent association of mycobacteria and FLA, as they are part of the most represented genera composing amoebae’s microbiome. This is the first time that an association between FLA and NTM is observed in water networks, highlighting the importance of FLA in the ecology of NTM.

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INTRODUCTION

ARB have the capacity to escape or modify the phagocytic process through amoebae, sometimes to the detriment of their host. These bacteria are thus potentially resistant to human macrophages, as they are ≪selected≫ to survive and escape phagocytosis, and can be considered as a potential public health concern.7,8 A wide range of bacterial species is known to be resistant against phagocytosis, some species being even able to multiply inside amoebae, like Legionella pneumophila. Among these ARB are numerous members of the Mycobacterium genus.6,9−12 Nontuberculous mycobacteria are environmental bacteria found ubiquitously in soil and freshwater, sharing thus the same ecological niches than amoebae. Many species are opportunistic pathogens, infecting mainly immunocompromised patients.13−15 They are causative agents of potentially severe

Free-living amoebae (FLA) are unicellular eukaryotes commonly found in soil and water.1 They feed mainly on bacteria and other microorganisms by phagocytosis. Amoebae represents a wide and polyphyletic group that can be found in at least two distinctive forms: the trophozoı̈te, a metabolically active and fragile form, and the cyst, a dormant and resistant form.2 As it has been reported multiple times, amoebae can colonise drinking water networks, due to resistance to chemical used for water disinfection, notably via their cystic form.3,4 The encystment process being reversible, amoebae can excyst when conditions become more favorable. The main species living in water networks belong to Vermamoeba (formerly Hartmannella), Acanthamoeba, Naegleria, Echinamoeba, and Vahlkampf ia. Interest for amoebae in drinking water networks started when it was considered to be the host for Legionella pneumophila multiplication.5 Nowadays, it is known that amoebae can carry bacteria, either in a transient way (then called amoeba resisting bacteria or ARB) or in a stable way (then called endosymbionts).6 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 11872

July 28, 2014 September 22, 2014 September 23, 2014 September 23, 2014 dx.doi.org/10.1021/es5036255 | Environ. Sci. Technol. 2014, 48, 11872−11882

Environmental Science & Technology

Article

according to the regulation, and usual parameters were documented (pH, Temperature, free chlorine, turbidity, conductivity, total flora). Amoebal Culture. Water samples were filtrated through a 3 μm nitrocellulosic membrane (Millipore). The membranes were placed on NNA plates (Non Nutrient Agar) seeded with either live bacteria or yeast as described previously34 (Escherichia coli strain K-12 cultured on LB-agar medium for 24h at 37 °C, and resuspended with Page’s amoeba saline buffer, or Saccharomyces cerevisiae grown for 48 h on Sabouraud plates). The plates were incubated at 30 °C and examined by contrast phase microscopy every 3 days, up to one month after cultivation. Samples were considered negative if no amoebal development could be observed by microscopy after one month. The presence of amoebal cells is characterized by the formation of a migration front, representing the amoebal movement upon agar plates, seeking for food. Once amoebae were observed, they were recovered by scrapping the agar plate at the migration front for collection or DNA extraction. Evaluation of Mycobacterial Presence. The presence of mycobacteria within samples was assessed by quantitative PCR targeting the genus specific atpe gene, using a Viia7 real time thermocycler (LifeTechnologies).35 qPCR mixtures contained 10 μL of 2 x reaction buffer including Taq buffer, dNTP and MgCl2, 500 nM of primers AtpeF (5′-CGGYGCCGGTATCGGYGA-3′) and AtpeR (5′-CGAAGACGAACARSGCCAT-3′), 100 nM of probe AtpeP (5′-ACSGTGATGAAGAACGGBGTRAA-3′), 2 μL of purified DNA and water in sufficient quantity for 20 μL. Amoebae and Mycobacteria Identification. All microorganisms collected from amoebal migration front were resuspended in 380 μL of MagNa Pure Lysis Buffer (Roche) and subjected to thermal shocks, consisting in 5 cycles of 30 s plunged in liquid nitrogen, and 1.5 min at 95 °C in a dry bath. Then, samples were treated with proteinase K (2 mg/mL) for 30 min at 40 °C. A mechanic lysis was performed using glass beads (diameter 212−300 μm, SIGMA). Finally, the samples went through a classical DNA extraction protocol using the MagNa Pure Compact extractor using the extraction protocol DNA Bacteria V3.2, following manufacturer’s recommendation. PCR reactions were carried out on an iCycler thermocycler (BioRad). The mixtures were prepared in a final volume of 25 μL, containing 2.5 μL of 10× reaction buffer, 400 nM of each deoxynucleotide triphosphate, 2.5 mM MgCl2, 2 U of Diamond Taq DNA polymerase (Eurogentec), 500 nM of each primer (Eurogentec; see below), 2.5 μL of the purified DNA and water in sufficient quantity for 25 μL. PCR mixtures were subjected to 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 56 °C for 30 s, and DNA elongation at 72 °C for 45−90 s. Every amplification program began with a denaturation step of 95 °C for 4 min and ended with a final elongation step of 72 °C for 5 min. Amplifications of a specific 365 bp portion of the rpoB gene were carried out with RPOID1 (5′-GACGACATCGACCACTTCGG-3′) and RPOID-2 (5′-GGGGTCTCGATCGGGCACAT-3′). Amplifications of a 600 to 800 bp amoebal portion of the 18S rRNA gene sequence were carried out with Ami6F1 (5′-CCAGCTCCAATAGCGTATATT-3′) and Ami9R (5′-GTTGAGTCGAATTAAGCCGC-3′) primer pair, as described previously.36 Sequencing and Phylogeny of Amoebae and Mycobacteria. PCR products were purified using QIAquick PCR Purification Kit (Qiagen), following manufacturer’s recommendations. The purified amplicons were then sized and quantified

pneumonia and health-care associated pathologies, including skin and soft tissues infections. Since 2004, the WHO (World Health Organization) classified several NTM species as emerging pathogens transmitted by water.16 Moreover, NTM have been identified as a causative agent of outbreaks linked to colonization from a common reservoir.17−19 The major species incriminated in human diseases belong to the Mycobacterium avium complex (MAC), M. chelonae, M. kansasii, M. xenopi, and Mycobacterium abscessus group.20 The particular and complex composition of mycobacteria’s cell wall, especially the presence of mycolic acids, allows them to resist against a wide range of bactericidal agents used in water disinfection.21,22 It is a fact that since the last decades, the number of declared cases of mycobacteria human infection is not diminishing despite more accurate treatments.23 In USA, the same phenomenon is observed with a prevalence comprised between 2 and 17.7 patients per 100 000 inhabitants depending on the epidemiological status.24,25 It is important to notice that mycobacteria infections are not systematically detected and notified, underlying that the census surely under evaluates the actual number of cases. In parallel, several surveys accomplished on drinking water networks gave evidence that drinking water could be the major contamination route in cases humans pathologies related to mycobacteria.26−29 Coherently with the fact that amoebae are shown to be “Trojan horses” for numerous bacteria,6 a strong interaction between amoebae and mycobacteria in the environment can be proposed as a possibility for mycobacteria to persist in water networks. Several studies highlighted the capacity of mycobacteria to survive in amoebae in vitro,30−32 suggesting the importance of amoebae in the protection and prevalence of mycobacteria in water. Moreover, it has been shown that mycobacteria-infected amoebae can spread viable germs via a nonlytic ejection process.33 Despite these arguments, research works in literature are focused on in vitro interaction and experimental cocultures using laboratory strains. In vitro interaction experiments give clues about the role of amoebae in mycobacteria persistence and replication, but at this level of knowledge, it is difficult to conclude that these interactions are taking place in environmental conditions. Thereby, our work aims to understand the implication of amoebae in the survival and the dispersion of mycobacteria using environmental samples and isolates. We investigated the relation between these microorganisms in Paris’ city drinking water network, through a water sampling campaign, and assessed the presence of NTM associated with amoebal isolates. We chose two different and complementary sequencing strategies in order to give more insight into these interactions. Thus, using classical Sanger’s sequencing techniques, we managed to identify amoebal and mycobacterial strains associated in our samples. In addition, we undertook a pyrosequencing analysis in order to evaluate the importance of NTM in the composition of FLA’s microbiota. Finally, microscopic observations were performed in order to visualize the potential association between these microorganisms.

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MATERIAL AND METHODS Water Sampling. Paris tap water is produced from two different origins, surface water and underground water. In this study, 25 sites of the end point water network provided by four reservoirs (Reservoirs A and B, supplied by surface water, Reservoirs C and D supplied by underground water) were selected. From September 2012 to September 2013, 251 end point water samples were collected: one liter was taken monthly for analyses, transported to the laboratory at 4 °C and processed within 6 h. All water samples were also monitored 11873

dx.doi.org/10.1021/es5036255 | Environ. Sci. Technol. 2014, 48, 11872−11882


Article

Figure 1. Amoebal phylogeny inferred by Maximum Likelihood, based on amoebal 18S rRNA gene sequences. 1000 bootstrap iterations were inferred and depicted at the nodes if >50%. The scale represents the average mutation rate per nucleotide in the alignment. Shapes indicate mycobacterial species associated with amoebal isolates; M. llatzerense (black square), M. chelonae (black triangle), M. aromaticivorans (black circle), M. phocaicum (white diamond), and M. mucogenicum (black diamond).

Staining kit and procedure (RAL Diagnostics). Slides were analyzed for the presence of acid-fast bacilli using light microscopy under a x100 magnification. Pyrosequencing of 16S rRNA Gene Sequences. DNA extractions from amoebal migration fronts were pooled regarding their providing reservoir, obtaining thus four conditions. DNA samples were amplified using bacterial 16S universal primers U341F (5′- CCTACGGGAGGCAGCAG −3′) and U926R (5′- CCGTCAATTCMTTTRAGT −3′) targeting variables regions V3−V5.39 Primers were synthesized with nucleotidic adaptators for pyrosequencing and a 10 nucleotides specific barcode for each DNA pool, placed on the reverse primer. The PCR mixtures were prepared as described above, except for the use of a high fidelity Platinum Taq DNA polymerase (Life Technologies). Amplifications were carried out with an initial denaturation step at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 50 °C for 30 s, elongation at 72 °C for 1 min and a final elongation step at 72 °C for 5 min. Amplicons for pyrosequencing were purified twice using Agencourt AMPure XP beads (Beckmann Coulter), as recommended by Roche. Purified amplicons were

using a bioanalyser coupled with DNA 1000 kit (Agilent Technologies). Sequencing reactions were carried out using the BigDye Terminator kit v1.1 (Life Technologies) following manufacturer’s recommendations. Sequencing products were then characterized on an ABI Prism 3130xl (Life Technologies). Sequences were cured and confronted to the nr/nt database from the NCBI using the BLASTn algorithm for identification.37 Accession numbers of all sequences identified in this study are available in supplementary data. Sequences were aligned using MEGA v5.38 Phylogenies were inferred using Maximum Likelihood (ML) as implemented in the MEGA v5 software. Topological support for branches was assessed from the consensus of 1000 ML bootstrap trees inferred in MEGA v5. Mycobacteria Staining. Amoebal cultures were recovered by cutting a piece of agar on the migration front. Cultures were then resuspended in phosphate buffer saline (PBS) by shaking vigorously for 1 min, and centrifuged for 15 min at 1000g, to pellet amoebae. The supernatant were discarded and the pellet were resuspended in 50 μL PBS; 35 μL were spread on a slide and heat-fixed for staining. Ziehl-Neelsen staining was processed with the RAL Stainer machine, using the Fast Cold 11874



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Phylogeny was inferred by 18S sequence analysis (Figure 1). Amoebae identified as Vermamoeba vermiformis were recovered from all sampled reservoirs and represent the most frequently found species in association with mycobacteria (Figure 2, Table 1).

then checked and sized using a bioanalyser coupled with DNA 1000 kit (Agilent Technologies). DNA concentration was assessed using Quant-It PicoGreen dsDNA kit (Life Tech), and each amplicon was adjusted at a concentration of 109 DNA molecules/μL before pooling. The following steps were conducted as recommended by the Amplicon Library Preparation manual provided by Roche, and samples were loaded on a GS junior Pyrosequencer (Roche). Raw sequences and quality files were then extracted for bioinformatic analyses. Bioinformatics Analyses. Bacterial 16S sequences data generated were processed with the QIIME v1.8.0 pipeline.40 Sequences shorter than 200 bp or longer than 1000 pb were automatically discarded, as well as sequences harboring an overall quality score below 25, more than 6 ambiguous bases, homopolymers exceeding six bases, and more than two mismatches in primers sequences. Chimeras were discarded using USEARCH 6.0.41 Sequences were then clustered into Operational Taxonomic Units (OTU) with a 97% similarity threshold. A representative sequence for each OTU was then picked for downstream analyses, based on the most abundant sequence. Representative sequences were aligned and filtered using pyNAST v1.2.2.42 Taxonomy was assigned up to the genus level, using UCLUST consensus taxonomy assigner coupled with SILVA database (release 111). OTUs files were generated using QIIME pipeline, and integrated into Cytoscape v3.1 software. To visualize the OTU network, the edge-weighed embedded algorithm was applied. OTUs were weighed depending on their sequences’ counts and contribution to microbial population.

Figure 2. Diversity of amoebal isolates associated with mycobacteria, as identified by 18S rRNA gene sequencing.

Table 1. Distribution of Amoebal Genera among Providing Reservoirs and Their Associated Mycobacterial Strains, As Identified by Sanger Sequencinga

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RESULTS Mycobacteria are Highly Associated with Amoebae. Our first aim was to assess the association between amoebae and mycobacteria in water. The sampling campaign conducted on Paris’ drinking water network allowed us to analyze 251 end point water samples over a year. We paid a particular attention to samples from the end of the water network, as these are more likely to harbor a richer microbial population, in terms of both concentration and diversity.43 Among 251 water samples of end point localization, 174 (69.3%) were positive for the presence of cultivable amoebae. Total DNA at the amoebal migration front were extracted from 129 out of 174 samples (DNA extractions from 45 samples were discarded for further analyses due to poor amoebal growth) and were checked for mycobacterial presence by qPCR. Most of the amoebal cultures recovered from the water network were positive for mycobacteria, as 113 out of 129 samples (87,6%) harbored a qPCR signal. These results indicate that FLA and mycobacteria are very frequently associated in drinking water network. As mycobacteria are nonmobile bacteria, its presence at the amoebal migration front is dependent on the amoebal movement upon the agar, meaning that amoebae carry mycobacteria. Identification of Amoebae and Mycobacteria. From samples harboring FLA and mycobacteria, as checked by culture and qPCR with DNA from the migration front, we undertook the identification of both types of microorganisms based on 18S (for amoebae) and rpoB (for mycobacteria) sequencing. We screened 42 samples harboring a single amplification for amoeba and mycobacteria identification. Amoebal identification based on 18S rRNA gene sequencing revealed the presence of various FLA affiliated to Vermamoeba vermiformis, Protacanthamoeba bohemica, Acanthamoeba spp., Echinamoeba exudans, Cryptodif f lugia operculata, Allovahlkampf ia sp. and Nuclearia thermophila

a Blanks in the table correspond to the absence of identified association in the corresponding reservoir.

All isolates related to Vermamoeba vermiformis, regardless of the sampling site, are closely related, testifying of a few genetic divergences (Figure 1). The same observations can be done with isolates related to Protacanthamoeba bohemica and Echinamoeba exudans. Isolates related to Acanthamoeba genus belong to the T4 genotype, one of the most frequently found in environment and implicated in human disease.44 Mycobacterial identification was performed without subculture step by using DNA extraction from amoebal migration fronts. Among 42 samples, most of identified strains are 11875



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Figure 3. Mycobacterial phylogeny inferred by Maximum Likelihood, based on rpoB gene sequences. 1000 bootstrap iterations were inferred and depicted at the nodes if values are >50%. The scale represents the average mutation rate per nucleotide in the alignment. Shapes indicate amoebal species associated with mycobacterial isolates; Acanthamoeba spp. (black square), Protacanthamoeba bohemica (black circle), Nuclearia thermophila (white triangle), Vermamoeba vermiformis (black triangle), Cryptodif f lugia operculata (white diamond); Echinamoeba exudans (black diamond), and Allovahlkampf ia sp. (white circle).

related to Mycobacterium llatzerense, followed by M. chelonae, M. aromaticivorans, M. phocaicum, and M. mucogenicum (Figure 3, Table 1). More than 50% of the isolates are closely related to Mycobacterium sp. isolate k328w. This strain was originally described from the analysis of a soil at an illegal dumping site in Japan and stated at the time as a potential new species.45 Comparison of partial rpoB, hsp65, and 16S coding genes present in GenBank database showed at least 99% of sequences identity between M. llatzerense and Mycobacterium sp. isolate k328w (data not shown). As the characterization of Mycobacterium sp. k328w is anterior to the description of M. llatzerense, we hypothesize here that the strain k328w is an actual member of M. llatzerense species. Phylogeny inferred by maximum likelihood on mycobacterial strains identified in this study confirmed the proximity between strains affiliated to M. llatzerense and Mycobacterium sp. Isolate k328w. In conclusion, more than 90% of amoebal isolates are associated with M. llatzerense (Figure 4). Mycobacteria Are a Part of FLA’s Core Microbiome. In order to better understand the association between amoebae and mycobacteria, a pyrosequencing approach was adopted,

Figure 4. Diversity of mycobacterial isolates associated with amoebae, as identified by rpoB gene sequencing.

based on V3−V5 variable 16S region characterization for all samples carrying FLA. This technique allowed us to identify 11876



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Figure 5. (A) Heatmap of OTUs containing more than 500 sequences associated with amoebae, for each reservoir. Values are expressed as log10 of OTU sequence count. (B) Network-based analysis of mycobacteria among samples pooled by their providing reservoirs. Big circles represent samples provided by one site (Reservoirs A, B, C, and D). Small circles represent OTUs affiliated to mycobacteria at a 97% similarity threshold. OTUs are linked to the reservoirs they have been identified by edges. The thickness of an edge is proportional to OTUs’ sequences counts.

the whole FLA microbiota, as described previously.34 Among 60955 16S sequences obtained, 1977 OTUs were identified at a 97% similarity threshold and 1.3% (33 OTUs) belonged to the Mycobacterium genus, representing 799 sequences. Mycobacteria

appeared to be part of the most represented OTUs within FLA microbiome (Figure 5A). This tends to confirm their important association with amoebae, as previously suspected. To visualize the repartition of mycobacterial OTUs among the sampling sites, 11877



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Figure 6. Mycobacterial phylogeny inferred by inferred by Maximum Likelihood, based on 16S rRNA sequences obtained by pyrosequencing. The length of the sequences was trimmed at 510 nucleotides. 1000 bootstrap iterations were inferred and depicted at the nodes if >50%. Reference sequences are indicated with gray squares.

Mycobacteria Are Found within Amoebae. Microscopic analyses were undertaken in this study in order to have a comprehensive view of the association between FLA and NTM in the environment. From samples representing the most frequently found amoeba genera, we performed a Ziehl-Neelsen staining, in order to clarify the nature of FLA and mycobacteria association (Figure 7). Ziehl-Neelsen staining is a classical tool used to confirm the presence of acid-fast bacilli (AFB) like mycobacteria, which will appear as pink bacilli, whereas other microorganisms will appear in a light blue color. Microscopic observations revealed the presence of AFB in all the 42 processed cultures. For Acanthamoeba and Protacanthamoeba cultures, numerous AFB were observed inside amoebae, in vacuole-like structures or in cytoplasm (Figure 7A, B, C, and D). For Vermamoeba cultures, intracellular AFB were rarely observed but a high density of bacteria was observed surrounding both trophozoites and cystic amoebal cells (Figure 7E and F). The density of AFB inside amoebae was highly variable, from one culture to another, as depicted in the figure (Figure 7). Independently from the nature of amoeba and mycobacteria, only a small fraction of the amoebal population was found to

a network was drawn using Cytoscape (Figure 5B). Among amoeba-associated mycobacteria, two OTUs are highly represented, in terms of both prevalence and sequences count; the OTU 1842 count for 75% of the sequences and is affiliated to M. llatzerense, while OTU 1814 count for 10% of the sequences, and is closely related to M. chelonae/M. abscessus. Most OTUs however correspond to yet undescribed environmental NTM, as reflected by phylogenic analysis inferred by maximum likelihood (Figure 6). The presence of mycobacteria among all sites indicates this genus is part of FLA’s core microbiota (Figure 5 A and B). This information confirms previously obtained results, indicating a frequent association of FLA with NTM. All OTUs affiliated to the Mycobacterium genus appear to belong to rapidly growing mycobacteria species (Figure 6). Pyrosequencing approach constitutes a deep analysis of mycobacterial species’ diversity associated with amoebae, revealing the potential presence of unclassified mycobacterial strains. Characterization of OTUs’ representative sequences corroborates identifications obtained with Sanger sequencing techniques, confirming the preponderance of M. llatzerense and M. chelonae in association with FLA. 11878



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Figure 7. Zhiel-Neelsen staining revealing mycobacteria associated within amoebal isolates; Acanthamoeba spp. (A and B), Protacanthamoeba bohemica (C and D), Vermamoeba vermiformis (E and F). Scale bar represents 10 μm.

mycobacteria. More generally, FLA from the Amoebozoa supergroup seem to be the major hosts for mycobacteria in water systems, and to a lesser extent, members of the Opisthokonta and Heterolobosoa supergroups. It is interesting to note that genera most frequently found in our study are also well described for their capacity to allow Legionella pneumophila replication.49 Among amoeba-associated mycobacteria, isolates related to M. llatzerense clearly prevailed, representing more than 90% of the isolates. Little is known about this recently described species, apart from the fact that it was recurrently isolated from freshwater environments.50 Lately, two case reports demonstrated the potential pathogenicity of M. llatzerense, relating pulmonary and skin diseases.51,52 Taken together, M. llatzerense could represent a potential emerging pathogen, largely disseminated by FLA through water systems. Among the less represented mycobacterial species associated with amoebae in our study are members of M. chelonae, M. aromaticivorans, M. phocaicum, and M. mucogenicum species. M. chelonae and M. mucogenicum are well described pathogenic environmental mycobacteria having the ability to survive inside Acanthamoeba polyphaga in coculture experiment.30 Their characterization in our study is in accordance with previous hypothesis indicating the potential role of FLA in the persistence and dissemination of these strains. M. phocaicum is a member of the M. mucogenicum group, along with M. aubagnense, recovered from cooling towers using amoebal cocultures experiments.53 Our findings confirm the ability of this species to use FLA as a niche for persistence. M. aromaticivorans species was so far only described as a polycyclic aromatic hydrocarbon (PAH) degrading strain, getting thus a peculiar attention in the context of contaminated-soils’ bioremediation.54 Our work represents here the first description of M. aromaticivorans isolated from a drinking water system, and in addition as associated with FLA. A pyrosequencing approach targeting 16S rRNA V3−V5 variable regions allowed us to recover nearly 800 mycobacterial sequences, composed of 33 OTUs at a 97% similarity threshold. This support the hypothesis that diverse mycobacterial species are able to interact with FLA, as it has been suggested before with laboratory strains.10,30 Results indicated that most of the OTUs affiliated to this genus could represent yet undescribed mycobacteria species.

be associated with mycobacteria. Most of the observed AFB are very likely to be Mycobacteria as the genus is by far the most represented genus of acid-fast-bacilli associated with amoebae found in our microbiome analysis.

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DISCUSSION Free-living amoebae and nontuberculous mycobacteria are classical inhabitants of water systems, sharing the same ecological niches. In vitro amoebal coculture experiments, conducted with many mycobacterial strains, demonstrated the ability of NTM to persist and in certain cases grow inside Acanthamoeba spp.10,30 However, it has not been shown whether this interaction could take place in environmental samples. Thanks to a large sampling campaign, we recovered hundreds of water samples from Paris’ drinking network system, and analyzed it for both amoebal and mycobacterial presence after an amoebal enrichment step. Cultivable FLA were present in 69.3% of the analyzed samples (174/251), and no significant correlation between the water quality descriptors (pH, Temperature, Free Chlorine···) and the amoeba/mycobacteria presence was found in our study. These results confirmed the ubiquitous nature of FLA in drinking water, as well as its resistance to water disinfection treatments as described before.3,4,36,46,47 Among the recovered amoebal cultures, 87.6% harbored mycobacteria as detected by qPCR. Although qPCR does not give information about the viability or integrity of bacteria, microscopic observation strengthen our molecular analysis and show that the interaction between these microorganisms did not end with the killing of mycobacteria. This strong association can be explained by the fact that FLA would transport mycobacteria either intracellularly or extracellularly. Both transports have been described by in vitro coculture experiments.30,48 These findings are in accordance with the knowledge concerning FLAmycobacteria interactions, and we demonstrate here that these interactions happen not only in vitro with laboratory strains, but also in the reality of water systems, with environmental strains directly isolated from the network. The characterization of protagonists of the FLA-mycobacteria interaction showed that amoebae affiliated to Vermamoeba, Protachantamoeba, Acanthamoeba, Echinamoeba, Nuclearia, Cryptodif f lugia, and Allovahlkampf ia were likely able to carry nontuberculous 11879



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These findings suggest that most of mycobacteria diversity found in water, and by extension, associated with amoebae, represent uncharacterized mycobacteria species, as suggested in previous studies.29 This powerful sequencing approach confirmed the predominance of M. llatzerense, representing 75% of sequences among FLA-associated mycobacteria, and showed that 10% of the sequences were affiliated to the well-known opportunistic pathogens M. chelonae and M. abscessus. Analysis of OTUs repartition revealed that mycobacteria are a part of FLA’s core microbiota, as it was found in every sampling site (Figure 5B). Interestingly, while Mycobacteria are frequently associated with Amoeba, only M. llatzerense was found ubiquitously in our sampling campaign. The presence of nontuberculous mycobacteria as an important component of amoebae’s core microbiota strongly suggests that these interactions are frequent and widespread in water systems. Studies regarding FLA-mycobacteria interactions have been so far exclusively done with Acanthamoeba spp. and Dictyostelium discoideum, likely because these amoebal genera are easy to grow in laboratory conditions. But these genera can represent a minor fraction of the amoebal population in water.34,46,55 Here, we describe for the first time, to our knowledge, the association of various amoebae with mycobacteria both isolated from the same environment. Vermamoeba vermiformis has been well studied for Legionella intracellular replication.49,56 Interestingly, microscopic observations show, as for Legionella pneumophila infection, that only a fraction of FLA’s population is effectively associated with NTM.57 Moreover, mycobacterial associations with FLA appeared to be slightly different depending on the amoebal genera implicated. This latter point is well illustrated by the fact that the majority of NTM observed in association with Centramoebida FLA group (comprising Acanthamoeba and Protacanthamoeba) were located intracellularly, whereas NTM associated with Tubulinea FLA group (comprising Vermamoeba, Echinamoeba, and Cryptodif f lugia) were mainly located extracellularly, surround̈ and cysts. We then hypothesize that the ing both trophozoites mechanism of interaction between mycobacteria and Tubulinea could be different than what is usually noted with members of Centramoebida, like Acanthamoeba spp.. These findings offer new perspectives on the way FLA and NTM interact. The newly documented interaction of M. llatzerense with V. vermiformis shows a saprozoic relationship, as suggested earlier with Acanthamoeba and M. avium.48 One could hypothesize that diffusible molecules secreted by V. vermiformis could enhance mycobacterial growth. The amoebal genus Protacanthamoeba is poorly known and has been described very recently in drinking water networks.58 In this study, we describe this amoebal genus as a host for mycobacteria, and, according to our findings, possible intracellular replication. Microscopic observations revealed trophozoite cells of P. bohemica filled with bacilli belonging to M. phocaicum species. A parallel can be drawn with what is observed in Legionella-infected amoebae.59,60 We therefore consider this amoeba species as an environmental host for mycobacterial persistence and with strong suspicions of enabling mycobacterial intracellular replication. To confirm this hypothesis, further work needs to be done regarding intra amoeba replication of environmental mycobacteria. The diversity of amoebae hosting NTM, belonging to three supergroups (Amoebozoa, Heterolobosoa, and Opisthokonta), tend to prove that NTM are able to benefit from a large spectrum of amoebal hosts, using different strategies for avoiding a baneful way.

In conclusion, this study demonstrates the very tight and frequent relationship between nontuberculous mycobacteria and free-living amoebae isolated from a drinking water network. Like for Legionella, we may hypothesize that FLA are a major niche for mycobacteria survival and replication in water networks or even in harsh conditions. Mycobacteria, like Legionella, could have evolved and selected peculiar survival strategy, as they do not grow rapidly in the environment, but take competitive advantage of a host for persistence and replication.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Laurent Raskine for his advice and help in identifying mycobacteria. We also thank the sampling department’s team of Eau de Paris for providing all water samples. Finally, we thank Jérôme Lesobre for his precious help during the pyrosequencing experiments.

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REFERENCES

(1) Rodríguez-Zaragoza, S. Ecology of free-living amoebae. Crit. Rev. Microbiol. 1994, 20, 225−241. (2) Fouque, E.; Trouilhé, M.-C.; Thomas, V.; Hartemann, P.; Rodier, M.-H.; Héchard, Y. Cellular, biochemical, and molecular changes during encystment of free-living amoebae. Eukaryot. Cell 2012, 11, 382−387. (3) Hoffmann, R.; Michel, R. Distribution of free-living amoebae (FLA) during preparation and supply of drinking water. Int. J. Hyg. Environ. Health 2001, 203, 215−219. (4) Thomas, V.; Loret, J.-F.; Jousset, M.; Greub, G. Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environ. Microbiol. 2008, 10, 2728−2745. (5) Rowbotham, T. J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 1980, 33, 1179−1183. (6) Greub, G.; Raoult, D. Microorganisms resistant to free-living amoebae. Clin. Microbiol. Rev. 2004, 17, 413−433. (7) Molmeret, M.; Horn, M.; Wagner, M.; Santic, M.; Abu Kwaik, Y. Amoebae as training grounds for intracellular bacterial pathogens. Appl. Environ. Microbiol. 2005, 71, 20−28. (8) Cateau, E.; Delafont, V.; Hechard, Y.; Rodier, M. H. Free-living amoebae: what part do they play in healthcare-associated infections? J. Hosp. Infect. 2014, 87, 131−140. (9) Pagnier, I.; Raoult, D.; La Scola, B. Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. Environ. Microbiol. 2008, 10, 1135−1144. (10) Ben Salah, I.; Ghigo, E.; Drancourt, M. Free-living amoebae, a training field for macrophage resistance of mycobacteria. Clin. Microbiol. Infect. 2009, 15, 894−905. (11) Thomas, V.; McDonnell, G.; Denyer, S. P.; Maillard, J.-Y. Freeliving amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol. Rev. 2010, 34, 231−259. (12) Garcia, A.; Goñi, P.; Cieloszyk, J.; Fernandez, M. T.; CalvoBeguería, L.; Rubio, E.; Fillat, M. F.; Peleato, M. L.; Clavel, A. Identification of free-living amoebae and amoeba-associated bacteria 11880



Article

from reservoirs and water treatment plants by molecular techniques. Environ. Sci. Technol. 2013, 47, 3132−3140. (13) Falkinham, J. O. Epidemiology of infection by nontuberculous mycobacteria. Clin. Microbiol. Rev. 1996, 9, 177−215. (14) Primm, T. P.; Lucero, C. A.; Falkinham, J. O. Health impacts of environmental mycobacteria. Clin. Microbiol. Rev. 2004, 17, 98−106. (15) Williams, M. M.; Armbruster, C. R.; Arduino, M. J. Plumbing of hospital premises is a reservoir for opportunistically pathogenic microorganisms: A review. Biofouling 2013, 29, 147−162. (16) Pedley, S.; Bartram, J.; Rees, G.; Dufour, A.; J. A. C Pathogenic Mycobacteria in Water; IWA, 2004; Vol. 4, pp 15−16. (17) Couderc, C.; Carbonne, A.; Thiolet, J. M.; Brossier, F.; Savey, A.; Bernet, C.; Ortmans, C.; Lecadet-Morin, C.; Coudière, I.; Aggoune, M.; et al. Infections à mycobactéries atypiques liées à des soins esthétiques en France, 2001−2010. Méd. Mal. Infect. 2011, 41, 379− 383. (18) Leão, S. C.; Viana-Niero, C.; Matsumoto, C. K.; Lima, K. V. B.; Lopes, M. L.; Palaci, M.; Hadad, D. J.; Vinhas, S.; Duarte, R. S.; Lourenço, M. C. S.; et al. Epidemic of surgical-site infections by a single clone of rapidly growing mycobacteria in Brazil. Future Microbiol. 2010, 5, 971−980. (19) Leao, S. C.; Tortoli, E.; Viana-Niero, C.; Ueki, S. Y. M.; Lima, K. V. B.; Lopes, M. L.; Yubero, J.; Menendez, M. C.; Garcia, M. J. Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the Mycobacterium chelonaeM. abscessus group is needed. J. Clin. Microbiol. 2009, 47, 2691−2698. (20) Griffith, D. E.; Aksamit, T.; Brown-Elliott, B. A.; Catanzaro, A.; Daley, C.; Gordin, F.; Holland, S. M.; Horsburgh, R.; Huitt, G.; Iademarco, M. F.; et al. An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am. J. Respir. Crit. Care Med. 2007, 175, 367−416. (21) Cangelosi, G. A.; Palermo, C. O.; Laurent, J.-P.; Hamlin, A. M.; Brabant, W. H. Colony morphotypes on Congo red agar segregate along species and drug susceptibility lines in the Mycobacterium avium−intracellulare complex. Microbiology 1999, 145, 1317−1324. (22) Falkinham, J. O.; Norton, C. D.; LeChevallier, M. W. Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems. Appl. Environ. Microbiol. 2001, 67, 1225−1231. (23) Hoefsloot, W.; van Ingen, J.; Andrejak, C.; Angeby, K.; Bauriaud, R.; Bemer, P.; Beylis, N.; Boeree, M. J.; Cacho, J.; Chihota, V.; et al. The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: A NTM-NET collaborative study. Eur. Respir. J. Off. J. Eur. Soc. Clin. Respir. Physiol. 2013, 42, 1604−1613. (24) Bodle, E. E.; Cunningham, J. A.; Della-Latta, P.; Schluger, N. W.; Saiman, L. Epidemiology of nontuberculous mycobacteria in patients without HIV Infection, New York City. Emerg. Infect. Dis. 2008, 14, 390−396. (25) Billinger, M. E.; Olivier, K. N.; Viboud, C.; Montes de Oca, R.; Steiner, C.; Holland, S. M.; Prevots, D. R. Nontuberculous mycobacteria-associated lung disease in hospitalized persons, United States, 1998−2005. Emerg. Infect. Dis. 2009, 15, 1562−1569. (26) Le Dantec, C.; Duguet, J.-P.; Montiel, A.; Dumoutier, N.; Dubrou, S.; Vincent, V. Occurrence of mycobacteria in water treatment lines and in water distribution systems. Appl. Environ. Microbiol. 2002, 68, 5318−5325. (27) Le Dantec, C.; Duguet, J.-P.; Montiel, A.; Dumoutier, N.; Dubrou, S.; Vincent, V. Chlorine disinfection of atypical mycobacteria isolated from a water distribution system. Appl. Environ. Microbiol. 2002, 68, 1025−1032. (28) Sebakova, H.; Kozisek, F.; Mudra, R.; Kaustova, J.; Fiedorova, M.; Hanslikova, D.; Nachtmannova, H.; Kubina, J.; Vraspir, P.; Sasek, J. Incidence of nontuberculous mycobacteria in four hot water systems using various types of disinfection. Can. J. Microbiol. 2008, 54, 891− 898. (29) Van der Wielen, P. W. J. J.; Heijnen, L.; van der Kooij, D. Pyrosequence analysis of the hsp65 gene of nontuberculous

Mycobacterium communities in unchlorinated drinking water in the Netherlands. Appl. Environ. Microbiol. 2013, 79, 6160−6166. (30) Adékambi, T.; Ben Salah, S.; Khlif, M.; Raoult, D.; Drancourt, M. Survival of environmental mycobacteria in Acanthamoeba polyphaga. Appl. Environ. Microbiol. 2006, 72, 5974−5981. (31) Drancourt, M.; Adékambi, T.; Raoult, D. Interactions between Mycobacterium xenopi, amoeba and human cells. J. Hosp. Infect. 2007, 65, 138−142. (32) Ovrutsky, A. R.; Chan, E. D.; Kartalija, M.; Bai, X.; Jackson, M.; Gibbs, S.; Falkinham, J. O., 3rd; Iseman, M. D.; Reynolds, P. R.; McDonnell, G.; et al. Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata. Appl. Environ. Microbiol. 2013, 79, 3185−3192. (33) Hagedorn, M.; Rohde, K. H.; Russell, D. G.; Soldati, T. Infection by tubercular mycobacteria is spread by nonlytic ejection from their amoeba hosts. Science 2009, 323, 1729−1733. (34) Delafont, V.; Brouke, A.; Bouchon, D.; Moulin, L.; Héchard, Y. Microbiome of free-living amoebae isolated from drinking water. Water Res. 2013, 47, 6958−6965. (35) Radomski, N.; Roguet, A.; Lucas, F. S.; Veyrier, F. J.; Cambau, E.; Accrombessi, H.; Moilleron, R.; Behr, M. A.; Moulin, L. atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples. BMC Microbiol. 2013, 13, 277. (36) Thomas, V.; Herrera-Rimann, K.; Blanc, D. S.; Greub, G. Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl. Environ. Microbiol. 2006, 72, 2428−2438. (37) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403−410. (38) Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011, 28, 2731−2739. (39) Baker, G. C.; Smith, J. J.; Cowan, D. A. Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 2003, 55, 541−555. (40) Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.; Costello, E. K.; Fierer, N.; Pena, A. G.; Goodrich, J. K.; Gordon, J. I.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335−336. (41) Edgar, R. C.; Haas, B. J.; Clemente, J. C.; Quince, C.; Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011, 27, 2194−2200. (42) Caporaso, J. G.; Bittinger, K.; Bushman, F. D.; DeSantis, T. Z.; Andersen, G. L.; Knight, R. PyNAST: A flexible tool for aligning sequences to a template alignment. Bioinformatics 2010, 26, 266−267. (43) Thomas, J. M.; Ashbolt, N. J. Do free-living amoebae in treated drinking water systems present an emerging health risk? Environ. Sci. Technol. 2011, 45, 860−869. (44) Maciver, S. K.; Asif, M.; Simmen, M. W.; Lorenzo-Morales, J. A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. Eur. J. Protistol. 2013, 49, 217−221. (45) Wang, Y.; Ogawa, M.; Fukuda, K.; Miyamoto, H.; Taniguchi, H. Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan. Microbiol. Immunol. 2006, 50, 513−524. (46) Rohr, U.; Weber, S.; Michel, R.; Selenka, F.; Wilhelm, M. Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance. Appl. Environ. Microbiol. 1998, 64, 1822−1824. (47) Dupuy, M.; Berne, F.; Herbelin, P.; Binet, M.; Berthelot, N.; Rodier, M.-H.; Soreau, S.; Héchard, Y. Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants. Int. J. Hyg. Environ. Health 2013, 217, 335−339. (48) Steinert, M.; Birkness, K.; White, E.; Fields, B.; Quinn, F. Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls. Appl. Environ. Microbiol. 1998, 64, 2256−2261. 11881



Article

(49) Fields, B.y S.; Sanden, G. N.; Barbaree, J. N. Intracellular multiplication of Legionella pneumophila in amoebae from hospital hot water tanks. Curr. Microbiol. 1989, 18, 131−137. (50) Dubrou, S.; Konjek, J.; Macheras, E.; Welté, B.; Guidicelli, L.; Chignon, E.; Joyeux, M.; Gaillard, J. L.; Heym, B.; Tully, T.; et al. Diversity, community composition, and dynamics of nonpigmented and late-pigmenting rapidly growing mycobacteria in an urban tap water production and distribution system. Appl. Environ. Microbiol. 2013, 79, 5498−5508. (51) Teixeira, L.; Avery, R. K.; Iseman, M.; Arrossi, A. V.; Harrington, S.; Stephens, K.; Winans, C. G. Mycobacterium llatzerense lung infection in a liver transplant recipient: Case report and review of the literature. Am. J. Transplant. 2013, 13, 2198−2200. (52) Cárdenas, A. M.; Gomila, M.; Lalucat, J.; Edelstein, P. H. Mycobacterium llatzerense abdominal abscess. J. Clin. Microbiol. 2014, 52, 1287−1289. (53) Pagnier, I.; Merchat, M.; Raoult, D.; La Scola, B. Emerging Mycobacteria spp. in cooling towers. Emerg. Infect. Dis. 2009, 15, 121− 122. (54) U.S. Environmental Protection Agency. Treatment Technologies for Site Cleanup: Annual Status Report; Washington, DC, 2007. (55) Valster, R. M.; Wullings, B. A.; Bakker, G.; Smidt, H.; van der Kooij, D. Free-living protozoa in two unchlorinated drinking water supplies, identified by phylogenic analysis of 18S rRNA gene sequences. Appl. Environ. Microbiol. 2009, 75, 4736−4746. (56) Wadowsky, R. M.; Wilson, T. M.; Kapp, N. J.; West, A. J.; Kuchta, J. M.; States, S. J.; Dowling, J. N.; Yee, R. B. Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis. Appl. Environ. Microbiol. 1991, 57, 1950−1955. (57) Dey, R.; Bodennec, J.; Mameri, M. O.; Pernin, P. Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila. FEMS Microbiol. Lett. 2009, 290, 10−17. (58) Buse, H. Y.; Lu, J.; Struewing, I. T.; Ashbolt, N. J. Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: Implications for health risks. Environ. Sci. Pollut. Res. Int. 2013, 20, 6351−6366. (59) Berk, S. G.; Ting, R. S.; Turner, G. W.; Ashburn, R. J. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl. Environ. Microbiol. 1998, 64, 279−286. (60) Kuiper, M. W.; Wullings, B. A.; Akkermans, A. D. L.; Beumer, R. R.; van der Kooij, D. Intracellular proliferation of legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl. Environ. Microbiol. 2004, 70, 6826−6833.

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