Vermamoeba vermiformis—Global Trend and Future Perspective Frederick Masangkay and Giovanni Milanez, Far Eastern University, Manila, Philippines Panagiotis Karanis, University of Cologne, Cologne, Germany; Qinghai University, Xining, China Veeranoot Nissapatorn, Walailak University, Nakhon Si Thammarat, Thailand

© 2018 Elsevier Inc. All rights reserved.

Vermamoeba vermiformis 1 Epidemiology 3 Pathogenesis 4 Endosymbionts 5 Bacteria 6 Fungus 7 Viruses 7 Isolation and Identification of Vermamoeba 7 Sample Collection and Processing of Samples Potentially Containing Vermamoeba 7 Microscopic and Molecular Protocols for Identification of Vermamoeba 7 Control and Disinfection of Vermamoeba 8 In Vitro and In Vivo Models 8 Conclusion Remark 9 References 9

Vermamoeba vermiformis

Free living amoebas (FLAs) are considered widespread in nature and therefore called ubiquitous (Lares-Villa et al., 2010); its spectrum of habitation extends from aquatic reservoir to terrestrial places (Scheikl et al., 2014). Of the organisms considered as free living amoebae; members of the genera Acanthamoeba, Naegleria, Hartmannella, and Balamuthia are known to infect humans (World Health Organization) and are considered medically important due to the fatal disease observed in their hosts. These organisms are considered free living in nature but are considered parasitic once they enter the hosts. Previously known as Hartmannella vermiformis, Vermamoeba vermiformis has been separated to a group under the order Echina- moebida as it is different from all other Hartmannella spp. in that it is worm-shaped and not clavate. Various sequences in GenBank isolated from environmental sequences points to multiple lineage (Smirnov et al., 2011). Page 1997 and Smirnov et al. (2011) provide morphologic descriptions which are key and invaluable in the identification of V. vermiformis coupled with the usual culture and molecular results. Widely dispersed in an array of environmental niches, viable V. vermiformis has been isolated from springs with recorded temperatures of 40C and snow samples of below 0C at altitudes of greater than 2700 m above sea level (Reyes- Batlle et al., 2015). Hospital environments have also shown to be positive for Vermamoeba isolates that are thermotolerant up to 46.5C–55.7C. It should be noted that in the identification of amoeba, species may be diverse even in a small area. It has been suggested that there is no correlation between geographical origin of the strain and their relatedness. Multiview approach as it is mentioned above remains the best practice for identification. Trophozoites should be studied for characteristic morphology, locomotion, nuclear characteristics, and cyst structure. Molecular identification is the best method for definitive identification of the target organisms up to the genetic level and is not hampered by polymorphisms that may be encountered in microscopic observation (Walochink et al., 2002). Microscopic observation is essential so as to provide phenotypic characteristics that can be coupled to genetic sequences that will be deposited in databases (De Jonckheere et al., 2012). Trophozoites length is 22.5–42.5 and 2.5–5.0 mm in width, are elongated, cylindrical, limax or slug-like in motility by the aid of a single pseudopodia as shown in Figs. 1 and 2; in some cases, extensively branching with 4–5 pseudopodia as observed in samples isolated from water, an example of which can be seen in Fig. 3 with the trophozoite extending lateral projections. Average speed of locomotion has been measured at 36.4 mm/min. Diameter of nucleus is 2–3 mm(Dykova et al., 1997). Transmission electron microscopy (TEM) investigations of V. vermiformis culture stages have demonstrated 6–8 mm trophozoites and scanning electron microscopy (SEM) has shown approximately 6 mm cystic stages (Fouque et al., 2012). V. vermiformis for effective grazing of prey bacteria requires that the bacterial prey be attached to the surface of the free-living amoeba (FLA) as it is known to be a surface feeder. Laboratory models have demonstrated that mere suspension of FLA surface feeder types with prey bacteria is insufficient to initiate feeding. Rather, it promotes starvation response, where encystment is observed in the case of Acanthamoeba castellanii and the formation of round bodies in Vermamoeba. Further, initiation of encystment in a single cell of A. castellanii promotes a cascade of encystment on the surrounding cells as well which suggests cell to cell signaling. Encysted cells transform to trophic stages once introduced to a surface with bacterial prey (Pickup et al., 2007). Cyst diameters are recorded at 6–9.5 mm(Dykova et al., 1997). Cyst wall has two layers (Fouque et al., 2015a). Studies are rare relative to Vermamoeba wall composition. Cyst walls are described as being composed of proteins and low density of glucose

Encyclopedia of Environmental Health, 2nd Edition https://doi.org/10.1016/B978-0-12-409548-9.11005-X 1 2 Vermamoeba vermiformis—Global Trend and Future Perspective

Fig. 1. Micrograph of isolated Vermamoeba vermiformis (MF716853). (A) Trophozoite with extension of cytoplasmic pseudopodia (black arrow) and (B) cystic stage.

Fig. 2 Vermamoeba trophozoite isolated from underground water.

polymer making it unsuitable to be stained by cellulose-specific markers. The bilayer cyst wall contains an endocyst 140 nm thick and an ectocyst measuring the same, which is composed of layers of filamentous material (Fouque et al., 2012). These structures points out to the resistance of this free-living-amoeba against disinfection particularly when it is in the cystic stage. In addition, studies relative to the ultrastructure and signaling pathways, which direct encystment and excystment are lacking; bridging this gap will enhance our knowledge on the inner workings of FLAs and may offer a better understanding in the development of appropriate and effective interventions in as far as disinfection and prevention of spread is concerned as well as the possible interactions in terms Vermamoeba vermiformis—Global Trend and Future Perspective 3

Fig. 3 Vermamoeba trophozoite isolated from well water with pseudopodia extensions. Nucleus is visible at the subterminal end (arrow in black).

of hazard or benefit when introduced to a suitable host. Mature cysts exhibit a natural fluorescence when subjected to wavelengths of 488 nm because of the presence of natural flavins and nicotinamide compounds from without and within the cyst structures (Dillon et al., 2014a). Some mature cyst may demonstrate binucleation (Cabello-Vilchez et al., 2014) and excystment is mediated by the organization of large vesicles proposed to contain hydrolases close to the cyst wall to aid in the transition. Encystation and excystation remains an area of Vermamoeba concern with gaps in research as this FLA is unique compared to Acanthamoeba and Naegleria (Fouque et al., 2015a). An immature cyst has a single nucleus with a single central nucleolus (Dykova et al., 1997). Chromatin bodies between the nucleolus and nuclear membrane have been noted in all stages especially during encystation (Dykova et al., 1997). Studies have shown that 100% encystment takes about 9 h in 25C–37C under alkaline pH (8–9) in moderate osmotic pressure (0.1 mol L 1 KCl). Further, encystment rate is directly proportional to cell concentration. It should be noted that variations of results were observed between environmental samples and reference strains (ATCC 50237) (Trouilhe et al., 2014). Viable strains of Vermamoeba have been isolated from filtered hot springs with recorded temperatures of 41C–53C and pH of 4.90–7.0. This is an evidence that filtration alone is insufficient for decontamination of water samples with this FLA (Solgi et al., 2012). Hence, improved methods of filtration and other disinfection methodologies appropriate to eliminate even cyst forms are of importance.

Epidemiology

Large concentrations of research and collaboration in the study of Vermamoeba are found in Europe and South East Asia and viable trophic and cystic forms have been isolated from a wide array of environmental conditions as well as in a variety of inanimate surfaces and living tissue. Fig. 4 maps and Fig. 5 outlines that Vermamoeba has been isolated from various sources such as natural water sources (Armand et al., 2016; Asiri et al., 1990; Hsu et al., 2011; Mears et al., 2016; Nazar et al., 2012; Tsvetkova et al., 2004; Park, 2016), soil (Cateau et al., 2014a; De Jonckheere et al., 2012), snow (Reyes-Batlle et al., 2015) and even in industrial compost heap, bio aerosols (Conza et al., 2013), and cooling towers (Scheikl et al., 2014). Hospital (Cateau et al., 2009, 2014b; Fields et al., 1990; Lasjerdi et al., 2015; Nahapetian et al., 1991; Pagnier et al., 2015; Henning et al., 2007; Ozcelik et al., 2017; Rohr et al., 1998; Sanden et al., 1992) and household samples (Gaëlle Reteno et al., 2015; Lu et al., 2017) have also been identified and are of concern relative to immunocompromised individuals. Mammals, birds and humans have also contributed to the isolation of this ubiquitous FLA. Human samples contaminated with Vermamoeba are rare and have only been reported so far in published articles with institutions originating from Costa Rica (Cabello-Vilchez et al., 2014), Scotland (Cabello-Vilchez et al., 2014), Spain (Cabello- Vilchez et al., 2014; Lorenzo-Morales et al., 2007), France (Bouchoucha et al., 2016), Iran (Abedkhojaste et al., 2013; Hajialilo et al., 2015), Japan (Inoue et al., 1998), and Peru (Cabello-Vilchez et al., 2014). Interestingly, Vermamoeba has even been isolated from bottled water (Montalbano Di Filippo et al., 2015). Isolation of Vermamoeba from a variety of natural and man-made water sources highlights the main source of spread of this FLA to other surfaces increasing the potential for infection. Vermamoeba demonstrates survival in cold environments and high altitudes like snow collected from mountains to thermotolerant activities of samples recovered from hot springs. Tap (Delafont et al., 2015; Loret and Greub, 2010; Niyyati et al., 2015; Lal et al., 2015), and tank water isolates (Dobrowsky et al., 2016; Qin et al., 2017; Wadowsky et al., 1991) are important concerns as this is indicative of both the level and methodology of sanitation employed by the water treatment facility (Smirnov and Michel, 1999; Thomas et al., 2008; Walochink et al., 2002) as well as the resistance to disinfection exhibited by this FLA. Its isolation in biofilms as well as the endosymbiotic relationship of bacteria, fungi, viruses and virions is of interest in terms of their interactions in biofilms with the amoeba cell. 4 Vermamoeba vermiformis—Global Trend and Future Perspective

Fig. 4 Vermamoeba Research Mapping (VRM) according to countries of origin.

Vermamoeba isolation from animals (Kadlec, 1978; Milanez et al., 2017; Chavatte et al., 2016; Mulec et al., 2016; Dyková and Tyml, 2016; Dykova et al., 2005) provides substantial knowledge in the biological pathway of spread of this organism through animate vectors. The explanation of how this FLA behaves inside its animal host may significantly contribute to literatures of its effective control and subsequently provide more models of infectious process to be observed and compared relatively to human pathogenesis. As human isolates of Vermamoeba have been documented (Inoue et al., 1998; Montalbano Di Filippo et al., 2015) but no clear proof of its exclusivity as the sole causative agent of the infectious or inflammatory process are put forth for the direct role of Vermamoeba as a mere hitchhiker, irritant, infectious agent or biological niche for increased virulence of endosymbionts remains elusive.

Pathogenesis

Vermamoeba isolated from Tilapia kidney tissues have been demonstrated to cause pathogenesis when introduced to other tilapias, gold fishes and carps (Dyková and Tyml, 2016). Highest incidence of proliferation of Vermamoeba in these animals models were observed in the liver, spleen pancreas and mesenteries in the form of granulomas. Carps were found to be most susceptible but mice models for pathogenicity studies failed to provide conclusive results (Dykova et al., 1997). Efforts to test Legionellosis via Vermamoeba vector utilized the inoculation of murine models. In vivo results show that inhalation of Vermamoeba with Legionella pneumophila endosymbiont can potentially cause pulmonary disease (Brieland et al., 1997a). Cell models for cytopathic effects of Vermamoeba have been conducted on keratocyte monolayers. Clinical isolates were tested and after 24 h of incubation with cell cultures, and showed cellular damage. Further, cytopathic products from Vermamoeba also induced cytopathic effects even without cellular contact. These observations are similar with those observed in studies of A. castellanii—the causative agent of Acanthamoeba keratitis (Kinnear, 2003). Vermamoeba has also been discovered from actual keratitis cases (Balczun and Scheid, 2017) as well as simultaneous isolation of both Vermamoeba and Acanthamoeba in a case of severe amoebic keratitis. The patient’s corneal scrapings and contact lens solution were both positive for Acanthamoeba and Vermamoeba cyst and trophozoites (Lorenzo-Morales et al., 2007; Hajialilo et al., 2015; Dyková and Tyml, 2016). Taking into consideration this particular case, it can be hypothesized that endosymbionts present in the Vermamoeba vermiformis—Global Trend and Future Perspective 5

Fig. 5 Vermamoeba Research Mapping (VRM) according to sampling sources. isolated FLAs may also play a role in increasing the gravity of the infection and can also influence the patient’s response to drug treatment or other types of intervention. Contact lens solutions from patients suffering from keratitis have been found positive for Vermamoeba isolates and bacteria such as Enterobacter cloacae, Stenotrophomonas maltophilia, Xanthobacter flavus, Pseudomonas aeruginosa, and Mycobacterium chelonae (Bou- choucha et al., 2016). This manifestation of a variety of bacterial cocultures may either be reflective of environmental or nosocomial contaminants or poor hygiene the patient was employing relative to contact lens care leading to keratitis. At present, there is only one published case of an exclusive isolation of Vermamoeba from a patient’s contact lens solution and storage case. The patient manifested with eye pain, redness, blurred vision, photosensitivity, tearing and a sensation of a foreign body in her eye. The infection was poorly responsive to anti-Acanthamoeba drugs but was resolved with Polyhexamethylene biguanide (PHMB) 0.02% treatment for 2 weeks (Abedkhojaste et al., 2013). This documented case of a single-species-amoeba isolate helps in understanding the pathogenicity that V. vermiformis can confer to a susceptible mammalian host and further identification of similar cases are necessary to establish the spectrum of conditions that may be observed in immunocompetent and in immunocompromised individuals. Hospital ward dust and biofilms have also been shown to harbor Vermamoeba in the same way that medical instruments and slit lamps have been positive for Acanthamoeba T4 genotype. Other isolates were Vahlkampfia, Naegleria, and Echinamoeba (Lasjerdi et al., 2015). Nasal swabs from an HIV patient demonstrated positivity for Vermamoeba with an endosymbiont of pathogenic M. chelonae (Cabello-Vilchez et al., 2014). What is interesting about this report aside from the discovery of yet another endosymbiont of Vermamoeba is that it shows how this FLA can thrive in the nasal mucosa of a mammalian host which leads us to hypothesize that V. vermiformis, a ubiquitous FLA can also be isolated in a variety of biological samples other than soft contact lenses and corneal scrapings. A recent study however, by Abedkhojaste et al. (2013) has isolated Vermamoeba from a cosmetic soft contact lens wearer in Iran highlighting its role as an agent of keratitis identified from a patient using morphological and molecular methods.

Endosymbionts

Various studies and models have shown how V. vermiformis can act as a “Trojan Horse” for pathogenic strains of bacteria the likes of Bacillus anthracis, P. aeruginosa, Legionella spp., Neochlamydia hartmannellae, Waddlia and other Chlamydia-like endosymbionts. This provision of intracellular niche for growth and replication is not limited to bacteria but are also extended to fungal species and viruses (Balczun and Scheid, 2017). Surprisingly, there is no such documentation on published articles yet relatively to preying of Vermamoeba on other FLAs (and FLA stages thereof ). 6 Vermamoeba vermiformis—Global Trend and Future Perspective

Identification of endosymbionts of FLA in particular endosymbionts of Vermamoeba is valuable not only in the perspective of the microbial, fungal, or viral tenacity but in the possibilities of these amoeba-resisting-organisms potential for pathogenicity. Supporting to this, it is of great curiosity to establish if their relationship with a suitable amoebic host influences acquisition of virulence factors and the forces this relationship exert on each organism’s survival, spread, development and evolution (Scheid, 2014). A variety of factors have been suggested to influence the predator-prey interaction between FLA and bacteria. Amoeba ingestion of bacteria can be inhibited by toxic pigments, outer membrane structures and other virulence factors unique for bacteria (Weekers et al., 1993). Furthermore, environmental strains and clinical isolates have been shown to behave differently compared to their laboratory control counterparts (Wadowsky et al., 1991). Hence, the importance of thoroughly understanding the predator-prey relationship not only in the research perspective of endosymbiosis but also on its effects to the environment like the release of nitrogen from soil and possibly water environments as a by-product of the digestion of bacteria and other microbes by amoeba. A cyst-forming FLA is a potential host for endosymbionts and offers a barrier of protection against adverse conditions such as temperatures in extreme, Ultra-violet radiation, ozone, chlorine dioxide, monochloramine and heavy metals like copper and silver (Muchesa et al., 2017). It is clearly demonstrated in several studies that low disinfectant concentrations have limited activity on FLA and allows for the formation of biofilms that in turn increases amoeba and bacteria persistence. The passage of endosymbionts in amoebal cells has potential in the alteration of virulence or antibiotic resistance which in turn may play a significant role in the bacterial survival within an infected host’s macrophages (Muchesa et al., 2017). FLA being hosts to bacteria provides a “biological gym” for pathogens to potentially enhance their disease causing potentials. Further, antibiotic resistant bacteria can exchange genetic materials with other intracellular pathogens in developing novel virulence traits for enhanced survival within macrophages (Rubenina et al., 2017). Although Vermamoeba as a causative agent of a disease state is still debatable, its viable isolation in human diseased tissues suggests a role in the inflammatory process (Muchesa et al., 2017). Hence, the importance of contributing to the growing body of research performed on Vermamoeba and a wider perspective of its implication not only to the environment but also to mammalian and nonmammalian host is necessary.

Bacteria

Modeling pathogenicity studies for V. vermiformis has found it useful in utilizing axenic cultures of Strain CDC 19 deposited as American Type Culture Collection (ATCC 50237). It is particularly useful for protozoan-bacterial interaction research (Fields et al., 1990). Evidence points that a 170-kD Gal/GalNAc is one lectin receptor of V. vermiformis exploited by L. pneumophila for its attachment and invasion and that other proteins and time-dependent tyrosine dephosphorylation are elements also involved in the process (Venkataraman et al., 1997). Legionella spp. has been observed to survive in encysted cells (Armand et al., 2016) and cyst walls as well (Greub and Raoult, 2003), fluorescent in situ hybridization (FISH) can be utilized in the intracellular identification of this bacteria within FLAs (Kuiper et al., 2004). It has been demonstrated that virulence of this bacteria is essential for the optimal intrapulmonary growth in mice models and supports the hypothesis that Vermamoeba potentially enhances bacterial growth and multiplication (Brieland et al., 1997b) and that temperature also plays a role in the endosymbionts replication within FLAs (Buse et al., 2017). Further, infection of mice host with Vermamoeba has been demonstrated to alter immune response which may be detrimental to the resolution of infections that may be caused by its corresponding endosymbiont which in turn may lead to more complicated inflammatory conditions. It has been demonstrated that cycloheximide interferes with the uptake of L. pneumophila (Abu Kwaik et al., 1994). V. vermiformis has also been demonstrated to harbor S. maltophilia, an opportunistic pathogen isolated in hospital water systems. S. maltophilia has also been recovered from amoebal structures after 28 days exposure to harsh conditions demonstrating the role V. vermiformis play in the survival, growth, and spread of this pathogen (Cateau et al., 2014b). In vitro models have shown that P. aeruginosa forms microcolonies restrictive of V. vermiformis grazing and that Escherichia coli and Staphylococcus aureus are favored as prey. However, clinical and environmental strains of the said bacteria behave differently when coinoculated with Vermamoeba (Rubenina et al., 2017; Cateau et al., 2008; Chavatte et al., 2014). N. hartmannellae has a Chlamydia-like life cycle within cells of V. vermiformis. But unlike Chlamydia spp. does not reside inside vacuoles (Horn et al., 2000; Bou Khalil et al., 2016). Vermamoeba harboring this organism grew faster compared to counterparts without the same endosymbionts and suggests possible upregulation of biological processes in the presence of a suitable intracellular resident. Conversely, downregulation of biological processes has also been observed in cocultures of Acanthamoeba spp. and Parachlamydia-related endocytobiont making these interplay of reactions an interesting area for research (Collingro et al., 2004). Relative to this alteration of biochemical processes, Vermamoeba infected with these bacteria have a lost activity for encystation compared to noninfected counterparts that may be significant in either prolonging the life span or making the FLA more susceptible to harsh conditions. This is another area of interest as the same interactions are not observed in Legionella infected Vermamoeba (Henning et al., 2007). Lastly, very interesting is the endosymbiotic relationships shared between organisms and how they ultimately affect these organisms behavior in general and its protein expression in particular such as in the case of V. vermiformis harboring intracellular Chlamydia-like bacteria of N. hartmannellae, which in turn is infected with viruses and acts not only as an endosymbiont but as a bacteriophage endosymbiont of this FLA (Schmid et al., 2001; Michel et al., 2001). Francisella novicida has been modeled in vitro in place of Francisella tularensis to demonstrate Francisella and Vermamoeba interactions. Results show that F. novicida replicates rapidly in Vermamoeba hosts compared to A. castellanii. F. novicida persists Vermamoeba vermiformis—Global Trend and Future Perspective 7 and replicates inside nonacidic vacuoles of the amoebal host compared to being in the cytosol when in mammalian cells which is demonstrative of survival advantages of endosymbionts from exposure to harsh environments. IgIC protein is suspected of being essential for intravacuolar proliferation and interference with phagolysosomal fusion (Santic et al., 2011). It has been demonstrated in this model that V. vermiformis infected with F. novicida behaved the same as those infected with Chlamydia-like bacteria wherein their encystation was delayed and trophic stages were encouraged to persist. Nasal swabs from HIV patients in Lima, Peru have been positive for V. vermiformis along with M. chelonae and encourages further studies in concepts of survival and propagation in the environment and host (Cabello-Vilchez et al., 2014). Mycobacterium bovis, the agent of Bovine tuberculosis survives within encysted cells of Vermamoeba for over 60 days which induces the development of pulmonary tuberculosis when inoculated in Balb/c mice, proof of bacterial viability within the amoebal cell in extended periods of time contributory to potential transmission of pathogenic organisms and causation of disease states to susceptible hosts (Sanchez- Hidalgo et al., 2017).

Fungus

Cocultures of fungi and amoebal supernatant has shown that V. vermiformis has a positive influence in Aspergillus fumigatus filamentation and may contribute to the severity of deep-seated infections with the organism (Maisonneuve et al., 2016). Candida yeast cells can also be internalized by Vermamoeba cells and are important considerations relative to immunocompromised host systems (Barbot et al., 2012). Exophiala dermatitidis and Fusarium oxysporum has been demonstrated to show increased growth potential in the presence of Vermamoeba trophozoites without observable detrimental effects to the FLA (Cateau et al., 2009, 2014a).

Viruses

Faustovirus is a new viral lineage discovered from Vermamoeba in the same manner as other giant viruses have been isolated from Acanthamoeba. These findings contribute to the knowledge Vermamoeba may be playing as viral protein factories within potential mammalian and nonmammalian systems (Gaëlle Reteno et al., 2015). In addition, some viruses may also act as virophages or virions which have been isolated from viral factories found within Acanthamoeba cells. These virions exploit the “host virus” machinery for replication similar to the manner of a bacteriophage (Bekliz et al., 2016). It is in this light that study of Vermamoeba and its endosymbionts are still further growing in that these research should encompass not only bacteria, fungi and viruses but virophages as well. At present, identification of virophages has been more dependent on metagenomics than culture methodologies.

Isolation and Identification of Vermamoeba Sample Collection and Processing of Samples Potentially Containing Vermamoeba Vermamoeba sp. has been successfully isolated from environmental samples through the analysis of subsurface water (Solgi et al., 2012), tap water samples (Wang et al., 2012), and hospital water systems (Maisonneuve et al., 2016). Although an established collection method (Method 1623) of 10 L of water sample for processing from the US Environmental Protection Agency (EPA) exists to guide researchers, there are a number of modifications that have been used in different studies in terms of the amount of water sample collected from sources ranging from 100 mL (Scheikl et al., 2014)to1L(Montalbano Di Filippo et al., 2015). It is important to note, however, that the volume of water sample collected at one time, though ample, may not necessarily mean successful isolation of Vermamoeba or FLAs of interest and that isolation rates may be increased by increasing the number of samples taken in terms of a wider and broader distribution of sampling areas and sampling frequencies from time of the day, days in a week and consideration of weather and seasonal conditions. Generally, there are two phases of processing collected samples namely: concentration (filtration Solgi et al., 2012; Montalbano Di Filippo et al., 2015; Niyyati et al., 2015 and ultracentrifugation Milanez et al., 2017) and cultivation (Pickup et al., 2007; Ozcelik et al., 2017; Abedkhojaste et al., 2013; Dykova et al., 2005; Fouque et al., 2015b; Scheikl et al., 2016) of resulting pellets after concentrating the samples. Cultivation protocols for the isolation of Vermamoeba sp. is relatively almost the same as compared with other FLAs which are cultured in Non nutrient agar lawned with either heat killed (Dykova et al., 1997; Cabello-Vilchez et al., 2014) or live colonies (Pickup et al., 2007)ofE. coli which may serve as food source for the growing trophozoites in the media.

Microscopic and Molecular Protocols for Identification of Vermamoeba Identification of Vermamoeba sp. cystic stages and trophozoite forms may pose a challenge to researchers due to the fact that cystic stages may have morphological similarities to other cystic forms of FLAs. Although a morphologic key devised by Page (Solgi et al., 2012; Dykova et al., 2005) is available as reference to identify one FLA to another through the use of either a regular light microscope (Montalbano Di Filippo et al., 2015; Dykova et al., 2005) it is still highly recommended to use alternative methods such as transmission electron microscopy (Dykova et al., 1997) for morphologic analysis from cultures or the use of in situ hybridization (Dykova et al., 2005) using probes from samples obtained from lesions of patients infected. 8 Vermamoeba vermiformis—Global Trend and Future Perspective

Due to these challenges of identifying Vermamoeba spp. through microscopic methods, it is highly encouraged to utilize molecular techniques in the exact identification of this FLA. Current trends in extracting the DNA of this amoeba includes the use of phenol chloroform (Abedkhojaste et al., 2013; Hajialilo et al., 2015), available DNA extraction kits and Chelex resin (Solgi et al., 2012), More so, a number of specific primer kits are available for use which targeting the 18SrRNA of the protozoan, nonspecific universal primers may also be used for the direct identification of the organism provided that DNA sequencing will be performed.

Control and Disinfection of Vermamoeba Relative to control of Vermamoeba in water samples, it has been shown that both cyst and trophozoites were susceptible to concentrations of chlorine ranging from 2.0–4.0 ppm. Concentrations above 10.0 ppm was shown to be lethal to Vermamoeba cocultured with L. pneumophila as it remained viable in concentrations of 4.0 ppm and further exposure to 60C made all trophic states of the FLA nonviable (Donlan et al., 2005; Dupuy et al., 2016; Kuchta et al., 1993). In vitro studies have demonstrated that German drinking water regulation (10 þ 100 mg/L Ag þ Cu) s insufficient to inactivate V. vermiformis relative to 1:10 silver/copper combination (Rohr et al., 2000). Presence of biofilms in water systems including premise plumbing requires special attention as it increases survival of organisms in its community as FLAs can re-colonize waters rapidly in as early as 24 h after disinfection (Wang et al., 2012; Scheikl et al., 2016). Heating and regular treatment of water with sodium hypochlorite, sodium bromide or isothiazolinones was not sufficient to significantly reduce occurrences of Acanthamoeba and Vermamoeba in water systems (Scheikl et al., 2014). Studies on reference and environmental strains of Vermamoeba show that cysts only became fully inactivated by the following intervention: 15 mg/L of chlorine for 10 min; 60 C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min; this methodology is in accordance to the French Circular relative to Legionella risk prevention in health facilities. In addition, subtilisin (0.625 U/mL for 24 h) has also shown promise in cyst inactivation (Fouque et al., 2015b). Modeling studies on dental unit water lines were also not successful in demonstrating susceptibility of Vermamoeba to commercial disinfectants (Costa et al., 2016). Studies conducted on river supplied water treatment facility recorded the presence of Vermamoeba even in the ozonated, sand- filtered and carbon-filtered water samples. In addition, several species of FLAs were present in the different stages of water treatment along with the presence of free bacteria and endosymbionts as well (Thomas et al., 2008). More interesting is that there was a higher diversity of FLA species isolated from the treatment process compared to the raw river water which suggests FLA colonization of the treatment facility. Amoeba infected with Amoeba-resisting-bacteria were mostly recovered from biofilm samples which suggests that biofilm environments facilitate effective interaction with and uptake of bacteria by FLAs. This study is evidence that even a well- operated treatment plant will not be an absolute barrier to contain these microbes and that continuous study is needed to further improve water treatment methodologies from a wide variety of microorganisms. Solar pasteurization has been shown to reduce gene copies of Vermamoeba and Naegleria spp. in high temperatures of 68C–93C and 74C–93C respectively and that Acanthamoeba spp. is more resistant to heat compared to Vermamoeba (Dobrowsky et al., 2016). There is no established intervention for Vermamoeba found in human hosts. Acanthamoeba trophozoites on the other hand are resolved by miconazole nitrate and natamycin while cystic stages are only sensitive to natamycin in as far as in vitro tests are concerned. Records show however that patients do not readily respond to these antiamoebic drugs (Inoue et al., 1998).

In Vitro and In Vivo Models

In vitro models for Vermamoeba and endosymbiont susceptibility have been modeled using dental unit water line systems (DUWL). This method is also useful in studying polymicrobial biofilms of bacteria, fungi, other FLAs and viruses or virions of interest from within and without the FLAs and bacteria (Dillon et al., 2014a,b; Donlan et al., 2005; Costa et al., 2016). Biofilm models are preferred when observing L. pneumophila interaction and survival within Vermamoeba cells in water systems (Buse et al., 2017). Spiking models are recommended in developing protocols for FLA recovery (Chavatte et al., 2014) and FLA starvation responses can be studied using suspended cells and agar systems with and without bacterial prey (Pickup et al., 2007) in addition, time lapse video, light and scanning electron microscopy are useful tools in cell culture studies like keratocyte-amoeba cocultured observations (Kinnear, 2003) as well as in other studies where the focus is image analysis at set time intervals. In vivo studies have exploited the availability of mice for murine models in demonstrating replication of L. pneumophila inside a host system (Brieland et al., 1997a) and A/J mice models of intratracheal inoculation have been successful in supporting hypotheses for intrapulmonary infection with Legionella strains living as endosymbionts within Vermamoeba (Brieland et al., 1997b). Giant viruses from arthropods can be isolated using V. vermiformis as described by Pagnier et al. (2015) with modifications that Vancomycin 10 mg/mL, Ciprofloxacin 20 mg/mL, Imipenem/Cilastatin 10 mg/mL, Doxycycline 20 mg/ML, and Voriconazole 20 mg/mL were added to the amoebal suspensions to prevent bacterial and fungal contamination (Temmam et al., 2015). Vermamoeba vermiformis—Global Trend and Future Perspective 9

Conclusion Remark

Vermamoeba being isolated in the environment is still a progressively growing body of research. Broader perspectives of its role in environmental ecology and possible direct role in the pathogenesis among nonmammalian and mammalian hosts are still being discovered, hence, the development of more efficient recovery and isolation techniques from environmental and biological samples for culture and molecular identification are necessary. Studies in the area of its viable recovery in biological hosts is still in its infancy and recent discoveries of Vermamoeba as being an ecosystem in itself which provides a suitable amoebological niche to a wide array of endosymbionts as well as furnishing a fortified cellular stronghold for environmental and chemical inclemencies further opens up gaps to be filled by the scientific community. Vermamoeba submits a plethora of wonder for researchers to ponder upon in the perspectives of this FLA as being ubiquitous in the environment ready to be exploited by potential endosymbionts but more so as how this seemingly harmless FLA can be a reliable host for the most drug-resistant bacteria as well as being a possible virulence and evolutionary cohort of a wide selection of prokaryotes, viruses, and virions.

References

Abedkhojaste H, Niyyati M, Rahimi F, Heidari M, Farnia S, and Rezaeian M (2013) First report of Hartmannella keratitis in a cosmetic soft contact Lens wearer in Iran. Iranian Journal of Parasitology 8(3): 481–485. Abu Kwaik Y, Fields B, and Engleberg C (1994) Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila. Infection and Immunity 62(5): 1860–1866. Armand B, Motazedian MH, and Asgari Q (2016) Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods. Parasitology Research 115(1): 63–68. Asiri S, Chinnis R, and Banta W (1990) Potentially pathogenic species of Acanthamoeba and Hartmannella (protozoa: amoebida) in sediment of the potomac river near Washington, DC. Journal of the Helminthological Society of Washington 57(1): 88–90. Balczun C and Scheid PL (2017) Free-living Amoeba as hosts for and vectors of intracellular microorganisms with public health significance. Virus 9(4): E65. Barbot V, Migeot V, Quellard N, Rodier MH, and Imbert C (2012) Hartmannella vermiformis can promote proliferation of Candida spp. in tap-water. Water Research 46(17): 5707–5714. Bekliz M, Colson P, and La Scola B (2016) The expanding family of Virophages. Virus 8: 317. Bou Khalil J, Benamar S, Baudoin JP, Croce O, Blanc-Tailleur C, Pagnier I, Raoult D, and La Scola B (2016) Developmental cycle and genome analysis of “Rubidus massiliensis,” a new Vermamoeba vermiformis pathogen. Frontiers in Cellular and Infection Microbiology 6: 31. Bouchoucha I, Aziz A, Hoffart L, and Drancourt M (2016) Repertoire of free-living Protozoa in contact Lens solutions. BMC Ophthalmology 16(1): 191. Brieland J, Fantone J, Remick D, LeGendre M, McClain M, and Engleberg C (1997a) The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of legionnaires’ disease. Infection and Immunity 65(12): 5330–5333. Brieland J, McClain M, LeGendre M, and Engleberg C (1997b) Intrapulmonary Hartmannella vermiformis: A potential niche for Legionella pneumophila replication in a murine model of Legionellosis. Infection and Immunity 65(11): 4892–4896. Buse HY, Ji P, Gomez-Alvarez V, Pruden A, Edwards MA, and Ashbolt NJ (2017) Effect of temperature and colonization of Legionella pneumophila and Vermamoeba vermiformis on bacterial community composition of copper drinking water biofilms. Microbial Biotechnology 10(4): 773–788. Cabello-Vilchez A, Mena R, Zuniga J, Cermeno P, Martin-Navarro C, Gonzalez A, Lopez-Arencibia A, Reyes-Batlle M, Pinero J, Valladares B, and Lorenzo-Morales J (2014) Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima Peru. Experimental Parasitology 145: S127–S130. Cateau E, Imbert C, and Rodier MH (2008) Hartmannella vermiformis can be permissive for Pseudomonas aeruginosa. Letters in Applied Microbiology 47: 475–477. Cateau E, Mergey T, Kauffmann-Lacroix C, and Rodier MH (2009) Relationships between free living amoebae and Exophiala dermatitidis: A preliminary study. Medical Mycology 47(1): 115–118. Cateau E, Hechard Y, Fernandez B, and Rodier MH (2014a) Free living amoebae could enhance Fusarium oxysporum growth. Fungal Ecology 8: 12–17. Cateau E, Maisonneuve E, Peguilhan S, Quellard N, Hechard Y, and Rodier MH (2014b) Stenotrophomonas maltophilia and Vermamoeba vermiformis relationships: Bacterial multiplication and protection in amoebal-derived structures. Research in Microbiology 165(10): 847–851. Chavatte N, Bare J, Lambrecht E, Van Damme I, Vaerewijck M, Sabbe K, and Houf K (2014) Co-occurrence of free-living Protozoa and foodborne pathogens on dishcloths: Implications for food safety. International Journal of Food Microbiology 191: 89–96. Chavatte N, Lambrecht E, Van Damme I, Sabbe K, and Houf K (2016) Free-living Protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living Protozoa. Veterinary Parasitology 30(225): 91–98. Collingro A, Walochnik J, Baranyi C, Michel R, Wagner M, Horn M, and Aspock H (2004) Chlamydial endocytobionts of free-living amoebae differentially affect the growth rate of their hosts. European Journal of Protistology 40(1): 57–60. Conza L, Pagani S, and Gaia V (2013) Presence of Legionella and free-living Amoeba in compost and bioaerosols from composting facilities. PLoS One 8(7)e68244. Costa D, Girardot M, Bertaux J, Verdon J, and Imbert C (2016) Efficacy of dental unit waterlines disinfectants on a Polymicrobial biofilm. Water Research 91: 38–44. De Jonckheere JF, Gryseels S, and Eddyani M (2012) Knowledge of morphology is still required when identifying new amoeba isolates by molecular techniques. European Journal of Protistology 48(3): 178–184. Delafont V, Samba-Louaka A, Bouchon D, Moulin L, and Héchard Y (2015) Shedding light on microbial dark matter: A TM6 bacterium as natural endosymbiont of a free-living Amoeba. Environmental Microbiology Reports 7(6): 970–978. Dillon A, Achilles-Day U, Singhrao S, Pearce M, Morton G, and Crean SJ (2014a) Biocide sensitivity of Vermamoeba vermiformis isolated from dental-unit-waterline systems. International Biodeterioration and Biodegradation 88: 97–105. Dillon A, Singhrao S, Achilles-Day U, Pearce M, Glyn Morton LH, and Crean SJ (2014b) Vermamoeba vermiformis does not propagate Legionella pneumophila subsp. pascullei in a simulated laboratory dental-unit waterline system. International Biodeterioration and Biodegradation 90: 1–7. Dobrowsky P, Khan S, Cloete T, and Khan W (2016) Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Harmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater. Parasites & Vectors 9(1): 539. Donlan RM, Forster T, Murga R, Brown E, Lucas C, Carpenter J, and Fields B (2005) Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi- species biofilm has reduced susceptibility to disinfectants. Biofouling 21(1): 1–7. Dupuy M, Binet M, Bouteleux C, Herbelin P, Soreau S, and Héchard Y (2016) Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila. FEMS Microbiology Letters 363(5)fnw022. 10 Vermamoeba vermiformis—Global Trend and Future Perspective

Dyková I and Tyml T (2016) Testate Amoeba Rhogostoma minus Belar, 1921, associated with nodular gill disease of rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 39(5): 539–546. Dykova I, Machackova B, and Peckova H (1997) Amoeba isolated from organs of farmed tilapias, Oreochromis niloticus. Folia Parasitologica 44: 81–90. Dykova I, Pindova Z, Fiala I, Dvorakova H, and Machackova B (2005) Fish-isolated strains of Hartmannella vermiformis page, 1967: Morphology, phylogeny and molecular diagnosis of the species in tissue lesions. Folia Parasitologica 52(4): 295–303. Fields BS, Nerad TA, Sawyer TK, King CH, Barbaree JM, Martin WT, Morrill WE, and Sanden GN (1990) Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis. Journal of Protozoology 37(6): 581–583. Fouque E, Trouilhe MC, Thomas V, Hartemann P, Rodier MH, and Hechard Y (2012) Cellular, biochemical, and molecular changes during encystment of free-living amoeba. Eukaryotic Cell 11(4): 382–387. Fouque E, Yefimova M, Trouilhé MC, Quellard N, Fernandez B, Rodier MH, Thomas V, Humeau P, and Héchard Y (2015a) Morphological study of the encystment and excystment of Vermamoeba vermiformis revealed original traits. The Journal of Eukaryotic Microbiology 62(3): 327–337. Fouque E, Hechard Y, Hartemann P, Humeau P, and Trouilhe MC (2015b) Sensitivity of Vermamoeba (Hartmannella) vermiformis cyst to conventional disinfectants and protease. Journal of Water and Health 13(2): 302–310. Gaëlle Reteno D, Benamar S, Khalil JB, Anreani J, Armstrong N, Klose T, Rossman M, Colson P, Raoult D, and La Scola B (2015) Faustovirus,anAsfarvirus-related new lineage of Giant viruses infecting amoebae. Journal of Virology 89(13): 6585–6594. Greub G and Raoult D (2003) Morphology of Legionella pneumophila according to their location within Hartmannella vermiformis. Research in Microbiology 154(9): 619–621. Hajialilo E, Niyyati M, Solaymani M, and Rezaeian M (2015) Pathogenic free-living Amoeba isolated from contact lenses of keratitis patients. Iranian Journal of Parasitology 10(4): 541–546. Henning K, Zoller L, Hauroder B, Hotzel H, and Michel R (2007) Hartmannella vermiformis (Hartmannellidae) Harboured a hidden Chlamydia-like endosymbiont. Endocytobiosis Cell Research 18: 1–10. Horn M, Wagner M, Müller KD, Schmid EN, Fritsche TR, Schleifer KH, and Michel R (2000) Neochlamydia hartmannella gen. nov., sp. nov. (Parachlamydiaceae), an Endoparasite of the Amoeba Hartmannella vermiformis. Microbiology 146(5): 1231–1239. Hsu BM, Huang CC, Chen JS, Chen NH, and Huang JT (2011) Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Research 45(16): 5171–5183. Inoue T, Asari S, Tahara K, Hayashi K, Kiritoshi A, and Shimomura Y (1998) Acanthamoeba keratitis with symbiosis of Hartmannella ameba. American Journal of Ophthalmology 125(5): 721–723. Kadlec V (1978) The occurrence of Amphizoic amebae in domestic animals. The Journal of Protozoology 25(2): 235–237. Kinnear FB (2003) Cytopathogenicity of Acanthamoeba, Vahlkampfia, and Hartmannella: Quantative & qualitative in vitro studies on keratocytes. The Journal of Infection 46(4): 228–237. Kuchta J, Navratil J, Sheperd M, Wadowsky R, Dowling J, States S, and Yee R (1993) Impact of chlorine and heat on the survival of Hartmannella vermiformis and subsequent growth of Legionella pneumophila. Applied and Environmental Microbiology 59(12): 4096–4100. Kuiper M, Wullings B, Akkermans A, Beumer R, and Van Der Kooij D (2004) Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Applied and Environmental Microbiology 70(11): 6826–6833. Lal S, Singhrao SK, Achilles-Day UE, Morton LH, Pearce M, and Crean S (2015) Risk assessment for the spread of Serratia marcescens within dental-unit waterline systems using Vermamoeba vermiformis. Current Microbiology 71(4): 434–442. Lares-Villa F and Hernández-Peña C (2010) Concentration of Naegleria Fowleri in natural waters used for recreational purposes in Sonora, Mexico (November 2007–October 2008). Experimental Parasitology 126(1): 33–36. https://doi.org/10.1016/j.exppara.2010.04.011. Lasjerdi Z, Niyyati M, Lorenzo-Morales J, Haghighi A, and Taghipour N (2015) Ophthalmology hospital wards contamination to pathogenic free living amoebae in Iran. Acta Parasitologica 60(3): 417–422. Lorenzo-Morales J, Martínez-Carretero E, Batista N, Alvarez-Marín J, Bahaya Y, Walochnik J, and Valladares B (2007) Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella. Parasitology Research 102(1): 167–169. Loret JF and Greub G (2010) Free-living amoebae: Biological by-passes in water treatment. International Journal of Hygiene and Environmental Health 213(3): 167–175. Lu J, Buse H, Struewing I, Zhao A, Lytle D, and Ashbolt N (2017) Annual variations and effects of temperature on Legionella spp. and other potential opportunistic pathogens in a bathroom. Environmental Science and Pollution Research International 24(3): 2326–2336. Maisonneuve E, Cateau E, Kaaki S, and Rodier MH (2016) Vermamoeba vermiformis-Aspergillus fumigatus relationships and comparison with other phagocytic cells. Parasitology Research 115(11): 4097–4105. Mears M, Karcs C, Capps D, Warang S, Aslan A, and Eremeeva M (2016) In:Detection of free-living amoebae and Amoeba-resisting Bacteria in beach waters in Southeastern Georgia. Environmental Health Sciences Faculty Presentations 12, Boston, MA: American Society for Microbiology Microbe Annual Conference (ASM). Michel R, Schmid E, Gmeiner G, Muller KD, and Hauroder B (2001) Evidence for bacteriophages within gram-negative cocci—Obligate Endoparasitic Bacteria of Naegleria sp. Acta Protozoologica 40: 229–232. Milanez GD, Masangkay FR, Thomas RC, Ordona MOGO, Bernales GQ, Corpuz VCM, Fortes HSV, Garcia CMS, Nicolas LC, and Nissapatorn V (2017) Molecular identification of Vermamoeba vermiformis from freshwater Fish in Lake Taal, Philippines. Experimental Parasitology 183: 201–206. Montalbano Di Filippo M, Santoro M, Lovreglio P, Monno R, Capolongo C, Calia C, Fumarola L, D’Alfonso R, Berrilli F, and Di Cave D (2015) Isolation and molecular characterization of free-living Amoeba from different water sources in Italy. International Journal of Environmental Research and Public Health 12(4): 3417–3427. Muchesa P, Leifels M, Jurzik L, Hoorzook KB, Barnard TG, and Bartie C (2017) Coexistence of free-living Amoeba and Bacteria in selected south African hospital water distribution systems. Parasitology Research 116(1): 155–165. Mulec J, Dietersdorfer E, Üstüntürk-Onan M, and Walochnik J (2016) Acanthamoeba and other free-living amoebae in bat guano, an extreme habitat. Parasitology Research 5(4): 1375–1383. Nahapetian K, Challemel O, Beurtin D, Dubrou S, Gounon P, and Squinazi F (1991) The intracellular multiplication of Legionella pneumophila in protozoa from hospital plumbing systems. Research in Microbiology 142(6): 677–685. Nazar M, Haghighi A, Taghipour N, Ortega-Rivas A, Tahvildar-Biderouni F, Nazemalhosseini Mojarad E, and Eftekhar M (2012) Molecular identificationofHartmannella vermiformis and Vannella persistens from man-made recreational water environments, Tehran, Iran. Parasitology Research 111(2): 835–839. Niyyati M, Lasgerdi Z, and Loranzo-Morales J (2015) Detection and molecular characterization of potentially pathogenic free-living Amoeba from water sources in Kish Island, Southern Iran. Microbiology Insights 8(S1): 1–6. Ozcelik S, Yunlu O, and Ozpinar N (2017) Isolation and Morphotyping of Acanthamoeba spp. and Vermamoeba spp. from hospital air-conditioning systems. Cumhuriyet Medical Journal 39(1): 369–373. Pagnier I, Valles C, Raoult D, and La Scola B (2015) Isolation of Vermamoeba vermiformis and associated bacteria in hospital water. Microbial Pathogenesis 80: 14–20. Park J (2016) First record of potentially pathogenic Amoeba Vermaoeba vermiformis (Lobosea: Gymnamoebia) isolated from a freshwater of Dokdo Island in the East Sea, Korea. Animal Systematics, Evolution and Diversity 32(1): 1–8. Pickup Z, Pickup R, and Paryy J (2007) A comparison of the growth and starvation responses of Acanthamoeba castellanii and Hartmannella vermiformis in the presence of suspended and attached Escherichia coli K12. FEMS Microbiology Ecology 59(3): 556–563. Qin K, Struewing I, Santo Domingo J, Lytle D, and Lu J (2017) Opportunistic pathogens and microbial communities and their associations with sediment and physical parameters in drinking water storage tank sediments. Pathogens 6(4)E54. Vermamoeba vermiformis—Global Trend and Future Perspective 11

Reyes-Batlle M, Niyyati M, Martin-Navarro C, Lopez-Arenciba A, Valladares B, Martinez-Carretero E, Pinero J, and Lorenzo-Morales J (2015) Unusual Vermamoeba vermiformis strain isolated from snow in mount Teide, Tenerife, Canary Islands, Spain. Novelty in Biomedicine 3(4): 189–192. Rohr U, Weber S, Michel R, Selenka F, and Wilhelm M (1998) Comparison of free-living Amoeba in hot water Systems of Hospitals with isolates from moist sanitary area by identifying genera and determining temperature tolerance. Applied and Environmental Microbiology 64(5): 1822–1824. Rohr U, Weber S, Selenka F, and Wilhelm M (2000) Impact of silver and copper on the survival of amoebae and ciliated protozoa in vitro. International Journal of Hygiene and Environmental Health 203(1): 87–89. Rubenina I, Kirjusina M, Berzins A, Valcina O, and Jahundicova I (2017) Relationships between free-living Amoeba and their intracellular Bacteria. Proceedings of the Latvian Academy of Sciences, Section B: Natural, Exact and Applied Sciences 71(4): 259–265. Sanchez-Hidalgo A, Obregón-Henao A, Wheat WH, Jackson M, and Gonzalez-Juarrero M (2017) Mycobacterium bovis hosted by free-living-amoebae permits their long-term persistence survival outside of host mammalian cells and remain capable of transmitting disease to mice. Environmental Microbiology 19(10): 4010–4021. Sanden G, Morrill W, Fields B, Breiman R, and Barbaree J (1992) Incubation of water samples containing Amoeba improves detection of Legionella by the culture method. Applied and Environmental Microbiology 58(6): 2001–2004. Santic M, Ozanic M, Semic V, Pavokovic G, Mrvcic V, and Kwaik YA (2011) Intra-vacuolar proliferation of F. novicida within Hartmannella vermiformis. Frontiers in Microbiology 18(2): 78. Scheid P (2014) Relevance of free-living amoebae as hosts for phylogenetically diverse microorganisms. Parasitology Research 113(7): 2407–2414. Scheikl U, Sommer R, Kirschner A, Rameder A, Schrammel B, Zweimüller I, Wesner W, Hinker M, and Walochnik J (2014) Free-living amoebae (FLA) co-occurring with Legionella in industrial waters. Protistology 50(4): 422–429. Scheikl U, Tsao HF, Horn M, Indra A, and Walochnik J (2016) Free-living Amoeba and their associated Bacteria in Austrian cooling towers: A 1-year routine screening. Parasitology Research 115: 3365–3374. Schmid E, Muller KD, and Michel R (2001) Evidence for bacteriophages within Neochlamydia hartmannella, an obligate Endoparasitic bacterium of the free-living Amoeba Hartmannella vermiformis. Endocytobiosis and Cellular Research 14: 115–119. Smirnov A and Michel R (1999) New data on the cyst structure of Hartmannella vermiformis page 1967 (Lobosea, Gymnamoebia). Protistology 1(2): 82–85. Smirnov AV, Chao E, Nassonova ES, and Cavalier-Smith T (2011) A revised classification of naked lobose amoebae (Amoebozoa: lobosa). Protist 162(4): 545–570. Solgi R, Niyyati M, Haghighi A, and Nazemalhosseini Mojarad E (2012) Occurrence of Thermotolerant Hartmannella vermiformis and Naegleria spp. in Hot Springs of Ardebil Province, Northwest Iran. Iranian Journal of Parasitology 7(2): 47–52. Temmam S, Monteil-Bouchard S, Sambou M, Aubadie-Ladrix M, Azza S, Decloquement P, Khalil JY, Baudoin JP, Jardot P, Robert C, La Scola B, Mediannikov OY, Raoult D, and Desnues C (2015) Faustovirus-like Asfarvirus in hematophagous biting midges and their vertebrate hosts. Frontiers in Microbiology 6: 1406. Thomas V, Loret JF, Jousset M, and Greub G (2008) Biodiversity of Amoeba and Amoeba-resisting Bacteria in a drinking water treatment plant. Environmental Microbiology 10(10): 2728–2745. Trouilhe C, Thomas V, Humeau P, and Hechard Y (2014) Encystment of Vermamoeba (Hartmannella) vermiformis: Effects of environmental conditions and cell concentration. Experimental Parasitology 145: S62–S68. Tsvetkova N, Schild M, Panaiotov S, Kurdova-Mintcheva R, Gottstein B, Walochnik J, Aspöck H, Lucas MS, and Müller N (2004) The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitology Research 92(5): 405–413. Venkataraman C, Haack B, Bondada S, and Abu Kwaik Y (1997) Identification of a gal/GalNac lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the legionnaires’ disease bacterium. Journal of Experimental Medicine 186(4): 537–547. Wadowsky R, Wilson T, Kapp N, West A, Kuchta J, States S, Dowling J, and Yee R (1991) Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis. Applied and Environmental Microbiology 57(7): 1950–1955. Walochink J, Michel R, and Aspock H (2002) Discrepancy between morphological and molecular biological characters in a strain of Hartmannella vermiformis Page 1967 (Lobosea, Gymnamoebia). Protistology 2(3): 185–188. Wang H, Edwards M, Falkinham J III, and Pruden A (2012) Molecular survey of the occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba hosts in two Chloraminated drinking water distribution systems. Applied and Environmental Microbiology 78(17): 6285–6294. Weekers P, Bodelier P, Wijen J, and Vogels G (1993) Effects of grazing by the free-living soil Amoeba Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on various Bacteria. Applied and Environmental Microbiology 59(7): 2317–2319.