Acanthamoeba

Species, Morphology, Life Cycle, Culture, Disease


Acanthamoeba is a genus of amoebae consisting of a number of species that are pathogenic in nature. They are ubiquitous in nature and are therefore widely distributed across the environment (fresh water, soil, etc). 


Examples of include:


  • A. polyphaga
  • A. astronyxis
  • A. rhysodes
  • A. lenticulat
  • A. divionensis



Protists (Protozoa)

Acanthamoeba belong to the subkingdom protozoa of the kingdom protista. As such, they belong to a group of unicellular organisms known as protists, which are eukaryotic organisms that do not fit in the other eukaryotic kingdoms. 


Some of the other eukaryotic kingdoms include:


  • Plant kingdom
  • Animal Kingdom
  • Fungi


* While members of this genus share various characteristics with other eukaryotes, they are not plants, animals or fungi.


Acanthamoeba polyphaga protozoa, CDC/ Catherine Armbruster; Margaret Williams acquired from Public Health Image Library (Website)Acanthamoeba polyphaga protozoa, CDC/ Catherine Armbruster; Margaret Williams acquired from Public Health Image Library (Website)


Eukaryotes

As eukaryotic organisms, cells of genus Acanthamoeba are organized into complex structures enclosed in a cell membrane. The nucleus of these cells is enclosed in a double layered membrane known as the nuclear envelope similar to that of other higher eukaryotic organisms.


Some of the other membrane bound organelles found in these cells include:



Habitat

(distribution of Acanthamoeba in the environment)

Species of the genus Acanthamoeba have been isolated in a wide variety of environments; both natural and man-made. For instance, while this specie can be found in such natural environments as water ponds, mud and streams (natural environments) they have also been identified in various factory discharges, air-conditioners, cooling towers as well as ventilation ducts among many others.

Apart from such common environments as oceans, beaches and soil, these species have also been isolated in a number of more extreme environments thus making the group an important focus for researchers.

One of the best examples of this was the isolation of these organisms in the Antarctica; an environment with extremely low temperatures and dry winds. This discovery proved that the organisms are present in virtually all environments across the world. 


Members of this genus have also been found in:


  • Dead and decaying animals
  • Droppings of such birds as pigeons
  • The digestive system of some reptiles


Morphology/Structure

Studies have identified more than 20 species of genus Acanthamoeba that are divided into three main groups based on their size and cyst morphology.

These include:



Group 1 Species

Species classified as group 1 are characterized by a large cyst (over 18um) and rounded ectocyst. Trophozoites of group 1 Acanthamoebas are also larger in size and have been shown to range from 25 to 35 um in size. While the cyst size has played an important role of identifying group 1, they have also been associated with the following characteristics:

They form ectocysts (outer wall) and endocysts (inner walls) that have a clear separation

Group 1 species are largely environmental organisms and cause little human and animal infections compared to the other groups.


Group 1 consists of 5 species including:

 

  • A.  astronyxis
  • A.  echinulata
  • A.  tubiashi
  • A.  comandoni



Group 2 Species

Group 2 consists of about 10 species. Compared to Group 1 species, species of Acanthamoeba in Group 2 have a smaller cyst (and smaller trophozoites) while the endocyst varies in shape.

Unlike Group 1 species, this Group has also been shown to cause a majority of human infections.

Some of the other characteristics of this Group include: 


·      The endocyst and ectocyst may be closer compared to those of Group 1 or clearly separated


·      The ectocyst may appear triangulate, round or polygonal in shape 


·      Cysts of Group 2 specie are less than 18um in size


Examples of Group 2 species include: 


  • A.  castellannii
  • A.  quina
  • A.  triangularis
  • A.  rhysodes
  • A.  hatchetti
  • A.  divionensis 
  • A.  griffini 


Group 3 Species

Compared to Group 1 species, species that belong to Group 3 are also smaller in size.


5 species have been classified in this group and they include: 


  • A.  culberlsoni
  • A.  pustules
  • A.  palestinensis
  • A.  royreba
  • A.  lenticulata



Genotypes

While its size and morphology have played an important role in the identification and classification of these organisms, recent studies have been able to group these species based on the sequence of their genomic DNA.

To date, about 17 genotypes of Acanthamoeba have been identified. This has proved to be a more effective way of identifying various species compared to using morphological features for identification purposes: The use of morphological features has been shown to be problematic due to inconsistencies and variants of the cyst morphology.

Some of its genotypes that have been clearly identified include, T1, T2a (and T2b), T3, T4*. T5, T6, T7, T8, T9, T10, T11, T12, T13, T14, T15. 

 

Genomic sequencing has shown there to be a 5 percent variance in the genomic sequence of these genotypes, making this a more effective method of identifying the different genotypes. These small differences between the genotypes (including the 4.9 percent variance between T2a and T2b) have made this method more effective and thus the method of choice for many researchers.

While many species have been shown to cause various opportunistic infections, Acanthamoeba keratitis infection (AK) and other non-keratitis infections have largely been associated with T4. These are serious infections that not only affect sight, but also various other organs in the body. 

 

* T4 is associated with about 90 percent of all keratitis infections. It is also associated with such non-keratitis infections as Acanthamoeba granulomatous encephalitis and other skin infections.


Genotype and Strains

Organ Infected

T1

Acanthamoeba sp. strain CDC V006

Acanthamoeba sp. strain OSU 03-022/CDC V329

Brain

T4

Acanthamoeba sp. strain CDC V168

 

Acanthamoeba sp. strain CDC V328

 

Acanthamoeba sp. strain OSU 03-009/CDC 12741:1

Skin




Brain




Lungs

Acanthamoeba sp. strain OSU 03-021/CDC V313


Nasal


Other strains like Acanthamoeba sp. strain OSU 03-029/CDC V501 and Acanthamoeba sp. strain OSU 03-030/CDC V503 (belonging to T4) have been isolated in the cerebrospinal fluid, which shows that they can affect the central nervous system. 



Life Cycle

This genus goes through two main life stages that include a trophozoite stage (active stage) and the dormant stage where cysts are formed. 


Trophozoite Stage 

 

This trophozoite stage is the vegetative and motile phase that occurs during favorable conditions (neutral pH, availability of sources of nutrition, optimal temperature- about 30 degrees etc).

Depending on the genotype, generation time ranges from 8 to 24 hours as the cells passe through the growth cycle. During this phase, the organisms actively feed on a variety of organic substrate particles as well as other unicellular organisms such as yeast and bacteria. 


* The trophozoite stage is the infective phase

 

* While they feed on various single celled organisms, it has also been shown to act as a reservoir for bacteria. According to studies, such bacteria as Mycobacterium avium, not only live in Acanthamoeba (as they are transported to the next host) but also multiply in these organisms.  


Cyst Stage


During the cyst stage, Acanthamoeba form a cyst, which allows the organisms to survive tough environmental conditions. For instance, the cyst is capable of surviving lack of food sources, desiccation, extreme pH and temperatures as well as ultraviolet irradiation among others. 


Growth Cycle


In favorable conditions, Acanthamoeba cells divide asexually through a process known as binary fission. This process produces two identical daughter cells that continues the life cycle.


The growth cycle is divided into the following phases:

 

·     Lag phase- This is the first phase in which the cells take time to adapt to their environment. It is also during this stage that the Acanthamoeba cells start maturing - Some of the molecules synthesized as the cells mature include enzymes and RNA. 


·      Exponential phase- With favorable conditions available (nutrients, temperature etc) the number of cells condition to increase in the exponential phase.  The cells continue to double until nutrients are depleted (and waste/toxins material increase). Depletion of nutrients and the buildup of toxins lead to the next phase in the cycle.


·      Stationary phase- The stationary phase is the result of high number of cells from the exponential phase, depletion of nutrients and increase of toxins. These conditions result in reduced rates of growth 


·      Cell death and encystation- As a result of depleted nutrients and high levels of toxins, the cells start to die or form cysts to ensure their survival. Cyst formation allows the cells to survive in various extreme conditions by remaining dormant until environment conditions improve. 


One of the other methods of describing its life cycle involves dividing the phases into two main stages.


These include:

 

Interphase - The interphase period is divided into the following phases:

 

  • G1 - In the G1 phase, various molecules including enzymes are synthesized as the cells prepare for the next phase of growth. Some of the cells start to divide in this phase.
  • S - DNA synthesis and replication takes place in the S phase. 
  • G2 - During this phase, DNA synthesized in the S phase contributes to high protein synthesis to produce microtubules. About 90 percent of cell division also takes place during this phase. 

 

* Microtubules produced during the G2 phase are important for mitosis (cell division) that occurs in the M phase

 

M phase - Nuclear division occurs in the M phase. Chromosomes are separated to form two daughter nuclei in preparation of cell division. While nuclear division (as well as cytoplasmic division) occurs during this phase, the M phase only accounts for about 10 percent of cell division in the growth cycle. 


Acanthamoeba Cell Biology

As mentioned, the life cycle of Acanthamoeba goes through two main stages (trophozoite and cysts). Being eukaryotic cells, the trophozoites contain various organelles that are present in higher animals and plants including a prominent nucleus, ribosome, mitochondria and a Golgi apparatus among others.  

In addition to these organelles, these cells also exhibit acanthopodia (pseudopodia), which are spike-like structures used for movement and trapping prey. The pseudopodia of these organisms are capable of adhering to various surfaces, which makes it possible for the cells to move and even capture prey.  

 

* Acanthamoeba use their acanthapodia to trap and capture various particles including single celled organisms like bacteria among other organisms to feed on. These particles are consumed through a process known as phagocytosis (or pinocytosis in some cases). Here, the surface of the cell invaginates and ultimately results in the particles being digested by various enzymes in the food vacuole. 


Infections

Acanthamoeba has been associated with a number of infections in both human beings and animals. However, some of the most common infections include:


Acanthamoeba Keratitis


Acanthamoeba keratitis is a serious infection of the eye that can result in blindness if not attended to. While it is rare, the infection has been shown to mostly affect those who wear contact lenses for a long period of time. However, it can also affect those who do not use such lenses in cases of poor personal hygiene.

Once the organism attaches to the cornea of the eye, it starts to breakdown the epithelial layer while depleting the keratocyte following stromal invasion.

If left untreated, the infection can ultimately cause stromal necrosis that can in turn cause visual impairment or complete blindness. 


Acanthamoeba Granulomatous Encephalitis (AGE)


Also referred to as Granulomatous Amebic Encephalitis, AGE is a serious infection of the brain and the spinal cord that typically affects those with a poor immune system.

In such cases, Acanthamoeba has been shown to enter through the respiratory system and ultimately reach the brain and spinal cord fluid through blood circulation. Because the immune system of the individual is compromised, the organisms are successfully transported to these organs where they can cause edema and hemorrhagic necrosis.


Treatment


Early intervention has been shown to be successful in the treatment of these infections. Whereas Acanthamoeba keratitis is treated using such drugs as chlorhexidine digluconate in combination with hexamidine or propamidine, the treatment or management of Acanthamoeba granulomatous encephalitis involves using such drugs as sulfadiazine, ketoconazole, itraconazole and amphotericin B among others.  


Culture


Requirements 

 

  • Non-nutrient agar 
  • Page's Saline 
  • Incubator
  • Agar plates
  • Pipette 
  • Centrifuge 
  • Test-tubes
  • Sample 
  • E. Coli

 

 

Procedure 

 

  • Add the non-nutrient agar in to two agar plates (clean sterile plates) and incubate at 35 degrees Celsius for 30 minutes
  • Using a tube of Page's saline, prepare a dense suspension of E. coli
  • Using a clean pipette, add a few drops of the suspension into the agar plates (2 to 3 drops)
  • Spread the suspension on the agar plate to ensure even distribution 

 

* E. coli agar allows for Acanthamoeba to be detected through feeding tracks in the media 

 

Sample preparation (of the Acanthamoeba)

 

Samples may be obtained from different sources including, water bodies, soil, cerebral spinal fluid and contact lenses among others. Depending on the source, the sample is prepared differently. This section will focus on soil sample preparation. 

 

Procedure 

 

  • Add about 2 grams of the sample into a tube of Page's Saline
  • Using a pipette, add a few drops of the suspension onto the agar plates (E. coli agar plates)
  • Incubate the plates for about 24 hours at about 35ºC
  • Seal the plates using paraffin strips and continue incubating (invert the plates)
  • Observe the plates under the inverted microscope

 

Observation 

 

Feeding tracks in the plates indicates the presence of Acanthamoeba 


Image – This image shows feeding tracks in the agar plate, which indicates the presence of Acanthamoeba (A. castellanii)


https://catalog.hardydiagnostics.com. A. castellanii (ATCC ® 30010) feeding tracks on Non-Nutrient Agar E. coli plate (Cat. no. G225). Trophozoites visible at ends of tracks. 100x phase-contrast.https://catalog.hardydiagnostics.com. A. castellanii (ATCC ® 30010) feeding tracks on Non-Nutrient Agar E. coli plate (Cat. no. G225). Trophozoites visible at ends of tracks. 100x phase-contrast.


Significance of Acanthamoeba

While this specie causes various infections in human beings and some animals, they have been shown to play an important role in controlling the population of certain micro-organisms in the environment as well as a role in the regulation of various nutrients and elements in soil and water bodies. 


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References

Naveed Ahmed Khan. Acanthamoeba: Biology and Pathogenesis. Originally published: 2009.

 

Naveed Ahmed Khan. Acanthamoeba : biologyand increasing importance in human health. School of Biological and Chemical Sciences, Birkbeck College, University of London, London, UK. Received 11 October 2005; revised 9 March.

 

Ruqaiyyah Siddiqui and Naveed Ahmed Khan. Biology and pathogenesis of Acanthamoeba. Parasites & Vectors 2012, 5:6


Links


https://en.vircell.com/diseases/21-acanthamoeba-castellanii/

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/FreeLivingAmebaeMedia.html



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