Research in Microbiology 165 (2014) 847e851 www.elsevier.com/locate/resmic
Brief note
Stenotrophomonas maltophilia and Vermamoeba vermiformis relationships: Bacterial multiplication and protection in amoebal-derived structures Estelle Cateau a,b,*, Elodie Maisonneuve b, Samuel Peguilhan a, Nathalie Quellard c, Yann Hechard b, Marie-Helene Rodier a,b b
a Laboratoire de parasitologie et mycologie, CHU La Miletrie, 86021 Poitiers Cedex, France Ecologie & Biologie des interactions, UMR CNRS 7267, Equipe Microbiologie de l'Eau, Universite de Poitiers, 1 rue Georges Bonnet, 86022 Poitiers Cedex, France c Laboratoire d'Anatomie et Cytologie pathologiques, CHU La Miletrie, 2 rue de la miletrie BP577, 86021 Poitiers Cedex, France
Received 14 April 2014; accepted 7 October 2014 Available online 22 October 2014
Abstract Stenotrophomonas maltophilia, a bacteria involved in healthcare-associated infections, can be found in hospital water systems. Other microorganisms, such as Free Living amoebae (FLA), are also at times recovered in the same environment. Amongst these protozoa, many authors have reported the presence of Vermamoeba vermiformis. We show here that this amoeba enhances S. maltophilia growth and harbors the bacteria in amoebal-derived structures after 28 days in harsh conditions. These results highlight the fact that particular attention should be paid to the presence of FLA in hospital water systems, because of their potential implication in survival and growth of pathogenic bacterial species. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Vermamoeba vermiformis; Free living amoebae; Stenotrophomonas maltophilia; Water; Environment
1. Introduction Stenotrophomonas maltophilia, first described as Pseudomonas maltophilia [1], is a gram-negative bacterium found ubiquitously in natural or artificial environments: plants, soil and water [9,17]. This bacterium is mainly a hospital-acquired pathogen in immunocompromised patients with intravascular catheters, ventilation tubes, exposure to broad spectrum antibiotics, and/or long hospital stays. Moreover, S. maltophilia has emerged as a new drug-resistant pathogen [13].
* Corresponding author. Laboratoire de parasitologie et mycologie, CHU La Miletrie, 86021 Poitiers Cedex, France. E-mail addresses: [email protected] (E. Cateau), elodie. [email protected] (E. Maisonneuve), samuel.peguilhan@ gmail.com (S. Peguilhan), [email protected] (N. Quellard), [email protected] (Y. Hechard), [email protected] (M.-H. Rodier).
The presence of S. maltophilia has been reported in different water sources: natural waters, water treatment plants, chlorinated distribution networks. In hospital, this microorganism has been implicated in nosocomial waterborne infections [2,13]. Free living amoebae (FLA) are also recovered in water. These mobile protozoa feed on bacteria, algae, fungi, protozoa or other organic particles. They may support bacterial growth and serve as reservoirs and vehicles for a number of pathogenic microorganisms [12]. Their life cycle consists of two stages: an actively feeding, dividing trophozoite form corresponding to the period of metabolic activity of the amoeba, and a dormant cyst that can resist hostile environmental conditions such as nutrient depletion, osmotic stress, temperature changes, pH variations, water disinfection [7,10]. Bacterial survival in environment has been associated with the production of cysts or vesicles [3]. Vermamoeba vermiformis (formerly Hartmanella vermiformis) is one of the most
http://dx.doi.org/10.1016/j.resmic.2014.10.004 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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E. Cateau et al. / Research in Microbiology 165 (2014) 847e851
common genera to have been isolated from environment and water networks [8]. Some bacteria, as Legionella pneumophila or Francisella novicida have been recovered in vacuoles within cytoplasm of V. vermiformis trophozoites [18] and/or in this amoeba cysts [11]. Rohr et al. have identified this amoebae as the main species in hot water samples of six hospitals contaminated with Legionella [16]. Thomas et al. also found V. vermiformis to be the predominant species in water of a Swiss hospital [19]. More recently, Ovrutsky et al. [14] recovered V. vermiformis from 18.8% of hospital water samples, mainly biofilms. The aim of this study is to evidence the relationships between V. vermiformis and S. maltophilia, both of which can be recovered in hospital water networks and, if appropriate, to investigate which amoebal structure is involved in the bacterial persistence.
Cocultures of V. vermiformis and S. maltophilia (SmW) were first carried out in flasks following the protocol previously described. After 24 h of coincubation, the medium was replaced by encystment medium to allow cyst formation and to mimic conditions of poor nutrient availability [3]. CFU of S. maltophilia were then numbered after 14 then 28 days of incubation at 27 C, after which, samples of suspension of coculture were examined by transmission electron microscopy.
2. Materials and methods
2.4. Electron microscopy
2.1. Strains and growth conditions
In order to visualize potential internalization of bacteria, transmission electron microscopy was performed on SmW/ amoebae cocultures. Samples obtained in filtered tap water (48 h or 96 h) or in encystment medium (28 days) were incubated for 1 h in 0.1 M phosphate buffer, containing 4% glutaraldehyde at 4 C. Cells were washed four times in PBS and post-fixed with 1% OsO4 in phosphate buffer 0.1 M for 1 h at 4 C. The samples were dehydrated in an acetone series and embedded in araldite resin. Sections were stained with uranyl acetate and lead nitrate before examination with a Jeol 1010 transmission electron microscope.
V. vermiformis (ATCC 50256) was grown in 150 cm2 tissue culture flasks at 27 C in PYNFH broth (in 1 L of distilled water: 10 g proteose peptone, 10 g yeast extract, 1 g ribonucleic acid, 15 mg folic acid, 1 mg hemin, 200 mg Na2HPO4.2H2O, 360 mg KH2PO4 and 100 mL FCS). When cells formed a monolayer, the trophozoites were harvested by tapping the flasks and washed three times in Page's modified Neff's amoeba saline (PAS, containing in 1 L of distilled water, 120 mg NaCl, 4 mg MgSO4.7H2O, 4 mg CaCl2.2H2O, 142 mg Na2HPO4 and 36 mg KH2PO4) or in filtered tap water (0.22 mm). For experiments carried out in 96-well microtiter plates, amoebae were used at a final cell concentration of 5 105/mL. Two strains of S. maltophilia were used, one isolated from a patient's blood culture (SmP), the other being isolated from hospital water (SmW). These strains were identified by mass spectrometry, Vitek MS® system (BioMerieux, France). Before experiments, these bacteria were grown on Mueller Hinton agar slants at 37 C for two days, then harvested and washed in PAS medium or in filtered tap water (0.22 mm) to a bacterial concentration of 5 104/mL. 2.2. Cocultivation of V. vermiformis and S. maltophilia The trophozoites were distributed into each well of a 96well microplate (5 104 cells/100 mL) and allowed to adhere to the wells for 2 h at 27 C. 100 mL containing 5 103 bacteria were then added in the wells to obtain a multiplicity of infection of 0.1, and incubation was carried out at 27 C. After 48 and 96 h of incubation, serial dilutions of the cocultures were plated on Mueller-Hinton medium and incubated at 37 C during 48 h to evaluate bacterial colonyforming units (CFU). Bacteria identification was confirmed, as previously, using Vitek MS® system. Controls were carried out by incubating bacteria in PAS or filtered tap water without amoebae. Microscopical examination of the cocultures using trypan blue staining was also carried out in order to determine the viability of amoebae.
All of the experiments were reproduced three times, each time in duplicate. The results presented are the mean of the series of 3 replicates of paired duplicates (n ¼ 3). 2.3. Incubation of infected amoebae in encystment medium
2.5. Statistical analysis Statistical analysis was carried out using non-parametric Wilcoxon test to determine statistical differences between groups. 3. Results and discussion 3.1. Cocultivation of V. vermiformis and S. maltophilia S. maltophilia is an emerging pathogen, responsible for considerable morbidity and mortality in immunocompromised patients. Having been recovered in water from different sources, this bacterium has, more particularly, been found in hospital tap water, and it has been proven that some outbreaks of S. maltophilia were linked to hospital water [5]. In these water networks, other microorganisms, such as FLA have also been frequently isolated. These protozoa colonize domestic and institutional water systems including domestic tap water or hospital water, swimming pools, cooling towers [16,20,21] and can interact with a wide variety of microorganisms. The majority of the published interactions have involved the genus Acanthamoeba [12], while few studies have reported S. maltophilia and Acanthamoeba spp. interactions [6,15]. As V. vermiformis is one of the most frequently recovered FLA in hospital tap water [19], we decided to investigate its interactions with S. maltophilia. In
E. Cateau et al. / Research in Microbiology 165 (2014) 847e851
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Fig. 1. Viable counts of Stenotrophomonas maltophilia (SmP: Patient strain or SmW: Hospital water strain) in coculture with V. vermiformis in PAS medium during 48 h or 96 h. Similar results were achieved with a coculture in tap water. ( p < 0.005).
the cocultures obtained in PAS medium, presence of the amoebae induced a major increase in growth of the two S. maltophilia strains after 48 h and 96 h of incubation in comparison with their growth without amoebae (Fig. 1). Results
were similar in experimentations carried out in filtered tap water (data not shown). In parallel, as shown by trypan blue exclusion experiments, V. vermiformis viability was not affected by the conditions of coculture, whatever the medium
Fig. 2. Transmission electron microscopy images of Vermamoeba vermiformis infected by Stenotrophomonas maltophilia (SmW): after 48 (A) and 96 (B) hours of coculture in tap water and after 28 days in encystment medium (with bacteria (C) or without bacteria (D)). Bacteria are indicated by arrows.
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Fig. 3. Viable counts of Stenotrophomonas maltophilia (SmW) alone or in coculture with V. vermiformis in encystment medium during 28 days. ( p < 0.01).
used (data not shown). Concerning other bacteria, Wadowsky et al. [22] have shown the multiplication of some Legionella strains in tap water containing V. vermiformis and more recently, Santic et al. [18] have reported an increased number of bacteria when V. vermiformis is cocultivated with F. novicida. We have previously shown how, in particular conditions, V. vermiformis can allow for Pseudomonas aeruginosa development in tap water [4]. In this study, the increase of S maltophilia growth in coculture with V. vermiformis is quite remarkable, in PAS (Fig. 1) as well as tap water. 3.2. Location of internalized bacteria In addition, electronic microscopy experiments have highlighted the presence of bacteria inside and around the amoeba after 48 h of incubation (Fig. 2A). After 96 h of incubation, bacteria are enclosed in vacuoles in the amoebal cytoplasm (Fig. 2B). These observations are in agreement with the previously reported location of F. novocida in intact vacuoles within V. vermiformis [18]. 3.3. Incubation of infected amoebae in encystment medium Amongst microorganisms related to FLA, some human bacterial pathogens can survive after internalization. In our experiments, we have evaluated the survival of S. maltophilia in a poor medium with and without amoebae and shown that the presence of V. vermiformis allowed for persistent bacterial viability after 14 and 28 days of incubation, while in the same medium, bacteria without amoebae cannot survive (Fig. 3). We have shown by electron microscopy that after 28 days of incubation, bacteria are located in specific forms, which do not correspond to trophozoites or cysts (Fig. 2C). These bacteriacontaining structures resemble the vesicles previously
described in other FLA species [3] but with greater size, evoking a ghost cell harboring bacteria and no longer including typical cell organelles. After a 28 days incubation in a poor medium without bacteria, amoeba precysts and cysts could be recovered (Fig. 2D), whereas same incubation carried out in presence of bacteria has led to disappearance of viable amoebae from the medium. So, we hypothesize that these ghost structures are able to protect bacteria from adverse conditions, thereby allowing them to survive in hostile environments. Complementary studies need to be conducted to test the sensitivity of the bacteria contained in these structures to biocides, primarily those used for disinfecting water networks. In conclusion, this study shows for the first time that the growth and survival of S. maltophilia, responsible for nosocomial waterborne infections, are enhanced by the presence of V. vermiformis in the same environment. The presence of FLA in hospital water networks can consequently represent an increased risk of healthcare-associated infections and justifies water filtration at the points of use. This study highlights the interest that should be paid to systematic detection of FLA in hospital water systems. Conflict of interest None. Acknowledgments We wish to thank Mr Jeffrey Arsham for revising the English text and Dr Marion Albouy-Llaty for statistical analysis. References [1] Adamek M, Overhage J, Bathe S, Winter J, Fischer R, Schwartz T. Genotyping of environmental and clinical Stenotrophomonas maltophilia isolates and their pathogenic potential. PLoS One 2011;6(11).
E. Cateau et al. / Research in Microbiology 165 (2014) 847e851 [2] Anaissie EJ, Penzak SR, Dignani MC. The hospital water supply as a source of nosocomial infections: a plea for action. Arch Intern Med 2002;162:1483e92. [3] Bouyer S, Imbert C, Rodier MH, Hechard Y. Long-term survival of Legionella pneumophila associated with Acanthamoeba castellanii vesicles. Environ Microbiol 2007;9:1341e4. [4] Cateau E, Imbert C, Rodier MH. Hartmanella vermiformis can be permissive for Pseudomonas aeruginosa. Lett Appl Microbiol 2008;47:475e7. [5] Cervia JS, Ortolano GA, Canonica FP. Hospital tap water as a source of Stenotrophomonas maltophilia infection. Clin Infect Dis 2008;46:1485e7. [6] Corsaro D, Mu¨ller KD, Michel R. Molecular characterization and ultrastructure of a new amoeba endoparasite belonging to the Stenotrophomonas maltophilia complex. Exp Parasitol 2013;133:383e90. [7] Critchley M, Bentham R. The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae. J Appl Microbiol 2009;106:784e9. [8] Da Rocha-Azevedo B, Tanowitz HB, Marciano-Cabral F. Diagnosis of infections caused by pathogenic free-living amoebae. Interdiscip Perspect Infect Dis 2009;2009. [9] Delafont V, Brouke A, Bouchon D, Moulin L, Hechard Y. Microbiome of free-living amoebae isolated from drinking water. Water Res 2013;47:6958e65. [10] Fouque E, Trouilhe MC, Thomas V, Hartemann P, Rodier MH, Hechard Y. Cellular, biochemical, and molecular changes during encystment of free-living amoebae. Eukaryot Cell 2012;11:382e7. [11] Greub G, Raoult D. Morphology of Legionella pneumophila according to their location within Hartmanella vermiformis. Res Microbiol 2003;154:619e21. [12] Greub G, Raoult D. Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 2004;17:413e33. [13] Looney WJ, Narita M, Mu¨hlemann K. Stenotrophomonas maltophilia : an emerging opportunist human pathogen. Lancet Infect Dis 2009;9:312e23.
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[14] Ovrutsky AR, Chan ED, Kartalija M, Bai X, Jackson M, Gibbs S, 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;79:3185e92. [15] Pagnier I, Raoult B, La Scola B. Isolation and identification of amoebaeresisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure. Environ Microbiol 2008;10:1135e44. [16] Rohr U, Weber S, Michel R, Selenka F, Wilhelm M. Comparison of freeliving amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance. Appl Environ Microbiol 1998;64:1822e4. [17] Ryan RP, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison MB, et al. The versatility and adaptation of bacteria from the genus stenotrophomonas. Nat Rev Microbiol 2009;7:514e25. [18] Santic M, Ozanic M, Semic V, Pavokovic G, Mrvcic V, Kwaik YA. Intravacuolar proliferation of F. novicida within H. vermiformis. Front Microbiol 2011;2:78. [19] Thomas V, Herrera-Rimann K, Blanc DS, Greub G. Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl Environ Microbiol 2006;72:2428e38. [20] Thomas V, Loret JF, Jousset M, Greub G. Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environ Microbiol 2008;10:2728e45. [21] Thomas V, McDonnel l G, Denyer SP, Maillard JY. Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol Rev 2010;34:231e59. [22] Wadowsky RM, Wilson TM, Kapp NJ, West AJ, Kuchta JM, States SJ, et al. Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis. Appl Environ Microbiol 1991;57:1950e5.
Brief note
Stenotrophomonas maltophilia and Vermamoeba vermiformis relationships: Bacterial multiplication and protection in amoebal-derived structures Estelle Cateau a,b,*, Elodie Maisonneuve b, Samuel Peguilhan a, Nathalie Quellard c, Yann Hechard b, Marie-Helene Rodier a,b b
a Laboratoire de parasitologie et mycologie, CHU La Miletrie, 86021 Poitiers Cedex, France Ecologie & Biologie des interactions, UMR CNRS 7267, Equipe Microbiologie de l'Eau, Universite de Poitiers, 1 rue Georges Bonnet, 86022 Poitiers Cedex, France c Laboratoire d'Anatomie et Cytologie pathologiques, CHU La Miletrie, 2 rue de la miletrie BP577, 86021 Poitiers Cedex, France
Received 14 April 2014; accepted 7 October 2014 Available online 22 October 2014
Abstract Stenotrophomonas maltophilia, a bacteria involved in healthcare-associated infections, can be found in hospital water systems. Other microorganisms, such as Free Living amoebae (FLA), are also at times recovered in the same environment. Amongst these protozoa, many authors have reported the presence of Vermamoeba vermiformis. We show here that this amoeba enhances S. maltophilia growth and harbors the bacteria in amoebal-derived structures after 28 days in harsh conditions. These results highlight the fact that particular attention should be paid to the presence of FLA in hospital water systems, because of their potential implication in survival and growth of pathogenic bacterial species. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Vermamoeba vermiformis; Free living amoebae; Stenotrophomonas maltophilia; Water; Environment
1. Introduction Stenotrophomonas maltophilia, first described as Pseudomonas maltophilia [1], is a gram-negative bacterium found ubiquitously in natural or artificial environments: plants, soil and water [9,17]. This bacterium is mainly a hospital-acquired pathogen in immunocompromised patients with intravascular catheters, ventilation tubes, exposure to broad spectrum antibiotics, and/or long hospital stays. Moreover, S. maltophilia has emerged as a new drug-resistant pathogen [13].
* Corresponding author. Laboratoire de parasitologie et mycologie, CHU La Miletrie, 86021 Poitiers Cedex, France. E-mail addresses: [email protected] (E. Cateau), elodie. [email protected] (E. Maisonneuve), samuel.peguilhan@ gmail.com (S. Peguilhan), [email protected] (N. Quellard), [email protected] (Y. Hechard), [email protected] (M.-H. Rodier).
The presence of S. maltophilia has been reported in different water sources: natural waters, water treatment plants, chlorinated distribution networks. In hospital, this microorganism has been implicated in nosocomial waterborne infections [2,13]. Free living amoebae (FLA) are also recovered in water. These mobile protozoa feed on bacteria, algae, fungi, protozoa or other organic particles. They may support bacterial growth and serve as reservoirs and vehicles for a number of pathogenic microorganisms [12]. Their life cycle consists of two stages: an actively feeding, dividing trophozoite form corresponding to the period of metabolic activity of the amoeba, and a dormant cyst that can resist hostile environmental conditions such as nutrient depletion, osmotic stress, temperature changes, pH variations, water disinfection [7,10]. Bacterial survival in environment has been associated with the production of cysts or vesicles [3]. Vermamoeba vermiformis (formerly Hartmanella vermiformis) is one of the most
http://dx.doi.org/10.1016/j.resmic.2014.10.004 0923-2508/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
848
E. Cateau et al. / Research in Microbiology 165 (2014) 847e851
common genera to have been isolated from environment and water networks [8]. Some bacteria, as Legionella pneumophila or Francisella novicida have been recovered in vacuoles within cytoplasm of V. vermiformis trophozoites [18] and/or in this amoeba cysts [11]. Rohr et al. have identified this amoebae as the main species in hot water samples of six hospitals contaminated with Legionella [16]. Thomas et al. also found V. vermiformis to be the predominant species in water of a Swiss hospital [19]. More recently, Ovrutsky et al. [14] recovered V. vermiformis from 18.8% of hospital water samples, mainly biofilms. The aim of this study is to evidence the relationships between V. vermiformis and S. maltophilia, both of which can be recovered in hospital water networks and, if appropriate, to investigate which amoebal structure is involved in the bacterial persistence.
Cocultures of V. vermiformis and S. maltophilia (SmW) were first carried out in flasks following the protocol previously described. After 24 h of coincubation, the medium was replaced by encystment medium to allow cyst formation and to mimic conditions of poor nutrient availability [3]. CFU of S. maltophilia were then numbered after 14 then 28 days of incubation at 27 C, after which, samples of suspension of coculture were examined by transmission electron microscopy.
2. Materials and methods
2.4. Electron microscopy
2.1. Strains and growth conditions
In order to visualize potential internalization of bacteria, transmission electron microscopy was performed on SmW/ amoebae cocultures. Samples obtained in filtered tap water (48 h or 96 h) or in encystment medium (28 days) were incubated for 1 h in 0.1 M phosphate buffer, containing 4% glutaraldehyde at 4 C. Cells were washed four times in PBS and post-fixed with 1% OsO4 in phosphate buffer 0.1 M for 1 h at 4 C. The samples were dehydrated in an acetone series and embedded in araldite resin. Sections were stained with uranyl acetate and lead nitrate before examination with a Jeol 1010 transmission electron microscope.
V. vermiformis (ATCC 50256) was grown in 150 cm2 tissue culture flasks at 27 C in PYNFH broth (in 1 L of distilled water: 10 g proteose peptone, 10 g yeast extract, 1 g ribonucleic acid, 15 mg folic acid, 1 mg hemin, 200 mg Na2HPO4.2H2O, 360 mg KH2PO4 and 100 mL FCS). When cells formed a monolayer, the trophozoites were harvested by tapping the flasks and washed three times in Page's modified Neff's amoeba saline (PAS, containing in 1 L of distilled water, 120 mg NaCl, 4 mg MgSO4.7H2O, 4 mg CaCl2.2H2O, 142 mg Na2HPO4 and 36 mg KH2PO4) or in filtered tap water (0.22 mm). For experiments carried out in 96-well microtiter plates, amoebae were used at a final cell concentration of 5 105/mL. Two strains of S. maltophilia were used, one isolated from a patient's blood culture (SmP), the other being isolated from hospital water (SmW). These strains were identified by mass spectrometry, Vitek MS® system (BioMerieux, France). Before experiments, these bacteria were grown on Mueller Hinton agar slants at 37 C for two days, then harvested and washed in PAS medium or in filtered tap water (0.22 mm) to a bacterial concentration of 5 104/mL. 2.2. Cocultivation of V. vermiformis and S. maltophilia The trophozoites were distributed into each well of a 96well microplate (5 104 cells/100 mL) and allowed to adhere to the wells for 2 h at 27 C. 100 mL containing 5 103 bacteria were then added in the wells to obtain a multiplicity of infection of 0.1, and incubation was carried out at 27 C. After 48 and 96 h of incubation, serial dilutions of the cocultures were plated on Mueller-Hinton medium and incubated at 37 C during 48 h to evaluate bacterial colonyforming units (CFU). Bacteria identification was confirmed, as previously, using Vitek MS® system. Controls were carried out by incubating bacteria in PAS or filtered tap water without amoebae. Microscopical examination of the cocultures using trypan blue staining was also carried out in order to determine the viability of amoebae.
All of the experiments were reproduced three times, each time in duplicate. The results presented are the mean of the series of 3 replicates of paired duplicates (n ¼ 3). 2.3. Incubation of infected amoebae in encystment medium
2.5. Statistical analysis Statistical analysis was carried out using non-parametric Wilcoxon test to determine statistical differences between groups. 3. Results and discussion 3.1. Cocultivation of V. vermiformis and S. maltophilia S. maltophilia is an emerging pathogen, responsible for considerable morbidity and mortality in immunocompromised patients. Having been recovered in water from different sources, this bacterium has, more particularly, been found in hospital tap water, and it has been proven that some outbreaks of S. maltophilia were linked to hospital water [5]. In these water networks, other microorganisms, such as FLA have also been frequently isolated. These protozoa colonize domestic and institutional water systems including domestic tap water or hospital water, swimming pools, cooling towers [16,20,21] and can interact with a wide variety of microorganisms. The majority of the published interactions have involved the genus Acanthamoeba [12], while few studies have reported S. maltophilia and Acanthamoeba spp. interactions [6,15]. As V. vermiformis is one of the most frequently recovered FLA in hospital tap water [19], we decided to investigate its interactions with S. maltophilia. In
E. Cateau et al. / Research in Microbiology 165 (2014) 847e851
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Fig. 1. Viable counts of Stenotrophomonas maltophilia (SmP: Patient strain or SmW: Hospital water strain) in coculture with V. vermiformis in PAS medium during 48 h or 96 h. Similar results were achieved with a coculture in tap water. ( p < 0.005).
the cocultures obtained in PAS medium, presence of the amoebae induced a major increase in growth of the two S. maltophilia strains after 48 h and 96 h of incubation in comparison with their growth without amoebae (Fig. 1). Results
were similar in experimentations carried out in filtered tap water (data not shown). In parallel, as shown by trypan blue exclusion experiments, V. vermiformis viability was not affected by the conditions of coculture, whatever the medium
Fig. 2. Transmission electron microscopy images of Vermamoeba vermiformis infected by Stenotrophomonas maltophilia (SmW): after 48 (A) and 96 (B) hours of coculture in tap water and after 28 days in encystment medium (with bacteria (C) or without bacteria (D)). Bacteria are indicated by arrows.
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Fig. 3. Viable counts of Stenotrophomonas maltophilia (SmW) alone or in coculture with V. vermiformis in encystment medium during 28 days. ( p < 0.01).
used (data not shown). Concerning other bacteria, Wadowsky et al. [22] have shown the multiplication of some Legionella strains in tap water containing V. vermiformis and more recently, Santic et al. [18] have reported an increased number of bacteria when V. vermiformis is cocultivated with F. novicida. We have previously shown how, in particular conditions, V. vermiformis can allow for Pseudomonas aeruginosa development in tap water [4]. In this study, the increase of S maltophilia growth in coculture with V. vermiformis is quite remarkable, in PAS (Fig. 1) as well as tap water. 3.2. Location of internalized bacteria In addition, electronic microscopy experiments have highlighted the presence of bacteria inside and around the amoeba after 48 h of incubation (Fig. 2A). After 96 h of incubation, bacteria are enclosed in vacuoles in the amoebal cytoplasm (Fig. 2B). These observations are in agreement with the previously reported location of F. novocida in intact vacuoles within V. vermiformis [18]. 3.3. Incubation of infected amoebae in encystment medium Amongst microorganisms related to FLA, some human bacterial pathogens can survive after internalization. In our experiments, we have evaluated the survival of S. maltophilia in a poor medium with and without amoebae and shown that the presence of V. vermiformis allowed for persistent bacterial viability after 14 and 28 days of incubation, while in the same medium, bacteria without amoebae cannot survive (Fig. 3). We have shown by electron microscopy that after 28 days of incubation, bacteria are located in specific forms, which do not correspond to trophozoites or cysts (Fig. 2C). These bacteriacontaining structures resemble the vesicles previously
described in other FLA species [3] but with greater size, evoking a ghost cell harboring bacteria and no longer including typical cell organelles. After a 28 days incubation in a poor medium without bacteria, amoeba precysts and cysts could be recovered (Fig. 2D), whereas same incubation carried out in presence of bacteria has led to disappearance of viable amoebae from the medium. So, we hypothesize that these ghost structures are able to protect bacteria from adverse conditions, thereby allowing them to survive in hostile environments. Complementary studies need to be conducted to test the sensitivity of the bacteria contained in these structures to biocides, primarily those used for disinfecting water networks. In conclusion, this study shows for the first time that the growth and survival of S. maltophilia, responsible for nosocomial waterborne infections, are enhanced by the presence of V. vermiformis in the same environment. The presence of FLA in hospital water networks can consequently represent an increased risk of healthcare-associated infections and justifies water filtration at the points of use. This study highlights the interest that should be paid to systematic detection of FLA in hospital water systems. Conflict of interest None. Acknowledgments We wish to thank Mr Jeffrey Arsham for revising the English text and Dr Marion Albouy-Llaty for statistical analysis. References [1] Adamek M, Overhage J, Bathe S, Winter J, Fischer R, Schwartz T. Genotyping of environmental and clinical Stenotrophomonas maltophilia isolates and their pathogenic potential. PLoS One 2011;6(11).
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