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FEMS Microbiology Ecology, 92, 2016, fiw025 doi: 10.1093/femsec/fiw025 Advance Access Publication Date: 8 February 2016 Research Article RESEARCH ARTICLE Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium discoideum: social amoebae can also package bacteria Valérie E. Paquet1,2 and Steve J. Charette1,2,3,∗ 1 Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, G1V 0A6, Canada, 2 Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Hôpital Laval, Quebec City, QC, G1V 4G5, Canada and 3 Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, QC, G1V 0A6, Canada ∗ Corresponding author: 1030 avenue de la medicine, Pavillon Marchand, local 4245, Université Laval, Quebec City, QC, G1V 0A6, Canada. Tel: +1-418-656-2131, ext. 6914; Fax: +1-418-656-7176; E-mail: [email protected] One sentence summary: This study shows that the social amoeba Dictyostelium discoideum can package bacteria, revealing a new aspect of microbial ecology. Editor: Rolf Kümmerli ABSTRACT Many bacteria can resist phagocytic digestion by various protozoa. Some of these bacteria (all human pathogens) are known to be packaged in multilamellar bodies produced in the phagocytic pathway of the protozoa and that are secreted into the extracellular milieu. Packaged bacteria are protected from harsh conditions, and the packaging process is suspected to promote bacterial persistence in the environment. To date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, have been shown to perform bacteria packaging. It is still unknown if social amoebae can do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the grazing of the social amoeba Dictyostelium discoideum and to be packaged by this amoeba was investigated in the present study. The 45 bacterial isolates displaying a resisting phenotype were tested for their capacity to be packaged. A total of seven isolates from Cupriavidus, Micrococcus, Microbacterium and Rathayibacter genera seemed to be packaged and secreted by D. discoideum based on immunofluorescence results. Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were formally packaged. These results show that social amoebae can package some bacteria from the environment revealing a new aspect of microbial ecology. Keywords: multilamellar bodies; Dictyostelium discoideum; packaged bacteria; amoeba-resisting bacteria; Cupriavidus; Rathayibacter INTRODUCTION Free-living amoebae (FLAs) like Acanthamoeba spp. are mobile unicellular protozoa that live in aquatic environments and feed on bacteria, fungi and algae (Rodriguez-Zaragoza 1994). FLAs can colonize many man-made infrastructures that provide a favorable environment for the proliferation of microorganisms, espe- cially where high bacterial population densities are found. Cooling towers (Pagnier, Merchat and La Scola 2009), air conditioners (Walker et al. 1986) and drinking water distribution systems (Thomas and Ashbolt 2011) are a few examples of man-made infrastructures where FLAs grow (reviewed in Siddiqui and Khan 2012 and Cateau et al. 2014) and regulate bacterial population densities. Received: 22 December 2015; Accepted: 2 February 2016 C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Ecology, 2016, Vol. 92, No. 3 FLAs capture bacteria by phagocytosis and transfer them to lysosomal compartments in the phagocytic pathway where they are usually digested by enzymes (Siddiqui and Khan 2012). However, some bacteria referred to as amoebae-resisting bacteria (ARBs) are able to avoid or withstand enzymatic degradation in the phagocytic pathway through various mechanisms and can survive amoeba predation and lodge inside amoebae (Loret et al. 2008). ARBs include human pathogenic bacteria such as Legionella, Chlamydia and Mycobacteria. It has also recently been shown that the ARB group includes non-pathogenic bacteria (Kebbi-Beghdadi and Greub 2014). ARBs can survive and grow within amoebae and may then escape by cell lysis or exocytosis as free bacteria, or by being packaged in fecal pellets, which are usually several concentric layers of lipid membranes known as multilamellar bodies (MLBs). The secretion of packaged bacteria has been confirmed only for a number of human pathogens (Legionella pneumophila, Salmonella enterica, Listeria monocytogenes, Helicobacter pylori and Escherichia coli O157:H7), but this process has been studied only with FLAs and protozoa of the ciliate group (reviewed by Denoncourt, Paquet and Charette 2014). Packaging provides bacteria with a number of advantages in unfavorable conditions (Berk et al. 1998; Brandl et al. 2005; Gourabathini et al. 2008; Raghu Nadhanan and Thomas 2014). For example, S. enterica bacteria packaged in MLBs by the ciliate Tetrahymena are more resistant to low concentrations of calcium hypochlorite than when they are in the planktonic state (Brandl et al. 2005). Salmonella enterica can even multiply inside pellets. The social amoeba Dictyostelium discoideum is a bacterial predator that lives in damp forest floors. The virulence traits and host–pathogen relationships of more than 20 pathogenic bacterial species have been studied using this amoeba as a model (Cosson and Soldati 2008; Bonifait et al. 2011; Dallaire-Dufresne, Paquet and Charette 2011). Dictyostelium discoideum is often compared to a macrophage-like organism that shares many proteins, such as lysosomal hydrolases involved in intracellular killing, that are found in specialized phagocytic cells in mammals (Cosson and Lima 2014). Dictyostelium discoideum produces (Mercanti et al. 2006) and secretes large amounts of MLBs when fed digestible bacteria (Paquet et al. 2013). While no studies on bacteria packaging by D. discoideum have been published, inert polystyrene beads can be packaged in D. discoideum MLBs in presence of digestible bacteria (Denoncourt, Paquet and Charette 2014). We propose that D. discoideum has also the capacity to package ARBs in MLBs. In the present study, 136 bacterial strains of various genera and environments were tested for their capacity to resist D. discoideum predation and to determine whether these newly identified ARBs are packaged in expelled MLBs. As expected, some ARBs were packaged in D. discoideum MLBs and were secreted into the extracellular milieu. Bacteria MATERIALS AND METHODS Production of packaged and secreted ARBs Amoebae Potential packaged bacteria deduced from the bacteria/amoebae co-culture results were mixed in a final volume of 300 μL with Ka using the best ratio determined from previous experiments and were plated on SM1/10 agar. Drops (5 μL) containing 100 000 D. discoideum cells were spotted on the bacterial lawns. The plates were allowed to dry and were incubated for 3 or 4 days at 21◦ C to obtain large phagocytic plaques. Samples from the peripheries of the phagocytic plaques were collected using sterile tips. The Dictyostelium discoideum DH1-10 cells (Cornillon et al. 2000) were grown at 21◦ C in HL5 medium supplemented with 15 μg mL−1 of tetracycline (Mercanti et al. 2006). The cells were subcultured twice a week in fresh medium to prevent the cultures from reaching confluence. They were also grown on bacterial lawns as described below. Klebsiella aerogenes was a kind gift from Pierre Cosson (Geneva University, Switzerland), 19 bacterial isolates were provided by Martin Filion (Moncton University, Canada) (Filion et al. 2004), and 78 bacterial isolates were provided by Janet Martha Blatny et al. (Norwegian University of Science and Technology, Norway) (Dybwad et al. 2012). All the other isolates used in the present study were from a drinking water distribution network model (Berthiaume et al. 2014) or were obtained from ATCC or USDA. Stock cultures were stored at −80◦ C in LB (EMD, Canada) supplemented with 15% glycerol. As needed, the stock cultures were thawed and were inoculated on Tryptic Soy Agar (TSA) (EMD, Canada) plates, which were incubated at 25◦ C, typically for two days, before being used for the experiments. Predation resistance assay Bacterial isolates grown on TSA plates were resuspended in 3 mL of LB, and the OD at 595 nm was adjusted to 1. The resuspended bacteria (300 μL) were plated on three different nutrient media (HL5: bacto peptone (Oxoid) 14.3 g L−1 , yeast extract 7.15 g L−1 , maltose monohydrate 18 g L−1 , Na2 HPO4 .2H2 O 0.65 g L−1 , KH2 PO4 0.5 g L−1 and bacto agar 20 g L−1 ); SM: bacto peptone 10 g L−1 , yeast extract 1 g L−1 , KH2 PO4 2.2 g L−1 , K2 HPO4 1 g L−1 , MgSO4 1 g L−1 and bacto agar 20 g L−1 ); or SM1/10 (the ingredients for SM were all diluted 1/10 except for the bacto agar). The plates were allowed to dry under sterile conditions to obtain bacterial lawns. The tetracycline from the amoeba cell culture maintenance was removed by medium replacement, and the D. discoideum cells were resuspended in fresh HL5 with no antibiotic before counting them in a hemacytometer chamber. Serial dilutions were prepared in HL5 medium to obtain the following D. discoideum cell concentrations: 500 000; 50 000; 5000; 500, 50 and 5 cells per 5 μL. The bacterial lawns were spotted with 5 μL of the serial D. discoideum dilutions. The plates were allowed to dry and were incubated at 21◦ C for 7 days. They were examined visually for plaque formation on days 1, 3 and 7. The isolates that did not allow the growth of amoebae were considered as ARBs. Bacteria/amoebae co-cultures The identified ARBs were co-cultured alone or were mixed in a final volume of 300 μL with digestible K. aerogenes (Ka), which is known to stimulate the production of MLBs (Paquet et al. 2013) and with 30 prewashed D. discoideum cells. The mixtures were spread on SM agar plates. Serial Ka:ARB ratios ([99:1], [9:1] [1:1], [1:9] and [1:99], in a total volume of 300 μL), based on an OD adjusted to 1, were used to determine the best conditions for D. discoideum growth on bacterial co-cultures. The plates were incubated at 21◦ C for 14 days and were examined visually for phagocytic plaque formation, bacterial colonies within the phagocytic plaques, or all other anomalous growth on days 3, 9 and 14. Paquet and Charette samples were gently diluted in fresh SM1/10 medium and were processed for immunofluorescence (IF) or transmission electron microscopy (TEM) as described below. Immunofluorescence The samples containing suspended cells and material from the peripheries of phagocytic plaques were allowed to adhere to glass coverslips for 3 h and were then fixed in 4% paraformaldehyde for 30 min. The coverslips were rinsed with PBS 1X (1.9 mM NaH2 PO4 + H2 O; 8.1 mM Na2 HPO4 + 2 H2 O; 154 mM NaCl, pH 7.4) containing 40 mM NH4 Cl to stop the fixation and then with PBS 1X. The cells were permeabilized for 2 min with methanol at −20◦ C, and the coverslips were rinsed with PBS 1X and then with PBS 1X containing 0.2% bovine serum albumin (PBS-BSA) at room temperature for at least 5 min to block non-specific binding sites. The adherent cells were then incubated for 45 min with the H36 antibody (Mercanti et al. 2006) diluted 1:1000 in PBS-BSA and then with Alexa 568-coupled anti-mouse IgG secondary antibody (diluted 1:400; Invitrogen, Canada) and 2.5 μg mL−1 of DAPI (4,6-di-amidino-2-phenylindole diluted in PBSBSA) for 30 min at room temperature in the dark. The coverslips were washed at least three times with PBS-BSA between each step. The coverslips were mounted on glass slides using Prolong Gold (Invitrogen). Images were acquired using an Axio Observer Z1 microscope equipped with an Axiocam camera (Carl Zeiss, Canada). Transmission electron microscopy Samples from the bacteria/amoebae co-cultures and material from the peripheries of the phagocytic plaques were collected using sterile tips and were fixed for 3 h in 0.1 M sodium cacodylate buffer (pH 7.3) containing 2% glutaraldehyde and 0.3% osmium tetroxide. They were washed three times with sodium cacodylate buffer and were dehydrated for 5 min in 30% ethanol, 5 min in 50% ethanol, 5 min in 70% ethanol, 10 min in 95% ethanol and 1 h in 100% ethanol. The samples were then embedded in Epon resin and were incubated overnight at 37◦ C followed by 3 days at 60◦ C. Very thin slices (60–80 nm) were cut and were stained for 8 min with 0.1% lead citrate and then for 5 min with 3% uranyl acetate. They were then examined using a transmission electron microscope (JEOL 1230) at 80 kV. 3 RESULTS AND DISCUSSION Predation resistance assay Dictyostelium discoideum is probably the simplest system for assessing bacterial virulence (Hilbi et al. 2007; Froquet et al. 2009). Because medium richness may have an impact on the results of predation resistance assays (Froquet et al. 2007; Filion and Charette 2014), our assays were performed using three different media of varying composition and richness (HL5, SM, SM1/10 ). Phagocytic plaques, which are bacteria-free zones due to amoeba grazing, are produced when amoebae are spotted on lawns of digestible bacteria (Fig. 1). Phagocytic plaques were not observed in the presence of ARBs or were observed only for the highest D. discoideum cell concentrations (Fig. 1C and D) (Filion and Charette 2014). Ka is used routinely in many phagocytic experiments to feed D. discoideum, which is why we used it as a positive control for amoeba predation (Fig. 1B) (Froquet et al. 2009). We considered that the isolates were ARBs when 500 or fewer D. discoideum cells were unable to produce phagocytic plaques on the bacterial lawn for at least one of the media tested. For example, it is the case for Cupriavidus sp. and Microbacterium sp. isolates shown in Fig. 1C and D. Isolates that allowed the growth of the amoebae with an initial inoculum of 500 D. discoideum cells per drop or less were considered sensitive to amoeba predation and were rejected for subsequent experiments. A total of 136 bacterial isolates were screened with the amoeba predation assay to identify those that were potential ARBs. All the experiments were performed twice, and 45 isolates were considered as D. discoideum resisting bacteria and, as such, potential candidates for the packaging process (see Table S1, Supporting Information). The newly discovered ARBs were not specific to one phylum but belonged to various clades distributed throughout the prokaryotes, which was in agreement with a study by Moliner, Fournier and Raoult (2010). Table 1 presents the ARBs discovered in the present study. Our results suggested that the adaptation of bacteria to avoid digestion during phagocytosis is widespread in bacteria. Moreover, the term ARB cannot be generalized and be applied to an entire genus or species since bacteria from the same genus or species did not display the same resistance to predation (Table 1). Figure 1. Predation resistance assay. (A) Serial dilutions of D. discoideum cells (500 000 to 5 cells/5 μL) were spotted counter clockwise on bacterial lawns on HL5 agar plates. The plates were incubated for 7 days. The negative control (HL5 medium only) was spotted in the middle of the lawn. (B) Klebsiella aerogenes is sensitive to predation by amoebae. It was used as a positive control for amoeba predation. Cupriavidus sp. (C) and Microbacterium sp. (D) were resistant to predation and were considered as potential ARBs. 4 FEMS Microbiology Ecology, 2016, Vol. 92, No. 3 Table 1. Taxonomic grouping of new ARBs identified by the predation assay. Gram Classa Positive Actino Order Actinomycetales Familia Microbacteriaceae Micrococcaceae Bacilli Negative Alpha Beta Gamma a Lactobacillales – Rhizobiales Burkholderiales Nocardiaceae Streptomycetaceae Promicromono-sporaceae Paenibacillaceae Staphylococcaceae Leuconostocaceae – Rhizobiaceae Burkholderiaceae Enterobacteriales Comamonadaceae Oxalobacteraceae Enterobacteriaceae Pseudomonadales Xanthomonadales Pseudomonadaceae Xanthomonadaceae Micrococcales Bacillales Genera Species No. of isolates tested Microbacterium Rathayibacter Kocuria Micrococcus Rhodococcus Streptomyces Cellulosimicrobium Paenibacillus Staphylococcus Weissella – Sinorhizobium Burkholderia Cupriavidus Comamonas Duganella Escherichia Serratia Pseudomonas Luteibacter Microbacterium sp. Rathayibacter tritici Kocuria sp. Micrococcus luteus Rhodococcus sp. Streptomyces luridiscabiei Cellulosimicrobium funkei Paenibacillus larvae Staphylococcus sp. Weissella confusa – Sinorhizobium sp. Burkholderia sp. Cupriavidus sp. Comamonas koreensis Duganella zoogloeoides Escherichia coli Serratia grimesii Pseudomonas sp. Luteibacter anthropi 8 1 17 23 6 1 1 1 9 1 1 2 3 5 1 1 3 1 13 1 No. of ARB isolates 3 1 2 9 6 1 1 1 1 1 1 1 3 4 1 1 2 1 4 1 Actino = Actinobacteria; Alpha = Alphaproteobacteria; Beta = Betaproteobacteria; Gamma = Gammaproteobacteria. Figure 2. Triple co-cultures. Example of potential ARB isolates co-cultured with digestible bacteria (Ka) and 30 D. discoideum cells on SM agar. (A) A lawn of Ka was used as positive control for phagocytic plaque formation (clear zones in the bacterial lawn; black arrow). (B) A lawn of co-cultured Ka and Luteibacter anthropic [ratio 1:9]. After the same incubation time, the amoebae were unable to farm the bacterial lawn, and the plaques (black arrow) were much smaller than those of the negative control. This bacterial species was not retained for subsequent analyses. (C) A lawn of co-cultured Ka and Cupriavidus sp. [ratio 1:9]. Pigmented colonies corresponding to the Cupriavidus sp. can be seen in the middle of the phagocytic plaques (upper black arrow). Pigmented colonies can also seen around the plaques (lower black arrow). This isolate was considered as an ARB. Triple co-cultures The 45 newly identified ARBs were co-cultured with digestible bacteria (Ka) and D. discoideum. The goal of this experiment was to assess the growth of amoebae on digestible bacteria (Ka) in the presence of ARBs to determine whether the ARBs were toxic for the amoebae, making it impossible for them to produce packaged bacteria. All the phagocytic plaques with a profile similar to the positive control, that is, with a large bacteria-free zone (black arrow, Fig. 2A) due to extensive amoeba growth, were rejected. Similarly, co-cultures where no amoeba growth occurred, as for the negative control, were also rejected. For example, all the Ka:Luteibacter anthropic ratios produced small phagocytic plaques compared to the plaques produced by amoebae grown only on Ka, suggesting that L. anthropic was toxic to the amoebae or markedly limited their growth (black arrow, Fig. 2B). Conversely, the presence of bacterial colonies in the middle of graz- ing plaques (black arrow at top, Fig. 2C) or substantial growth of the ARB around phagocytic plaques (black arrow at the bottom, Fig. 2C) indicated that the ARB was resistant to predation and had no obvious toxicity for D. discoideum. One possibility is that the bacteria passed through the phagocytic pathway and were expelled as packaged bacteria, which then began to grow and form colonies. Three Cupriavidus and 17 other isolates displayed this profile (Table 2). Thus based on the unusual growth pattern of amoebae on their lawns, 20 isolates were considered as ARBs and were retained in order to determine whether they were packageable. Bacteria packaging by D. discoideum The next step was to determine whether D. discoideum cells were able to package ARBs. Based on previous packaging assays by Gourabathini et al. with E. coli O157:H7 and the ciliate Paquet and Charette 5 Table 2. ARBs identified after co-culture assays as potential candidates for bacteria packaging. Strains Ratio KA:ARB Cupriavidus basilensis 1:1 Cupriavidus sp. Micrococcus luteus (Norway) Micrococcus luteus US4 9:1 9:1 1:9 Rathayibacter tritici Rhodococcus erythropolis US1 Rhodococcus erythropolis US2 Rhodococcus fascians US1 Rhodococcus fascians US2 Cupriavidus necator US1 9:1 1:9 9:1 9:1 1:1 1:1 Duganella zoogloeoides Kocuria kristinae Microbacterium oxydans US1 Micrococcus luteus Micrococcus luteus 8 4 14 × 2 Micrococcus luteus D 1 6 × 2 Micrococcus luteus US3 Rhodococcus erythropolis Rhodococcus pyridinovorans Cellulosimicrobium funkei 1:1 1:9 9:1 9:1 1:9 1:9 1:9 1:1 1:9 1:1 Tetrahymena pyriformis (Gourabathini et al. 2008), packaged bacteria released on a rich medium are able to grow inside the package and break out. Indeed, packaged bacteria are likely a transitory state, allowing the bacteria to survive in harsh conditions (Berk et al. 1998; Marciano-Cabral and Cabral 2003) until they are released into an environment that is more favorable for bacterial growth. Packaged ARBs were not observed during the triple coculture experiments using rich medium even after a long period of time probably due to growth of potentially packaged bacteria. On the other hand, starvation media (Smith et al. 2010), which contains only few nutriments to prevent bacterial growth have been also tried, but they induce the multicellular development of amoebae despite the presence of digestible bacteria (data not shown). Again, no packaged bacteria were seen because active vegetative D. discoideum cells are required for the packaging process to occur. The stimulation of bacteria packaging and secretion was also studied using diluted nutrient agar (SM1/10 ) to avoid rapid bacterial growth following exocytosis that could break up the packages. We observed amoebae on mixed bacterial lawns of digestible bacteria and ARBs (see ratios and strains in Table 2). Samples collected at the peripheries of the phagocytic plaques were examined by IF with the H36 antibody (Mercanti et al. 2006) and by TEM. A sample containing potential packaged bacteria had to display combined DAPI and H36 antibody-positive staining for structures smaller than amoebae but bigger than free-living bacteria (data not shown) due to packaging of bacteria. DAPI would reveal the presence of bacteria in the structures. On its side, H36 antibody has been shown in a previous study to be a specific marker of MLBs by binding to a protein still not characterized (Paquet et al. 2013). The magenta arrows in Fig. 3 point to bacteria packages measuring 2–3 μm in diameter, and the black arrow indicates a D. discoideum cell. Of the 20 potential candidates tested by IF, three Cupriavidus isolates, two Micrococcus luteus isolates Observations and comments Based on the morphology and color of the colonies at the center and periphery of the phagocytic plaques A few fruiting bodies, with colored spores at the top. Several colonies within the phagocytic plaques. Unusual growth on agar. and one isolate each of Rathayibacter tritici and Microbacterium oxydans presented features suggesting that they were packaged by D. discoideum. The same co-culture protocol was performed on several samples to formally confirm the presence of expelled packaged bacteria by TEM. For the control condition shown on Fig. 4, D. discoideum produced (white arrow, Fig. 4B) and secreted empty MLBs (black arrow, Fig. 4C) in the presence of digestible bacteria on SM1/10 . However, D. discoideum produced fewer MLBs on SM1/10 than on rich HL5 medium (Paquet et al. 2013). Despite this, Cupriavidus sp. and R. tritici were found inside secreted MLBs when they were co-cultured with amoeba and digestible bacteria (Fig. 4E, F and I). The TEM observations revealed that some of the tested bacteria could be packaged by D. discoideum. Interestingly, R. tritici accumulated inside the amoebae, with up to 50 undigested bacteria visible inside each D. discoideum cell (Fig. 4H). It is not clear whether the accumulation was due to rapid bacterial growth inside the amoebae, the inhibition of the exocytic process, or a combination of both. While the mechanism involved is not known, this result suggested that bacteria can also survive in harsh environments by residing inside amoebae. The intracellular survival in protozoa of many bacteria has been described in the past (reviewed in Denoncourt, Paquet and Charette 2014). Many bacteria of the genus Rathayibacter are phytopathogens of terrestrial plants (Hahn et al. 2003; Schaad and Schuenzel 2010), and it is likely that amoebae and these soil bacteria interact. We showed that the packaging of bacteria is possible by D. discoideum amoeba model and that the phenomenon is not restricted to specific genera. Indeed, both Gram-negative and positive bacteria from various environments, including soil and water, were trapped inside the MLBs. Moreover, the outcome of various isolates from a same genera or even a same species regarding packaging is fairly variable. For example, 23 strains of M. luteus were tested using the predation assay and 9 were 6 FEMS Microbiology Ecology, 2016, Vol. 92, No. 3 Figure 3. Immunofluorescence of bacteria packaged by D. discoideum. Material from the peripheries of phagocytic plaques on lawns of co-cultured bacteria (see ratio in Table 2) on SM1/10 agar spotted with D. discoideum were processed for IF and were observed under an epifluorescence microscope. For each ARB tested, the differential interference contrast (DIC) is shown on the left while DAPI (blue), which targets the DNA of bacteria and amoebae, and the H36 antibody (red), which targets MLBs and the amoeba membrane, staining are presented on the right. Dictyostelium discoideum (black arrow in A) produced and secreted a few packaged Cupriavidus sp. (magenta arrow) into the extracellular milieu. The bacteria shown on the images (A and B) were coated and recognized by the H36 antibody. In (C) and (D), only a fraction of the M. luteus and R. tritici cells were in H36-positive structures. identified as ARBs, two of which were packaged in MLBs based on the IF results. A total of 13 Pseudomonas strains were also tested using the predation assay. While four displayed an ARB phenotype, none was packaged in MLBs. These results indicated that bacterial adaptive evolution with respect to protozoa is complex, as has been shown by the farming of different strains of Burkholderia sp. by non-farmer D. discoideum (DiSalvo et al. 2015). Given this, it would be difficult to predict whether a given bacterial isolate can be packaged or can resist predation by a specific protozoan without in vitro testing. It would thus be interesting to determine whether the same ARBs are packaged by different wild-type strains of D. discoideum or other protozoa. Lastly, the present study showed that some ARBs are packaged in MLBs and are secreted by D. discoideum in laboratory conditions. Amoeba/bacteria interactions are ubiquitous in natural as well as in man-made environments such as in municipal drinking water storage tank sediments (Lu et al. 2015), the floating and fixed biofilms of spring recreation areas (Hsu et al. 2011), and the surface water of warm water systems and cooling towers (Kuiper et al. 2006). As such, it is likely that Paquet and Charette 7 Figure 4. Transmission electron microscopy of bacteria packaged and secreted by D. discoideum. The peripheries of phagocytic plaques from co-cultured bacteria (see ratio in Table 2) on SM1/10 agar spotted with D. discoideum were processed and were observed by TEM. (A), (D), and (G) Bacteria grown alone on rich medium. (B) and (C) Dictyostelium discoideum produces (white arrow) and secretes (black arrow) MLBs with digestible bacteria on SM1/10 . No Ka were seen inside the MLBs. (E), (F), and (I) Cupriavidus sp. and R. tritici were packaged by D. discoideum and were exocytosed into the extracellular milieu. H. More than 50 undigested R. tritici can be seen inside a D. discoideum cell. bacteria packaging occurs in real conditions, not just in the laboratory. SUPPLEMENTARY DATA Supplementary data are available at FEMSEC online. CONCLUSION The resistance to predation of 136 bacterial isolates was assessed using a standardized D. discoideum predation assay. A total of 45 of these isolates displayed an ARB phenotype and were co-cultured with digestible bacteria to stimulate MLB production. Twenty potential candidates were retained based on this screening. The bacteria packaging of seven isolates by D. discoideum was suggested by IF and confirmed for two isolates by TEM. This is the first study to show that D. discoideum can package bacteria. These results open the way to a better understanding of the role of ARBs in microbial ecology and their persistence in many environments. ACKNOWLEDGEMENTS We are grateful to P. Cosson (University of Geneva, Switzerland) for the antibodies and bacterial strains. We warmly thank the teams of J. M. Blatny (FFI, Norway) and M. Filion (University of Moncton, Canada) as well as the USDA, who provided many bacterial strains. We thank A. Denoncourt and A. Vincent (Université Laval, Canada) for their critical reading of the manuscript and Richard Janvier (Plateforme de microscopie, IBIS, Université Laval, Canada) for acquiring the transmission electron microphotographs. 8 FEMS Microbiology Ecology, 2016, Vol. 92, No. 3 FUNDING This work was supported by grants to SJC from the Fonds de la Recherche du Québec – Nature et Technologies (FRQNT) [2014-PR-173418], the Chaire de pneumologie de la fondation J.-D. Bégin de l’Université Laval, the Fonds Alphonse L’Espérance de la fondation de l’IUCPQ, and the Establishment of young researchers - Juniors 1 program of the Fonds de la Recherche du Québec en Santé (FRQS) [20004]. Conflict of interest. None declared. REFERENCES Berk SG, Ting RS, Turner GW et al. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microb 1998;64:279–86. Berthiaume C, Gilbert Y, Fournier-Larente J et al. Identification of dichloroacetic acid degrading Cupriavidus bacteria in a drinking water distribution network model. J Appl Microbiol 2014;116:208–21. Bonifait L, Charette SJ, Filion G et al. Amoeba host model for evaluation of Streptococcus suis virulence. Appl Environ Microb 2011;77:6271–3. Brandl MT, Rosenthal BM, Haxo AF et al. Enhanced survival of Salmonella enterica in vesicles released by a soilborne Tetrahymena species. Appl Environ Microbiol 2005;71:1562–9. Cateau E, Delafont V, Hechard Y et al. Free-living amoebae: what part do they play in healthcare-associated infections? J Hosp Infect 2014;87:131–40. Cornillon S, Pech E, Benghezal M et al. Phg1p is a ninetransmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis. J Biol Chem 2000;275:34287–92. Cosson P, Lima WC. Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien? Cell Microbiol 2014;16:816–23. Cosson P, Soldati T. Eat, kill or die: when amoeba meets bacteria. Curr Opin Microbiol 2008;11:271–6. Dallaire-Dufresne S, Paquet VE, Charette SJ. Dictyostelium discoideum: a model for the study of bacterial virulence. Can J Microbiol 2011;57:699–707. Denoncourt AM, Paquet VE, Charette SJ. Potential role of bacteria packaging by protozoa in the persistence and transmission of pathogenic bacteria. Front Microbiol 2014;5:240. DiSalvo S, Haselkorn TS, Bashir U et al. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria. P Natl Acad Sci USA 2015;112:E5029–37. Dybwad M, Granum PE, Bruheim P et al. Characterization of airborne bacteria at an underground subway station. Appl Environ Microb 2012;78:1917–29. Filion G, Charette SJ. Assessing Pseudomonas aeruginosa virulence using a nonmammalian host: Dictyostelium discoideum. Methods Mol Biol 2014;1149:671–80. Filion M, Hamelin RC, Bernier L et al. Molecular profiling of rhizosphere microbial communities associated with healthy and diseased black spruce (Picea mariana) seedlings grown in a nursery. Appl Environ Microb 2004;70:3541–51. Froquet R, Cherix N, Burr SE et al. Alternative host model to evaluate Aeromonas virulence. Appl Environ Microb 2007;73: 5657–9. Froquet R, Lelong E, Marchetti A et al. Dictyostelium discoideum: a model host to measure bacterial virulence. Nat Protoc 2009;4:25–30. Gourabathini P, Brandl MT, Redding KS et al. Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach. Appl Environ Microb 2008;74: 2518–25. Hahn MW, Lunsdorf H, Wu Q et al. Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia. Appl Environ Microb 2003;69:1442–51. Hilbi H, Weber SS, Ragaz C et al. Environmental predators as models for bacterial pathogenesis. Environ Microbiol 2007;9:563–75. Hsu BM, Huang CC, Chen JS et al. Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res 2011;45:5171–83. Kebbi-Beghdadi C, Greub G. Importance of amoebae as a tool to isolate amoeba-resisting microorganisms and for their ecology and evolution: the Chlamydia paradigm. Environ Microbiol Rep 2014;6:309–24. Kuiper MW, Valster RM, Wullings BA et al. Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR. Appl Environ Microb 2006;72:5750–6. Loret JF, Jousset M, Robert S et al. Amoebae-resisting bacteria in drinking water: risk assessment and management. Water Sci Technol 2008;58:571–7. Lu J, Struewing I, Yelton S et al. Molecular survey of occurrence and quantity of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in municipal drinking water storage tank sediments. J Appl Microbiol 2015;119: 278–88. Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 2003;16:273–307. Mercanti V, Charette SJ, Bennett N et al. Selective membrane exclusion in phagocytic and macropinocytic cups. J Cell Sci 2006;119:4079–87. Moliner C, Fournier PE, Raoult D. Genome analysis of microorganisms living in amoebae reveals a melting pot of evolution. FEMS Microbiol Rev 2010;34:281–94. Pagnier I, Merchat M, La Scola B. Potentially pathogenic amoebaassociated microorganisms in cooling towers and their control. Future Microbiol 2009;4:615–29. Paquet VE, Lessire R, Domergue F et al. Lipid composition of multilamellar bodies secreted by Dictyostelium discoideum reveals their amoebal origin. Eukaryot Cell 2013;12:1326–34. Raghu Nadhanan R, Thomas CJ. Colpoda secrete viable Listeria monocytogenes within faecal pellets. Environ Microbiol 2014;16:396–404. Rodriguez-Zaragoza S. Ecology of free-living amoebae. Crit Rev Microbiol 1994;20:225–41. Schaad N, Schuenzel E. Sensitive molecular diagnostic assays to mitigate the risks of asymptomatic bacterial diseases of plants. Crit Rev Immunol 2010;30:271–5. Siddiqui R, Khan NA. Biology and pathogenesis of Acanthamoeba. Parasite Vector 2012;5:6. Smith EW, Lima WC, Charette SJ et al. Effect of starvation on the endocytic pathway in Dictyostelium cells. Eukaryot Cell 2010;9:387–92. Thomas JM, Ashbolt NJ. Do free-living amoebae in treated drinking water systems present an emerging health risk? Environ Sci Technol 2011;45:860–9. Walker PL, Prociv P, Gardiner WG et al. Isolation of free-living amoebae from air samples and an air-conditioner filter in Brisbane. Med J Aust 1986;145:175.