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Review

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

by
Binod Rayamajhee
1,2,*,
Dinesh Subedi
3,
Hari Kumar Peguda
1,
Mark Duncan Willcox
1,
Fiona L. Henriquez
4 and
Nicole Carnt
1
1
School of Optometry and Vision Science, University of New South Wales (UNSW), Sydney, NSW 2052, Australia
2
Department of Infection and Immunology, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur 44700, Nepal
3
School of Biological Sciences, Monash University, Clayton, VIC 3800, Australia
4
Institute of Biomedical and Environmental Health Research, School of Health and Life Sciences, University of the West of Scotland (UWS), Paisley PA1 2BE, UK
*
Author to whom correspondence should be addressed.
Pathogens 2021, 10(2), 225; https://doi.org/10.3390/pathogens10020225
Submission received: 31 January 2021 / Revised: 13 February 2021 / Accepted: 15 February 2021 / Published: 18 February 2021
(This article belongs to the Special Issue New Insights in Acanthamoeba)
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Abstract

:
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.

Graphical Abstract

1. 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 also classified into at least 22 (T1–T22) genotypes based on their 18S rRNA sequences, with species, such as A. castellanii and A. polyphaga, within the T4 genotype frequently associated with corneal infection [12,13,14].
The Acanthamoeba trophozoite feeds on other microbes, such as bacteria, fungi, algae, and viruses, and can carry them intracellularly acting as “Trojan horse” [15,16]. Therefore, Acanthamoeba can act as a vector of potentially pathogenic microorganisms and, hence, play a role in pathogen dissemination as well as acting as a pathogen itself [17,18,19]. Both clinical and environmental isolates of Acanthamoeba harbour pathogenic prokaryotes as endosymbionts [20,21,22]. The term “endosymbiont” has been described as “a regulated, harmonious cohabitation of two nonrelated partners, in which one of them lives in the body of the other”, and a bacterium is considered to be an endosymbiont when it is able to institute a replicative niche within, for example, eukaryotic cells [23]. However, another generic term “endocytobiont” has been coined to name the intracellular microbes that are associated with free-living amoeba to overcome any suggestion that the intracellular microbes might show mutualism, symbiosis, parasitism, phoresy, or zoochory [24,25]. Throughout the remainder of this review, the term “intracellular” will be used to encompass endosymbionts, endocytobionts, and other forms intracellular microbes within Acanthamoeba spp.
The detailed molecular pathways and strategies of intracellular interactions between Acanthamoeba and other microbes are unexplored at present. In a more generalised context, Acanthamoeba shares similar morphological and ultrastructural features to macrophages and they have a similar mechanism of interaction with microbes [26]. Amoeba may possess universal classes of receptors which bind with a wide array of microbial receptors facilitating adhesion and engulfment of a diverse range of microbes, such as Gal/GalNAc on Legionella pneumophila [27] or type III secretion structures on Vibrio parahaemolyticus [28] and E. coli K1 [29,30] (Figure 1). If the engulfed microbes can then escape the normal phagosome-associated feeding pathway, they may exist intracellular [18]. The ability of microbes to set up an intracellular lifestyle in Acanthamoeba and remain viable has been hypothesised to allow them to subsequently live intracellularly in mammalian cells [31,32]. The intracellular survival mechanisms of bacteria in the amoebal cytoplasm differ between species and this, coupled with analysis of phylogenetic lineages of intracellular bacteria, indicates the ability that has developed with time over the microbe’s evolution [33]. For instance, V. cholerae can escape degradation by applying an intricate neutralising program that effectively neutralises changes to the pH, digestive enzyme functions, and the production of reactive oxygen radicals that may otherwise destroy the bacteria [34]. On the other hand, L. pneumophila forms a membrane-enclosed microenvironment within the Acanthamoeba that is produced via the endoplasmic reticulum, membrane transporters, and fusion with other membrane-bound vesicles [35,36]. The intracellular survival and proliferation of bacteria in amoebal cells has been associated with enhanced resistance of bacteria to antimicrobials and increased bacterial pathogenicity [37]. Acanthamoeba containing intracellular bacteria, such as Pseudomonas, Mycobacterium, and Chlamydia, has demonstrated a more rapid cytopathic effect (CPE) in in vitro as compared to isolates without intracellular bacteria [21,38], showing enhanced amoebal pathogenic potential.
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.

2. Results

2.1. 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.

2.2. 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.

2.3. Laboratory Techniques Used for the Isolation and Identification of Intracellular Microbes in Acanthamoeba spp.

Microbial culture, fluorescence in situ hybridization (FISH), microscopy, polymerase chain reaction (PCR), gene sequencing, and gas liquid chromatography were the laboratory techniques used for the identification of Acanthamoeba and associated intracellular microbes [21,22,33,48,50,55,56,61,72,74,76]. Two studies used gas–liquid chromatography to detect cellular fatty acids of intracellular bacteria and the identification was performed using Microbial Identification Inc. protocols (MIDI) (Newark, DE, USA) [44,46]. PCR (33/43, 76.7%), gene sequencing (30/43, 69.8%), and microscopy (transmission and scanning electron microscopy, confocal laser scanning, and phase-contrast microscopy) (29/43, 67.4%) were the most commonly used techniques to identify the amoeba and intracellular microbes, followed by fluorescence in situ hybridization (12/43, 27.9%) (Figure S1) [21,49,52,53,62,68,74,77]. Two studies observed intracellular bacteria in Acanthamoeba cysts [52,80].

2.4. 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 (MgSO4 × 7H2O, sodium citrate dihydrate × 2H2O, Na2HPO4 × 7H2O, KH2PO4, Fe(NH4)2(SO4)2 × 6H2O) [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 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 co-culture [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.

2.5. Species and Genotypes of Acanthamoeba spp.

The most commonly reported genotypes of Acanthamoeba are T4 [11,52,57,64,73,74,76,77,81], followed by T3 [58,69,71,72,75,80], T5 [69,72,80,110], and T2 [33,58,62,74] (Figure S2). A. polyphaga was detected in eight studies [20,33,47,53,54,65,70,78] and A. castellani was observed in five studies [20,33,56,66,70]. A. hatchetti (T11, T4) [20,22] and A. palestinensis (T2, T6) [20,67] were observed in two studies. Additionally, A. culbertsoni, A. astronyxis (T7) [20], A. lugdunesis [41], A. mauritaniensis [42], and Acanthamoeba T7 [58] and T11 [75] strains were also reported by single studies.

2.6. The Types of Microorganisms Commonly Found Inside Acanthamoeba spp.

Bacteria were the most commonly identified intracellular microorganism in Acanthamoeba followed by viruses and fungi (Figure S3). Unidentified bacteria, termed Candidatus, were reported in 1/3rd of included studies [22,33,42,47,50,51,52,53,57,61,65,69,72,77,78,80] and Chlamydia species were detected in 11 studies [21,33,42,49,57,62,64,69,70,76,78]. Five studies found Legionella spp. [21,44,64,75,79], another five studies reported Mycobacterium [21,55,66,78,79] or Pseudomonas spp. [21,71,74,79,81], four studies found Rickettsia spp. [48,77,78,111], three studies detected Cytophaga spp. [46,52,56], and E. coli [73,81] or Stenotrophomonas maltophilia [68,81] were detected in two studies. Burkholderia pickettii [43], Agrobacterium tumefaciens [74], Brevibacillus sp. [81], Flavobacterium sp. [52], Brevundimonas vesicularis, or Microbacterium sp. [81] were also reported in single studies. Three studies only reported the morphology of intracellular “bacteria” present in Acanthamoeba [20,40,41]. An archaea-like organism was detected in the cytoplasm of Acanthamoeba recovered from a potable water reservoir [45].
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].
The presence of more than one intracellular microbe was reported in ten studies [21,22,57,62,65,70,71,78,79,81]. For example, Chlamydia and Legionella have been observed in a clinical isolate of Acanthamoeba, an environmental isolate that harboured Legionella and Mycobacterium [21], and Procabacter and Parachlamydia were found in Acanthamoeba (OEW1) isolated from a saline lake in Austria [57]. A study from Iran reported three intracellular microorganisms, P. aeruginosa, Aspergillus spp. and HAdV in a clinical isolate of Acanthamoeba T4 (ICS7) [81]. A. polyphaga isolated from a keratitis patient hosted four intracellular prokaryotes: Deltaproteobacterium, Alphaproteobacterium, mimivirus Lentille, and the virophage Sputnik 2 [65].

2.7. 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].
Irrespective of the place of isolation, Acanthamoeba hosts many different pathogens [18] but endemically important human pathogens, such as E. coli, Pseudomonas spp. and Mycobacterium spp., were more commonly identified in Acanthamoeba cultured from clinical specimens, whereas giant viruses (mimivirus and Pandoravirus), Legionella spp., and unnamed bacteria of genus Candidatus were often detected in environmental Acanthamoeba. This suggests that most intracellular microbes interact with Acanthamoeba in their natural environment [117]. Acanthamoeba may act as a “Trojan horse” for microbes, providing them with the opportunity to colonise or infect different environments [118]. The ability of Acanthamoeba to host several different intracellular microbes suggests that these may interact with each other and lead to highly complex differences in the pathogenesis of Acanthamoeba [21].

3. 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]. Multiple-species 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.

4. Methods

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

4.1. 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.

4.2. 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.

4.3. 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.

4.4. 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 meta-analysis 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).

4.5. 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.

5. 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.

6. 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.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-0817/10/2/225/s1. Supplementary file I (S1): Table S1: The Newcastle-Ottawa Scale (NOS) for assessing the quality of studies included in this review; Supplementary file II (S2): List of supplementary figures (S1–S4); Supplementary file III (S3): PRISMA 2009 checklist.

Author Contributions

Conceptualization of the study, B.R., N.C., M.D.W., F.L.H.; data selection and extraction, B.R.; analysis, B.R., analysis, editing, M.D.W., F.L.H., N.C., D.S., H.K.P.; writing—original draft preparation, B.R., D.S., M.D.W., N.C., F.L.H.; review and editing; M.D.W., N.C., F.L.H., D.S., H.K.P.; supervision: M.D.W., N.C. and F.L.H. All authors have read and agreed to the published version of the manuscript.

Funding

None of the authors received external funding for this research.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in Table 1, Table 2 and Table 3, Table 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.

Abbreviations

ARMsAmoeba-resistant microorganisms
ARBAmoeba-resistant bacteria
CPECytopathic effect
FLAFree living amoeba
HAdVHuman adenovirus
PYGPeptone-yeast-glucose medium
PRISMAPreferred Reporting Items for Systematic reviews and Meta-Analyses
MATEMultidrug and toxin extrusion
MBPMannose-binding protein
NOSNewcastle-Ottawa Scale
NNANon-nutrient agar
NTMNontuberculous Mycobacterium
PYGPeptone-yeast-glucose
SCGYESerum-casein glucose yeast extract
TSBTryptic soy-yeast extract broth
VBMCViable but non-culturable
WoSWeb of Science

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Figure 1. Representation of the different microorganisms as food of Acanthamoeba and interaction with bacteria. (a) attachment: possible receptor-mediated adhesion of bacteria; (b) entry: ingestion of bacteria using pseudopods and phagocytosis; (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.
Figure 1. Representation of the different microorganisms as food of Acanthamoeba and interaction with bacteria. (a) attachment: possible receptor-mediated adhesion of bacteria; (b) entry: ingestion of bacteria using pseudopods and phagocytosis; (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.
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Figure 2. Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) flow diagram for selection of articles.
Figure 2. Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) flow diagram for selection of articles.
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Figure 3. Laboratory procedures for the isolation and identification of Acanthamoeba and associated intracellular microorganisms from clinical and environmental samples. Adapted from Thomas et al. (2010) [37]. PAS: Page’s Amoeba Saline, NNA: Non-nutrient agar, TEM = Transmission Electron Microscopy, SEM = Scanning Electron Microscope. Created with BioRender.com (accessed on 20 January 2021).
Figure 3. Laboratory procedures for the isolation and identification of Acanthamoeba and associated intracellular microorganisms from clinical and environmental samples. Adapted from Thomas et al. (2010) [37]. PAS: Page’s Amoeba Saline, NNA: Non-nutrient agar, TEM = Transmission Electron Microscopy, SEM = Scanning Electron Microscope. Created with BioRender.com (accessed on 20 January 2021).
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Table
Table 1. Intracellular microorganisms identified in environmental and clinical isolates of Acanthamoeba species.
Table 1. Intracellular microorganisms identified in environmental and clinical isolates of Acanthamoeba species.
Country, Date of StudyAnalysed Sample (Clinical/Environmental)Laboratory InvestigationPositive Samples for Intracellular MicrobesSpecies and Genotypes of AcanthamoebaIdentified Intracellular Microbes
USA, 1993 [20]Clinical (corneal-19, and contact lens-4), environmental specimens (soil, forest detritus, lake and stream sediments, pond water, tree bark, potting soil, 25), and ATCC strains (9)Culture, electron microscopy, staining14 of 57ATCC strains:
A. culbertsoni 30886, 30011, and 30868
A. rhysodes 30973,
A. polyphaga 30871 and 30461
A. astronyxis 30137,
A. hatchetti 30730,
A. palestinensis 30870,
Acanthamoeba strain 30173		Gram-negative rods and cocci and non-acid fast non-motile bacteria
Philippines, 1995 [40]PondCulture, PCR, electronic microscopy1 of 1Acanthamoeba spsGram-negative rod-shaped bacteria, 1.3 × 0.43 µm in size
South Korea, 1997 [41]Contact lens storagePCR, TEM1 of 1A. lugdunesisRod-shaped bacteria, 1.38 × 0.5µm in size
Germany, 1997
[42]		Nasal mucosa of humansCulture, electron microscopy, in situ hybridization2 of 2Acanthamoeba spp. and A. mauritaniensisCoccoid shaped related to Chlamydia spp.; Ca. Parachlamydia acanthamoebae (proposed name for strain Bn9)
Germany, 1997 [43]Wet area of a physiotherapy unitCulture, light, and electron microscopy, biochemical tests1 of 2Acanthamoeba spp. Group IIBurkholderia pickettii (biovar 2)
Germany, 1998 [44]Cold water tap of a hospital plumbing systemCulture, electron microscopy, gas-liquid chromatography1 of 1Acanthamoeba spp. Group II (K62)Legionella-like slender rods
Germany, 1998 [45]Potable water reservoirCulture, electron microscopy1 of 1Acanthamoeba sps Group IIArchaea like (short rod shaped, 1–1.5 μm length) endoparasite
Germany, 1999 [46]Drinking water system of a hospitalCulture, phase contrast and electron microscopy, gas-liquid chromatography, Gram staining, biochemical tests1 of 1Acanthamoeba spp. Group IICytophaga spp. (K69i)
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)3 of 3Acanthamoeba spp. (UWC9 and UWE39); A. polyphaga (HN-3) [20]Ca. Caedibacter acanthamoebae (proposed name); Ca. Paracaedibacter acanthamoebae (proposed name); Ca. Paracaedibacter symbiosus (proposed name)
USA, 1999 [48]Corneal scrapingCulture, Gram and Giemsa staining, confocal laser-scanning microscopy, PCR amplification, sequencing of 16S rRNA gene, EM2 of 2Acanthamoeba species (UWC8 and UWC36)Phylogenetically related to members of the order Rickettsiales branch of the alpha subdivision of the Proteobacteria (99.6% sequence similarity to each other), Ca. Midichloriaceae family in Rickettsiales
USA, 2000
[49]		Clinical (corneal tissues—1), and environmental isolates (soil samples from the USA—1, and sewage sludge from Germany—1)Culture, Giemsa staining, FISH, electron microscopy, PCR, sequencing4 of 4Acanthamoeba spp.Gram-negative cocci, may represent distinct species of Parachlamydiaceae
Ca. Protochlamydia amoebophila (UWE25) [50]
Greece, 2000 [51]Water sample collected from the drip-tray of the air conditioning unit of a hospitalCulture, Gimenez
Staining, microscopy, PCR, 16S rRNA sequencing		1 of 1Acanthamoeba spsCa. Odyssella thessalonicensis’ gen. nov., sp. nov. [gram negative, rod, and motile] (proposed name); Note: The phylogenetic position, inferred from comparison of the 16S rRNA gene sequence, is within the α-Proteobacteria.
Germany, 2001 [52]Drinking water in a hospital, corneal scrapings of a keratitis patients (Germany) and eutrophic lake sediment (Malaysia)Culture, phase contrast and electron microscopy, PCR, 16S rRNA sequencing3 of 3Acanthamoeba spp. T4Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity); Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (<82% 16S rRNA sequence similarity); Ca. Amoebophilus asiaticus (proposed name)
Germany, 2002 [53]Clinical and environmental isolates from the USA and MalaysiaCulture, Gram, Giemsa and DAPI staining, electron microscopy, FISH, PCR, 16S and 23S rDNA-based sequencing6 of 6A. polyphaga strain Page 23 and Acanthamoeba spp.Rod-shaped Gram-negative obligate bacterial endosymbionts, related to the β-Proteobacteria: Ca. Procabacter acanthamoebae’ gen. nov., sp. nov. (proposed name)
France, 2003 [54]Water of cooling towerGram staining, electronic microscopy, genome sequencing1 of 1A. polyphagaMimivirus
South Korea, 2007 [55]Contact lens storage caseCulture, MtDNA RFLP analysis, TEM, PCR, sequencing, AFB, and fluorescent staining1 of 1A. lugdunensisMycobacterium spp.
South Korea, 2007 [56]From the infected corneas of Korean patientsCulture, orcein staining, RFLP, TEM, PCR, sequence analysis of 16S rDNA of endosymbiontsand 18S rDNA of Acanthamoeba4 of 4Strains of Acanthamoeba spp. belonging to the A. castellanii complex T4Caedibacter caryophilus (proposed name); Cytophaga-Flavobacterium-Bacteroides (CFB) phylum
Austria, 2007 [57]LakeCulture, FISH, TEM, PCR, 16S rRNA sequences1 of 1Acanthamoeba sps T4Ca. procabacter sp. OEW1 (proposed name); Parachlamydia acanthamoebae Bn9
Spain, 2007 [58]Tap water samplesCulture, PCR34 of 236Acanthamoeba spp. T2; T3; T4; T6 and T7Human adenoviruses (HadV); serotypes HadV-1, 2, 8, and 37
Germany, 2008 [59]Contact lens and storage case fluidCulture, light and electron microscopy1 of 11. A. triangularis
2. Not yet determined, with polygonal cysts		Pandoravirus inopinatum [60]
Austria, 2008 [33]Soil and lake sediment samples from Austria, Tunisia, and Dominica (N=10)Culture, TEM and confocal laser scanning microscopy, PCR, genotyping, sequencing8 of 10Acanthamoeba spp. (isolates EI1, EI2, EI3, 5a2, EIDS3, and EI6) = T4 and (isolates EI4 and EI5) = T2Parachlamydia sp. isolate Hall’s coccus; Protochlamydia amoebophila UWE25; Ca. Paracaedibacter acanthamoebae (proposed name); Ca. Amoebophilus asiaticus TUMSJ-321 (proposed name); Ca. Procabacter acanthamoebae Page23 (proposed name); Parachlamydia sp. isolate UV-7
South Korea, 2009 [61]Tap waterCulture, TEM and phase-contrast light microscopy, PCR, 16S r DNA sequencing5 of 17Acanthamoeba spp.Ca. Amoebophilus asiaticus (proposed name); Ca. Odyssella thessalonicensis (α-Proteobacteria) (proposed name); Methylophilus spp.
Japan, 2010
[62]		Environmental samples (41 soil samples: 19 river water samples, 4 lake water samples and 2 pond water samples)Culture, PCR, sequencing, FISH, TEM5 of 41Acanthamoeba spp. T2;T4; T6 and T13Rod-shaped belonging to α- and β-Proteobacteria phyla; sphere/crescent-shaped belonging to the order chlamydiales
Protochlamydia; Neochlamydia [63]
USA, 2010 [21]Acanthamoeba isolates (N=37) recovered from the cornea and contact lens paraphernalia of 23 patients, 1 environmental (water) isolateCulture, PCR, sequencing, FISH, TEM22 of 38Acanthamoeba spp.Legionella sp.; Pseudomonas sp.; Mycobacterium sp.; Chlamydia sp.
Spain, 2010 [64]Three different water treatment plantsAxenic culture, sequencing a portion of the 18S rRNA gene for amoeba and specific 16S rRNA gene PCR for endosymbionts5 of 9Acanthamoeba T4 strainChlamydiae; Legionellae
France, 2011 [65]Corneal scraping of AK patient, contact lens storage case liquidCulture, slit-lamp examination, PCR, sequencing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry1 of 1A. polyphagaCa. Babela massiliensis/ Deltaproteobacterium (proposed name); Alphaproteobacterium bacillus; mimivirus strain Lentille; virophage Sputnik 2
USA, 2011 [66]Eye infection, A. castellanii strain Ma (ATCC 50370), culture collectionCulture, light microscopy, PCR, sequencing1 of 1A. castellanii (ATCC 50370)Species of Mycobacterium avium complex (MAC) (M. timonense; M. marseillense and
M. chimaera).
UK, 2011
[67]		Sewage sludgeCulture, PCR, sequencing of Amoeba only1 of 1A. palestinensis (22/25 clones) within the T6 cladeMimivirus-like particles
Germany, 2013
[68]		From biofilm of a flushing cistern in a lavatoryCulture, PCR, sequencing, electron microscopy1 of 1Acanthamoeba spp.Stenotrophomonas maltophilia complex (96.5% sequence similarity)
Japan, 2014 [69]Hot Spring in JapanCulture, FISH, TEM, confocal laser and phase-contrast microscopy, PCR, sequencing1 of 1Acanthamoeba spp. T5Protochlamydia
Austria, 2014 [70]Three environmental samplesAxenic culture, PCR, FISH, sequencing7 of 10Acanthamoeba spp. (closely related to A. castellanii Neff GenBank Acc. U07416, A. polyphaga)Paraceadibacter; Neochlamydia; Protochlamydia; Procabacter; Rickettsiales; Amoebophilus
Brazil, 2015 [71]Seven samples from air-condition units, and five from contact lens casesCulture, FISH, semi nested-PCR, DGGE, sequencing3 of 12Acanthamoeba spp. T3; T4 and T5Paenibacillus spp., Ca. Protochlamydia amoebophila, (uncultured γ-Proteobacterium) (prposed name)
Brazil, 2015 [72]Seven samples from air-condition units, and five from contact lens casesAxenic culture, conventional PCR, amplicon sequencing12 of 12Acanthamoeba spp. T3; T4 and T5Pseudomonas spp.
Japan, 2015 [73]Isolated from a patient with AKCulture, Gram staining, MicroScan autoSCAN-4 system, PCR1 of 1Acanthamoeba strain T4E. coli
Iran, 2015 [74]Recreational water sourcesAxenic culture, staining, PCR, genotyping, microscopy5 of 16Acanthamoeba spp. T4 and T5P. aeruginosa; Agrobacterium tumefaciens
Spain, 2015
[75]		Seventy water samples (three DWTP, three wastewater treatment plants and five natural pools)Culture, PCR, genotyping, sequencing43 of 54Acanthamoeba T3, T4 and T11Legionella spp.
Japan, 2016 [76]Smear samples from University HospitalCulture, PCR, sequencing3 of 21Acanthamoeba spp. T4Protochlamydia spp.; Neochlamydia spp.
Austria, 2016 [22]Corneal scraping of AK patientAxenic culture, PCR, sequencing, FISH, TEM1 of 1A. hatchetti, T4Parachlamydia acanthamoebae; Candidatus Paracaedibacter acanthamoebae (proposed name)
Austria, 2016 [77]Seventy-eight water samples (66 cooling tower water: 2 cooling towers of hospital, 1 cooling tower of company, and 12 tap water)Culture, FISH, real-time PCR, genotyping, and sequencing3 of 53Acanthamoeba spp. T4Paracaedibacter acanthamoebae; Rickettsiales; L. pneumophila
Canada, 2017 [78]Five clinical isolates (human cornea, nasal swab, monkey kidney tissue
Culture) and four environmental isolates (lake sediment, soil, and water reservoir); all ATCC strains		Axenic culture, amplifying and sequencing of bacterial 16S DNA3 of 9A. polyphaga ATCC 30173 and 50495; Acanthamoeba spp. PRA-220Holosporaceae (Rickettsiales); Mycobacterium spp.; Parachlamydia spp.; Ca. procabacter sp. (proposed name)
Malaysia, 2017
[79]		Isolates from air-conditioning outlets in wards and operating theatres (dust particles)Axenic culture, PCR, genotyping, sequencing29 of 36Acanthamoeba spp.Mycobacterium spp. (M. fortuitum, M. massiliense, M. abscessus, M. vanbaalenii, M. senegalense, M. trivial and M. vaccae); Legionella spp. (L. longbeachae, L. wadwaorthii, L. monrovica, L. massiliensis and L. feeleii); Pseudomonas spp. (P. stutzeri; P. aeruginosa; P. denitrificans; P. chlororaphis and P. knackmussi)
Malaysia, 2018 [80]Air-condition (11 isolates), and keratitis isolates (2)Axenic culture, PCR, sequencing, FISH (double), TEM6 of 13Acanthamoeba spp. T3; T4 and T5Ca. Caedibacter acanthamoebae/Ca. Paracaedimonas acanthamoeba and Ca. Jidaibacter acanthamoeba (proposed name)
Iran, 2019 [81]Corneal scrapes and contact lenses isolate of keratitis patientsCulture, light microscopy, gram staining, PCR, sequencing7 of 15Acanthamoeba spp. T4E. coli; Achromobacter sps; P. aeruginosa; Aspergillus sp.; Mastadenovirus sp.; Microbacterium sp.; Stenotrophomonas maltophilia; Brevundimonas vesicularis and Brevibacillus sp.
Key: AFB = Acid Fast Bacilli, ATCC = American Type Culture Collection, AK = Acanthamoeba keratitis, PCR = Polymerase Chain Reaction, TEM = Transmission Electron Microscopy, SEM = Scanning Electron Microscope, FISH =Fluorescence in situ Hybridization, DWTP = Drinking Water Treatment Plant, DAPI = 4′,6-diamidino-2-phenylindole, MtDNA = Mitochondrial DNA, RFLP = Restriction Fragment Length Polymorphism, DGGE = Denaturing Gradient Gel Electrophoresis, Ca. = Candidatus.
Table
Table 2. The types of microbes isolated from Acanthamoeba spp. using different culturing techniques.
Table 2. The types of microbes isolated from Acanthamoeba spp. using different culturing techniques.
Culture TypeSource of AcanthamoebaIdentified Intracellular Organism in AcanthamoebaStudy
Axenic culture on PYG, KCM agar, NNA
(n= 12)		Clinical isolatesMycobacterium avium complex[66]
Escherichia coli[73]
Parachlamydia acanthamoebae and Ca. Paracaedibacter acanthamoebae[22]
Environmental isolatesCandidatus 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.[79]
Clinical and environmental (both) isolatesRickettsiales; Mycobacterium spp.; Parachlamydia spp. and Ca. procabacter sp.[78]
Candidatus spp.[80]
Axenic culture in presence of antibiotics
(n = 3)		Environmental isolatesHuman adenoviruses[58]
Paenibacillus spp.; Ca. Protochlamydia amoebophila; γ-Proteobacterium[71]
Pseudomonas spp.[72]
NNA with live/inactivated/killed bacteria (n= 18)Clinical isolatesE. coli; Achromobacter sps; P. aeruginosa; Aspergillus sps; Mastadenovirus; Microbacterium sps; Stenotrophomonas maltophilia; Brevibacillus sps and Brevundimonas vesicularis[81]
Caedibacter caryophilus and Cytophaga-Flavobacterium-Bacteroides[56]
Environmental isolatesCa. Babela massiliensis, Alphaproteobacterium bacillus, Mimivirus (Lentille), Virophage (Sputnik 2)[65]
Mimivirus-like particles[67]
Stenotrophomonas maltophilia complex[68]
Legionella spp.[75]
Ca. procabacter sp. and Parachlamydia acanthamoebae[57]
Protochlamydia spp. and Neochlamydia spp.[76]
Paracaedibacter acanthamoebae; Rickettsiales; L. pneumophila[77]
Pandoravirus[59]
Parachlamydia sp.; Protochlamydia amoebophila; Candidatus spp.[33]
Candidatus spp.[61]
α- and β-Proteobacteria and chlamydiales[62]
Chlamydiae; Legionellae[64]
Clinical and environmental (both) isolatesGram-negative; rods and coccus; non-acid fast; non-motile[20]
Parachlamydiaceae and Ca. Protochlamydia amoebophila[49]
Ca. Procabacter acanthamoebae’ gen. nov., sp. nov. (proposed)[53]
Legionella; Pseudomonas; Mycobacterium; Chlamydia[21]
Live/inactivated/killed bacteria on NNA/SCGYE/TSB/PYG with antibiotics (n= 7)Clinical isolatesChlamydia spp. and Ca. Parachlamydia acanthamoebae[42]
Rickettsiales spp.[48]
Environmental isolatesArchaea like organism[45]
Gram-negative, rod-shaped bacteria[40]
Paraceadibacter; Neochlamydia; Protochlamydia; Procabacter; Rickettsiales; Amoebophilus[70]
Clinical and environmental (both) isolatesCandidatus spp.[47]
Flavobacterium spp. and Ca. Amoebophilus asiaticus[52]
Key: PYG = Peptone-yeast-glucose, KCM agar = KCM buffer (KCl, CaCl2 and MgSO4.H2O) in Bacto agar, NNA = non-nutrient agar, TSB = Tryptic soy-yeast extract broth, SCGYE = Serum-casein glucose yeast extract.
Table
Table 3. Interactions of fungi or viruses with Acanthamoeba spp.
Table 3. Interactions of fungi or viruses with Acanthamoeba spp.
S.N.MicroorganismsInteraction with Acanthamoeba spp.Reference
1.Fungi
Histoplasma capsulatumCo-culture with A. castellanii (ATCC 30324), cell lysis[106]
C. neoformansIntracellular multiplication in A. castellanii strain 30324[107]
Sporothrix schenckiiCo-culture with A. castellanii (ATCC 30324), cell lysis[106]
Fusarium conidiaCo-culture with different strains of A. castellanii (ATCC 30010, 50492), germinate in amoebal cells[108]
2.Viruses
HAdVCo-culture with different isolates of Acanthamoeba, intracellular survival[58]
Coxsackie virusIntracyst and intracellular survival in a clinical isolate of A. castellanii[109]
MimivirusIntracellular multiplication in A. polyphaga isolated from the water sample of a cooling tower[54]
Table
Table 4. Intracellular microbes identified in Acanthamoeba spp. from clinical or environmental sources.
Table 4. Intracellular microbes identified in Acanthamoeba spp. from clinical or environmental sources.
Sample TypeAnalysed SampleAmoebal HostIdentified Intracellular Pathogenic Microbes in Acanthamoeba spp.Study
Clinical
specimens		Corneal specimensAcanthamoeba spp.Legionella, Pseudomonas;
Mycobacterium;
Chlamydia		[21]
A. castellanii (ATCC 50370)Mycobacterium avium complex (MAC)[66]
A. polyphaga (ATCC 50495)Mycobacterium spp.[78]
Acanthamoeba spp.Rickettsiales[48,111]
Acanthamoeba spp. T4P. aeruginosa; Aspergillus spp.;
Mastadenovirus spp.		[81]
A. castellanii T4Caedibacter caryophilus; Cytophaga-Flavobacterium-Bacteroides (CFB)[56]
A. hatchetti T4Parachlamydia acanthamoebae[22]
Acanthamoeba T4E. coli[73,81]
Human nasal mucosaAcanthamoeba spp.Chlamydia sps; Candidatus Parachlamydia acanthamoebae[42]
A. polyphaga (ATCC 30173)Rickettsiales[78]
Contact lens and fluidAcanthamoeba spp.
(A. triangularis)		Pandoravirus inopinatum[59,60]
Environmental
samples		Tap waterAcanthamoeba (T2, T3, T4, T6, and T7)Human adenoviruses[58]
Recreational water sourcesAcanthamoeba (T4, T5)P. aeruginosa; A. tumefaciens[74]
Water treatment plant, natural poolsAcanthamoeba (T3, T4, T11)Legionella spp.[64,75]
Sewage sludge and cooling tower waterA. palestinensis;
A. polyphaga		Mimivirus[54,67]
Contact lens storage case/liquidA. lugdunensisMycobacterium spp.[55]
A. polyphagaDeltaproteobacterium; Mimivirus Lentille;
Virophage Sputnik 2;
Alphaproteobacterium bacillus		[65]
Soil and lake sedimentA. castellanii and A. royreba T4; A. pustulosa and A. polyphaga T2Parachlamydia sp.;
Protochlamydia amoebophila;
Ca. Paracaedibacter acanthamoebae;
Ca. Amoebophilus asiaticus,
Ca. Procabacter acanthamoebae		[33]
Biofilm of a flushing cistern in a lavatoryAcanthamoeba spp.Stenotrophomonas spp.[68]
Hot SpringAcanthamoeba spp. T5Protochlamydia[69]
Hospital environmentAcanthamoeba spp. T4Protochlamydia spp.; Neochlamydia spp.[76]
Tap waterAcanthamoeba spp.Ca. Amoebophilus asiaticus; α-Proteobacteria; Methylophilus sps[61]
Recreational water sourcesAcanthamoeba spp. T4 and T5P. aeruginosa and Agrobacterium tumefaciens[74]
Lake waterAcanthamoeba sps T4Parachlamydia acanthamoebae;
Ca. procabacter sp.		[57]
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Rayamajhee, B.; Subedi, D.; Peguda, H.K.; Willcox, M.D.; Henriquez, F.L.; Carnt, N. A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection. Pathogens 2021, 10, 225. https://doi.org/10.3390/pathogens10020225

AMA Style

Rayamajhee B, Subedi D, Peguda HK, Willcox MD, Henriquez FL, Carnt N. A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection. Pathogens. 2021; 10(2):225. https://doi.org/10.3390/pathogens10020225

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Rayamajhee, Binod, Dinesh Subedi, Hari Kumar Peguda, Mark Duncan Willcox, Fiona L. Henriquez, and Nicole Carnt. 2021. "A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection" Pathogens 10, no. 2: 225. https://doi.org/10.3390/pathogens10020225

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