A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection

Acanthamoeba, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. Acanthamoeba feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that Acanthamoeba acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within Acanthamoeba following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in clinical isolates of Acanthamoeba, whereas Legionella, adenovirus, mimivirus, and unidentified bacteria (Candidatus) were often identified in environmental Acanthamoeba. Increasing resistance of Acanthamoeba associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in Acanthamoeba, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of Acanthamoeba infections.


Introduction
Acanthamoeba, a ubiquitously distributed free-living amoeba, is known to cause a rare, but potentially sight-threatening, painful, often misdiagnosed, and difficult to treat corneal infection, keratitis, and meningoencephalitis, a fatal infection of the central nervous system (CNS) [1][2][3][4][5]. Acanthamoeba spp. can also cause sinusitis and cutaneous lesions in immunocompromised individuals, such as AIDS patients [3,4,6]. It has two distinct stages in its life cycle, an active phagotrophic trophozoite and a quiescent double walled cyst stage, with the cyst stage enabling the amoeba to remain viable for many years, even in harsh conditions, including chlorine treated water [7,8]. The infective form is the trophozoite stage, although both trophozoites and cysts can gain entry into the human body via different routes, such as debrided skin, cornea, and nasal passages [9]. Based on their morphology, Acanthamoeba species have been broadly classified into three groups (I, II, and III) [10] and pathogenic strains are common of group II [11]. Acanthamoeba species are This systematic review examines the intracellular microorganisms in Acanthamoeba and compares the types of microbial species that were identified in environmental and clinical isolates of Acanthamoeba, and potential impact of intracellular microorganisms on Acanthamoeba keratitis. The major aims of this review are: (a) to determine the laboratory techniques that have been used for the isolation and identification of intracellular microbes in Acanthamoeba spp.; (b) to assess whether different ways of culturing Acanthamoeba affect the types of intracellular bacteria; (c) to examine which microbes are most commonly found inside Acanthamoeba spp.; and (d) to determine whether environmental and clinical isolates of Acanthamoeba harbor the same intracellular prokaryotes.

Results of the Search
The electronic search identified 1331 articles (PubMed = 234, Scopus = 704, WoS = (c) trafficking: prevention of phagosome-lysosome fusion by bacteria helps them evade lysosomal degradation and prevents acidification of the phagosomes [39]; (d) spread: vacuoles containing microbes disperse throughout the amoebal cytoplasm; and (e) replication: intraphagosomal replication of bacteria possible eventual escape into the amoebal cytoplasm.

Results of the Search
The electronic search identified 1331 articles (PubMed = 234, Scopus = 704, WoS = 393). After the removal of duplicates (n = 138), 1193 articles were screened based on their titles and abstracts. The outcome was that 43 studies met the inclusion criteria. Figure 2 depicts the screening process.

Included Studies
In total, 43 studies were analysed. The study location, sample type, laboratory methods used, species and genotypes of Acanthamoeba strains, types of intracellular microbes, and co-occurrence of multiple microorganisms were examined. Brief details of each study included in the analysis are mentioned in Table 1. Germany, 1999 [47] Two clinical isolates (HN-3 and UWC9) and one environmental isolate (UWE39) Culture, PCR, Gram and Giemsa staining, sequencing, electron microscopy, FISH, confocal laser scanning microscopy (CLSM)

Included Studies
In total, 43 studies were analysed. The study location, sample type, laboratory methods used, species and genotypes of Acanthamoeba strains, types of intracellular microbes, and co-occurrence of multiple microorganisms were examined. Brief details of each study included in the analysis are mentioned in Table 1.

Culture Techniques Used to Isolate and Identify Acanthamoeba
Acanthamoeba can be axenically cultured [82], which means a culture in which only a single species is present entirely free from other contaminating organisms, i.e., with no food organisms, or by adding live or dead microbes to stimulate the growth of trophozoites [15,83,84]. Samples (clinical or environmental) are cultured on non-nutrient agar (NNA) covered with bacteria where amoebae graze and move away from the inoculation point in order to recover the symbiont with its natural amoeba host [85]. Axenic culture medium that supports Acanthamoeba growth consists of protease peptone, yeast extract, glucose (PYG), and inorganic salts ( [86,87]. A wide range of bacteria have been used in co-culture with Acanthamoeba. The most common microbes used to culture Acanthamoeba are E. coli, Klebsiella aerogenes [88][89][90] and Enterobacter spp. (E. cloacae and E. aerogenes) [8,25,59] on NNA or in saline [83] (Figure 3). It is not entirely clear why E. coli or K. aerogenes are the most commonly used as food supplement for culturing Acanthamoeba spp. There are only a few studies examining whether Gram negative or Gram positive are preferred or whether bacterial preference is dependent on amoebal species or genotypes [88]. One such study has shown that Acanthamoeba grows better on E. coli, Salmonella enterica serovar Typhimurium, or Bacillus subtilis than Enterococcus faecalis or Staphylococcus aureus [91].  The bacteria used are commonly heat-killed [86,92] or heat-inactivated [56,62] and spread upon NNA plates [70]. The use of bacteria, even dead bacteria, to grow Acanthamoeba trophozoites could potentially affect the types of intracellular microbes that can be grown from the Acanthamoeba. Twelve studies have examined the presence of intracellular bacteria using axenic culture [22,43,46,51,66,69,71,72,[78][79][80], where three studies [58,71,72] have used antibiotics (streptomycin, penicillin, and gentamicin) in PYG to grow amoebae axenically, 18 studies have used NNA with live/inactivated or killed bacteria (E. coli, E. cloacae, S. cerevisiae, E. aerogenes), followed by axenic culture, to recover the intracellular microbes harbouring Acanthamoeba [20,21,49,53,56,57,59,61,62,64,65,67,68,[75][76][77]81] and antibiotics (penicillin, streptomycin, ampicillin, and amphotericin B) were added in culture media (NNA, TSB, SCGYE, PYG) to make the growth contamination free and axenic in another seven studies [40,42,45,47,48,52,70] (Table 2). Some studies have used PYG without inorganic salts to maintain axenic growth of amoeba [69,72]. In the absence of established method for the recovery and identification of intracellular microbes of amoeba, different methods have been used to cultivate intracellular microorganisms carrying Acanthamoeba, which has shown inconsistent results. Pathogenic bacteria, such as Mycobacterium spp. [55,66,79] and Pseudomonas spp. [72,74,79], were often detected by axenic culture technique, whereas pathogenic intracellular bacteria belonging to the genera Legionella, Pseudomonas, Mycobacterium, and Chlamydia in clinical isolates of Acanthamoeba were detected by culturing on NNA pre-seeded with heat killed E. coli followed by axenic culture in 1X Page's saline solution [21]. Ten studies have used antibiotics at some point of cultivation to maintain the axenic culture and they have reported limited intracellular microorganisms as compared to studies grown Acanthamoeba on NNA supplemented with bacteria, where phylogenetically varied intracellular bacteria were re- The bacteria used are commonly heat-killed [86,92] or heat-inactivated [56,62] and spread upon NNA plates [70]. The use of bacteria, even dead bacteria, to grow Acanthamoeba trophozoites could potentially affect the types of intracellular microbes that can be grown from the Acanthamoeba. Twelve studies have examined the presence of intracellular bacteria using axenic culture [22,43,46,51,66,69,71,72,[78][79][80], where three studies [58,71,72] have used antibiotics (streptomycin, penicillin, and gentamicin) in PYG to grow amoebae axenically, 18 studies have used NNA with live/inactivated or killed bacteria (E. coli, E. cloacae, S. cerevisiae, E. aerogenes), followed by axenic culture, to recover the intracellular microbes harbouring Acanthamoeba [20,21,49,53,56,57,59,61,62,64,65,67,68,[75][76][77]81] and antibiotics (penicillin, streptomycin, ampicillin, and amphotericin B) were added in culture media (NNA, TSB, SCGYE, PYG) to make the growth contamination free and axenic in another seven studies [40,42,45,47,48,52,70] (Table 2). Some studies have used PYG without inorganic salts to maintain axenic growth of amoeba [69,72]. In the absence of established method for the recovery and identification of intracellular microbes of amoeba, different methods have been used to cultivate intracellular microorganisms carrying Acanthamoeba, which has shown inconsistent results. Pathogenic bacteria, such as Mycobacterium spp. [55,66,79] and Pseudomonas spp. [72,74,79], were often detected by axenic culture technique, whereas pathogenic intracellular bacteria belonging to the genera Legionella, Pseudomonas, Mycobacterium, and Chlamydia in clinical isolates of Acanthamoeba were detected by culturing on NNA pre-seeded with heat killed E. coli followed by axenic culture in 1X Page's saline solution [21]. Ten studies have used antibiotics at some point of cultivation to maintain the axenic culture and they have reported limited intracellular microorganisms as compared to studies grown Acanthamoeba on NNA supplemented with bacteria, where phylogenetically varied intracellular bacteria were repeatedly detected. In addition, axenic culture has been frequently used for clinical specimens (5/8) and NNA with pre-seeded bacteria was preferred to culture environmental samples (22/26). Four serotypes of Adenovirus (Ad1, Ad2, Ad8, and Ad37) were detected in water-isolated Acanthamoeba by growing amoeba in PYG with gentamicin (50 µg/mL) [58].
The co-culture of environmental samples with symbiont-free Acanthamoeba as a surrogate host is being used as a new method to grow and recover facultative or obligate intracellular bacteria [93][94][95], but this method is not appropriate for isolating symbiont bacteria together with natural host.
Some bacteria have been examined for their ability to survive co-culture with Acanthamoeba. S. aureus can grow within A. polyphaga strain [91]. Shigella dysenteriae and S. sonnei were able to survive in co-culture with A. castellanii for >3 weeks [96] and mycobacterial strains related to M. intracellulare and M. avium for six years without any amoebal cytopathic effects [55]. Co-culture of C. jejuni with amoebal cells resulted in longer survival times as compared to bacteria grown alone [97]. C. jejuni and L. pneumophila were able to be resuscitated from a viable-but-nonculturable (VBNC) state when co-cultured with A. polyphaga or A. castellanii, respectively [97,98]. Acanthamoeba co-culture has been used to enrich low bacterial concentrations of four Campylobacter species, C. jejuni, C. lari, C. coli, and C. hyointestinalis [99]. VBNC P. aeruginosa can become culturable and active within 2 h of Acanthamoeba ingestion [100]. In vitro studies have shown A. castellanii can act as an important environmental reservoir of highly infectious bacteria, such as Francisella tularensis and V. cholerae [101,102]. Furthermore, V. cholerae survives within the contractile vacuole of amoeba, even upon the encystment and F. tularensis grows faster in co-culture with amoeba when compared to bacteria grown alone and causes rapid amoebal encystment [103]. Similarly, viable and intact growth of Helicobacter pylori is increased when co-cultured with A. castellanii [104]. Spores of a virulent B. anthracis (Ames strain with both pX01 and pX02 virulence plasmids, and Sterne strain with only pX01), an agent of bioterrorism, have shown a 50-times increase in spore count after 72 h of co-culture with A. castellanii. In addition, the spores were germinated within phagosomes of amoeba, with the Sterne strain showing less growth [105]. Pathogenic bacteria, such as A. baumannii, K. pneumoniae, and E. coli have been recovered from water samples by A. polyphaga coculture [93]. Acanthamoeba also promotes the survival and growth of fungi and viruses ( Table 3), suggesting that Acanthamoeba can act as an environmental incubator for medically important prokaryotes and fungi. Escherichia coli [73] Parachlamydia acanthamoebae and Ca. Paracaedibacter acanthamoebae [22] Environmental isolates Candidatus spp. [51] Protochlamydia [69] Burkholderia pickettii (biovar 2) [43] Cytophaga spp. [46] Mycobacterium spp. [55] P. aeruginosa and Agrobacterium tumefaciens [74] Mycobacterium spp. and Pseudomonas spp.
Giant mimivirus was detected in three studies [54,65,67], and human adenovirus (HAdV) was isolated in two studies [58,81]. The virophage sputnik 2 [65] and pandoravirus [59] were detected in the contact lens of AK patient in one study. Aspergillus was found in Acanthamoeba recovered from corneal scrapes and contact lenses of a keratitis patient in one study [81].

Differences between the Intracellular Prokaryotes Found in Environmental and Clinical Isolates of Acanthamoeba
Twenty-six studies (60.5%) analysed environmental samples that were collected from soil, sewage sludge, water treatment plants, household tap water, recreational water sources, air conditioning units, hospital areas, such as operating theatres, and contact lens storage cases. Eight (18.6%) studies processed specimens from patients, such as nasal or mucosal swabs, corneal scrapes/swabs or tissue, and AK patient's contact lenses, and these were grouped as clinical samples. Another nine studies (20.9%) examined both types of samples ( Figure S4 and Table 1).
Pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in Acanthamoeba strains that were cultured from clinical specimens [21,66,73,81] ( Table 4). Acanthamoeba spp. obtained from the corneas of patients contained obligate intracellular bacteria of the order Rickettsiales [48,111], E. coli [73], Pseudomonas, Chlamydia [21], Caedibacter caryophilus and Cytophaga-Flavobacterium-Bacteroides (CFB) [56]. The presence of bacteria in Acanthamoeba has been shown to exacerbate keratitis [21,112] and influence the virulence, pathogenicity, and susceptibility of keratitis causing amoeba to therapeutic drugs [55,75]. Chlamydia was observed in Acanthamoeba isolated from the nasal mucosa of volunteers [42] and presence of Pandoravirus inopinatum was confirmed in Acanthamoeba strain recovered from pieces of contact lenses worn by a keratitis patient [59,60].
Acanthamoeba carrying mimivirus and Legionella spp. were isolated from environmental samples that were collected from air-conditioning units, water treatment plants, and sewage sludge [44,54,64,67,75]. Contact lens cases, often cultured when a keratitis case presented for treatment, have been a rich source of intracellular microbes. Mimivirus strain Lentille, Sputnik 2 [65] and Mycobacterium sp. [55] have been isolated from contact lens storage cases. Even though contact lens cases are frequently exposed to disinfectants, several studies have shown that these disinfectants often have poor activity against Acanthamoeba spp. [113][114][115]. Hospital floor and sink swabs were found to be positive for Acanthamoeba with Chlamydia (14.3%) showing the possibility of pathogen transmission via amoeba in the hospital setting [76]. Four different serotypes of human adenovirus (HAdV-1, 2, 8, 37) were found in 14.4% (34/236) of amoeba isolated from tap water [58]. P. aeruginosa and A. tumefaciens were detected in Acanthamoeba strains cultured from recreational water samples [74]. Acanthamoeba trophozoites and cysts are highly resistant to disinfectants used to decontaminate water supplies and the intracellular bacteria may be protected from these external disinfectants [37,74,116].

Discussion
This study systematically analysed 43 published studies assessing the reported intracellular microorganisms that were associated with clinical and environmental isolates of Acanthamoeba. PCR followed by gene sequencing and microscopy were the most common laboratory techniques used to identify the intracellular microbes. Potentially pathogenic bacteria, such as Mycobacterium spp., P. aeruginosa, Rickettsiales, and E. coli, were often detected in clinical isolates, while Legionella, human adenovirus, mimivirus, and uncategorised bacteria (Candidatus) were found in environmental isolates. It appeared that the niche from which Acanthamoeba had been isolated affected the types of intracellular microbes present, or perhaps affected the ability of particular Acanthamoeba strains to cause infections. This latter hypothesis is presented based on previous investigations that domestic water supplies and contact lenses that are exposed to water are risk factors for Acanthamoeba keratitis [5,[119][120][121]. This suggests that water is the source of the infecting Acanthamoeba [122] and, perhaps, those strains that harbour particular intracellular microbes are more able to instigate corneal (or other) infections [21]. However, not all Acanthamoeba isolated from infections have been shown to harbour intracellular microbes, perhaps because their presence has not been analysed. Alternatively, the Acanthamoeba may expel resident intracellular microbes during the infectious process. These hypotheses require scientific investigation.
NNA with live/heat-inactivated/killed E. cloacae/E. coli was the most common method (25/43) used for the recovery and identification of Acanthamoeba associated microorganisms [21,33,56,68]. A higher proportion of clinical specimens were cultivated using axenic (PYG, NNA, KCM agar) media, while NNA with bacteria was often used to culture environmental samples. Environmental samples may consist of more promiscuous microbes, thus the culture media with Acanthamoeba could enhance the recovery and isolation of intracellular bacteria [95]. The use of different bacterial strains to cultivate amoebal trophozoites could affect the intracellular bacteria that can be recovered from the Acanthamoeba since different bacteria affect trophozoite growth and encystment [83]. In addition, antibiotics have been used to eliminate live bacteria for the axenic cultivation of Acanthamoeba. However, this review supports that use of antibiotics in culture media to grow clinical or environmental Acanthamoeba axenically could inhibit amoebal symbionts and limits the recovery of multiple intracellular bacteria. Therefore, before the adaptation to axenic growth, Acanthamoeba spp. should be sub-cultured several times on NNA plates that were covered with heat-killed E. coli [70], even though Acanthamoeba may grow better with live bacteria than heat killed [83]. The use of live E. coli tolC knockout mutants on NNA without antibiotics improved the axenic growth of Acanthamoeba spp. and these amoebae had phylogenetically distinct intracellular bacteria [70]. There is a definite need to understand whether the food preferences of Acanthamoeba depend on its resident sites/species/genotypes or intracellular microbes or change the intracellular community of microbes. Information such as preference for bacterial consumption on growth of amoeba, time for cyst formation, and intracellular survival of bacteria during the cultivation of Acanthamoeba have not yet been reported. These dynamics of Acanthamoeba-bacteria interaction should be taken into consideration in future studies.
Phylogenetically unrelated intracellular microbes were found within the same isolate of Acanthamoeba in ten studies. The diversity of intracellular microbes suggests that their ability to exploit Acanthamoeba as a host has developed continually, independent of the phylogenetic lineage [31]. Intracellular microbes can be either in a stable or transient association. Long-term stable interactions have been observed between Acanthamoeba and α/β-Proteobacteria, chlamydiae, M. avium subsp. paratuberculosis, and Bacteroidetes [51,52,123]. However, amoeba can release intracellular microbes in suitable environments [124]. Transient association has been reported for bacteria, such as E. coli O157:H7, L. pneumophila, among others [39,125]. Intracellular survival of enterohaemorrhagic E. coli O157:H7 in A. castellanii was reduced by Shiga toxins (Stx) that were produced by the bacterium [125]. Co-occurrence of phylogenetically different bacterial species in Acanthamoeba can provide an opportunity for lateral gene transfer between intracellular bacteria [57,126]. Multiplespecies association within the same host cell poses challenges to all intracellular microbes, such as competition for nutrients obtained from the host cell, while the interplay between intracellular microbes needs to be balanced to ensure the stability of the association [57]. In depth biochemical and genomic analysis are needed in future research to understand the details of the interactions.
Intracellular microbes have been detected in Acanthamoeba isolates that belong to genotypes T2-T7, T11, and T13 [33,47,56,58,62,69,75], whether the occurrence of intracellular microbial strains is, in some way, dependent with amoebal genotypes is still an unanswered question. Acanthamoeba hosts for a wide range of microbial species that can presumably, and especially if they are permanent residents, resist phagocytosis, survive, multiply, and endure intracellularly [127]. Whether this can train these intracellular bacteria to survive in other cells, such as human macrophages [31,128,129], perhaps by the exchange of genes with other intracellular microbes [130] or by genetic mutation requires further investigation. This hypothesis is further supported by Chlamydia species, which use the same strategies to interact with various different host cells and that likely evolved years ago during interaction with primitive unicellular eukaryotes [31]. From a clinical viewpoint, a better understanding of molecular mechanisms by which pathogenic bacteria can resist amoebal phagocytosis may allow for the design of future antibiotics and vaccines in the treatment of intracellular human bacterial pathogens.

Methods
The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were followed for this systematic review [131].

Search Strategy and Data Sources
A systematic search was conducted using three electronic databases, PubMed (Medline), Scopus, and Web of Science (WoS), to identify peer-reviewed articles providing information on the types of intracellular microbes associated with Acanthamoeba spp. The literature search was performed using the key terms, "Free-living amoeba" OR "FLA" OR "Acanthamoeba" AND "Bacterial endosymbiont"/"Bacterial endocytobiont" OR "Intracellular Acanthamoeba Endosymbiosis" OR "Amoeba symbiosis" OR "Amoeba-resisting bacteria" as Combinations of Medical Subject Headings (MeSH). This results in searches of articles containing the words 'Acanthamoeba' AND "Endosymbiont"/"Endocytobiont" OR "Acanthamoeba endosymbiosis" OR "Intracellular" OR "Symbiosis" OR "Free-living amoeba" OR "FLA" in their titles and/or abstracts. Additionally, a snow-ball sampling approach was applied while using the reference lists of the selected articles to expand the search. The search was limited to studies that were published in English language and full text articles published between 1 February 1993 to 30 July 2019.

Inclusion Criteria
For an article to be included in this study, it had to be peer-reviewed, available in full text, with its primary objective to isolate and identify intracellular microbes in clinical or environmental isolates of Acanthamoeba spp. However, case reports of Acanthamoeba with symbionts were included. A narrative review was performed for all of the selected studies.

Exclusion Criteria
Articles that were published in languages other than English, conference abstracts, institutional protocols, other review papers, in vitro studies on the co-culture of Acanthamoeba species with bacteria, or other microorganisms for the analysis of symbiosis and isolation of intracellular microbes from amoeba other than Acanthamoeba were excluded from the study. Additionally, the coincidental finding of Acanthamoeba and microbes in the same sample, but with no evidence of the other microbes being intracellular, were not included in this study.

Data Abstraction, Quality Assessment, and Appraise Risk of Bias in Individual Studies
At first, two members of the review team screened all of the articles, as per the inclusion and eligibility criteria following PRISMA guidelines and excluded inappropriate articles after consultation with the other authors. Following the database search, studies were pooled and uploaded sequentially into EndNote version X9 (Clarivate Analytics, Philadelphia, PA, USA), then duplicate studies were removed from the list. The authors reviewed a selection of the articles to verify the selection methodology. Any discrepancies between the reviewers were resolved by consensus discussion amongst all of the reviewers. Variables of interest in the included studies were laboratory techniques that were used for the identification of microorganisms, detection and types of Acanthamoeba and associated intracellular microbial species, study location, type of sample analysed (clinical or environmental), co-occurrence of multiple intracellular microbes within a Acanthamoeba cell, and sequence similarity of detected microbes with reference strains.
The potential risk of bias was assessed with a raw score of quality, as per the Newcastle-Ottawa Scale (NOS) guidelines (adapted for cross-sectional and observational studies) for the appropriateness and aims of the study, method of sample collection, and laboratory identification of Acanthamoeba and intracellular microbes [132]. A final score was assigned to each study after consensus between the reviewers. NOS scores can vary from 0 to 9, and studies, with an average score of ≥6 were included for this review (Table S1) [133]. A metaanalysis of the studies was not performed due to a high level of heterogeneity. Therefore, a systematic analysis was performed. Relevant data were extracted from each study in customised datasheets. Because of the diversity in variables in each study, the assessment scale was primarily based on the methodological quality, Acanthamoeba identification and evidence of intracellular microbes. Figures were created using Origin Lab, Version 2018 (Northampton, MA, USA).

Outcome Measurements
The main outcome measure of this review was the types of intracellular microbes that were identified dwelling in Acanthamoeba species. The secondary outcome measures were the effect of culture techniques on the types of intracellular microbes recovered from Acanthamoeba and the type of intracellular microbes from environmental and clinical sources.

Conclusions
This study systematically reviewed articles on the types of intracellular microorganisms in Acanthamoeba. Acanthamoeba acts as an incubator and carrier of a wide range of microorganisms. The niche or home of the Acanthamoeba appears to affect the types of intracellular microbes. Chlamydia spp., E. coli, Rickettsiales, Pseudomonas spp., and Mycobacterium spp. were the most commonly reported microbes in Acanthamoeba that were cultured from clinical specimens and Legionella, human adenovirus, mimivirus, and bacteria of Candidatus group were detected in environmental Acanthamoeba. Human macrophage and Acanthamoeba share significant cellular and functional features, particularly phagocytic activity, so amoebal cells might train and serve as a preparatory arena for the pathogens to onset diseases in mammalian cells. Molecular-based future studies are expected to assess the microbiome composition residing in Acanthamoeba to view the role of amoeba as a universal host and evolutionary trigger of phylogenetically varied microorganisms.

Limitations of the Study
The major limitation of this review was the lack of meta-analysis due to heterogeneous variables among the included studies. Although the study used multiple search engines using keywords, the query string may not have short-listed all the relevant studies given the disparity in terminology, such as "endosymbiont", "endocytobiont", "endosymbiosis", "amoeba symbiosis", "intracellular bacteria", and "amoeba-resisting bacteria". Additionally, the use of different laboratory techniques to identify the intracellular microbes in the included studies may have biased the reported microbes. Many studies applied protocols to isolate and identify particular prokaryotes, rather than assessing the whole microbiome residing in Acanthamoeba, which may not represent all of the microorganisms present within the amoebal cell. This suggests the use of deep sequencing technique could help to identify the composition of amoebal microbiome.

Data Availability Statement:
The data presented in this study are available in Tables 1-3 and S1, and Figures S1-S4.
Acknowledgments: Authors acknowledge the assistance of an academic librarian of the University of New South Wales (UNSW), Sydney, Australia for guiding the search strategy in the databases. B.R. is recipient of the Tuition Fee Scholarship (UNSW, Sydney) for his doctor degree, with which this review was completed.

Conflicts of Interest:
The authors declare no conflict of interest.