Acanthamoeba Genotype T4 Research Articles

Identification of Acanthamoeba genotype T4 in three drinking water treatment plants in Menofia Governorate.

Identification of Acanthamoeba genotype T4 in three drinking water treatment plants in Menofia Governorate.

First report on the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples stored at room temperature

ABSTRACT Acanthamoeba is an opportunistic, free-living amoeba ubiquitous in the environment. Despite reports of its wide distribution in the Philippines’ freshwater resources, more information on the long-term viability of the Acanthamoeba species is needed. This study aimed to define the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples after 3 years of storage at room temperature. Stored water samples from 15 study sites were filtered through a 1.2-μm pore size glass microfiber filter, cultured in non-nutrient agar (NNA) lawned with Escherichia coli, and observed for amoebic growth for 14 days using light microscopy. Isolates from positive NNA culture were subjected to polymerase chain reaction (PCR) using JDP1 and JDP2 Acanthamoeba-specific primers. The study site positivity was 33% (5/15). Acanthamoeba genotype T4 and Acanthamoeba lenticulata were isolated from Luzon; Acanthamoeba divionensis was isolated from Visayas; and Acanthamoeba sp. and genotype T20 were isolated from Mindanao. The long-term viability of Acanthamoeba species is an added risk factor for the sustained contamination of aquatic resources and other sample matrices. This heightens the risk of transmission to humans and animals. This study demonstrated that water samples fated for Acanthamoeba studies can be stored unpreserved at room temperature for several years.

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

First Report of Acanthamoeba Genotype T4 from the Newly Formed Tajogaite Volcano Tephra (La Palma, Canary Islands).

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli. Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain's pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano.

Occurrence of Free-Living Amoebae in Non-Human Primate Gut.

The gut microbiome reflects health and predicts possible disease in hosts. A holistic view of this community is needed, focusing on identifying species and dissecting how species interact with their host and each other, regardless of whether their presence is beneficial, inconsequential, or detrimental. The distribution of gut-associated eukaryotes within and across non-human primates is likely driven by host behavior and ecology. To ascertain the existence of free-living amoebae (FLA) in the gut of wild and captive non-human primates, 101 stool samples were collected and submitted to culture-dependent microscopy examination and DNA sequencing. Free-living amoebae were detected in 45.4% (46/101) of fecal samples analyzed, and their morphological characteristics matched those of Acanthamoeba spp., Vermamoeba spp., heterolobosean amoeboflagellates and fan-shaped amoebae of the family Vannellidae. Sequence analysis of the PCR products revealed that the suspected amoebae are highly homologous (99% identity and 100% query coverage) with Acanthamoeba T4 genotype and Vermamoeba vermiformis amoebae. The results showed a great diversity of amoebae in the non-human primate's microbiome, which may pose a potential risk to the health of NHPs. To our knowledge, this is the first report of free-living amoebae in non-human primates that are naturally infected. However, it is unknown whether gut-borne amoebae exploit a viable ecological niche or are simply transient residents in the gut.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020–2022

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as “free-living”. The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.

Morphological Characterization and Genotyping of Acanthamoeba Isolates From Oral and Nasal Samples of Cancer Patients in Kashan, Iran

Background: Acanthamoeba​ species are recognized as the most prevalent free‐living amoebae (FLA). They can cause granulomatous amebic encephalitis (GAE) and pulmonary and ocular infections. The present study aimed to isolate and identify Acanthamoeba genotypes in cancer patients referred to Kashan’s hospitals in Central Iran.Methods: This cross‐sectional study was conducted with oral and nasal swab samples collected from a hundred cancer patients referred to Kashan’s Beheshti and Yasrebi hospitals during 2019–2020. The samples were cultured in 1.5% non‐nutrient agar (NNA) with heat‐killed Escherichia coli and examined for “FLA.” A polymerase chain reaction (PCR) assay amplifying the 18S rRNA gene was performed, and Acanthamoeba‐positive isolates were subjected to nucleotide sequencing to identify their genotypes.Results: The prevalence of Acanthamoeba infection was 51% in the oral cavity and 38% in the nasal samples of cancer patients. The most frequent Acanthamoeba cysts were (51.3%) wrinkled polygonal and sized 9.55‐11.5 μm (Group II). Acanthamoeba genotypes T4, T11, and T5 were identified in the oral cavity samples, whereas T4 and T11 were detected in the nasal samples.Conclusion: The prevalence of Acanthamoeba infection in oral and nasal cancer patients was higher in Kashan, Iran, compared to other countries. Due to the high rate of oral Acanthamoeba contamination, oral sampling is recommended for better detection of this protozoan. Since T4 is the predominant genotype that can cause serious diseases in high‐risk groups, increasing physicians’ awareness of infections associated with Acanthamoeba and preventive and control measures are strongly suggested.

Isolation and genotyping of Acanthamoeba species and Vahlkampfiidae in the harsh environmental conditions in the centre of Iran.

Different species of free-living amoeba (FLA) have been abundantly isolated in harsh environmental conditions such as hot springs and brackish water. The present study aimed to isolate, genotype, and evaluate the pathogenicity of FLAs in Qom Roud, a large river, in the centre of Iran. About 500 mL of water samples (n = 30) were collected from each sampling site and were investigated for the presence of FLAs using morphological and molecular characters. Genotype identification was performed using DNA sequencing and a phylogenetic tree was constructed with the MEGA X software. The pathogenic potential of all positive isolates was evaluated using the tolerance ability test. Morphological and molecular analysis indicated that 14 (46.66%) and two (6.66%) water samples were positive for Acanthamoeba species and Vahlkampfiidae, respectively. According to sequence analysis, Acanthamoeba isolates related to the T4 genotype and Vahlkampfiidae sequences were similar to Naegleria philippinensis. In the next step, thermo- and osmotolerance tests indicated four Acanthamoeba strains are extremely pathogenic. Our data showed the presence of potentially pathogenic Acanthamoeba T4 genotype and N. philippinensis in the super harsh Qom Roud. Contamination of water with virulent T4 genotype of Acanthamoeba may pose risk factors for contact lens users, children, and immunocompromised people.

Molecular identification of Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri in soil samples using quantitative real-time PCR assay in Turkey; Hidden danger in the soil!

Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri are pathogenic free-living amoeba (FLA) and are commonly found in the environment, particularly soil. This pathogenic FLA causes central nervous system-affecting granulomatous amebic encephalitis (GAE) or primary amebic meningoencephalitis (PAM) and can also cause keratitis and skin infections. In the present study, we aimed to determine the quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in soil samples collected from places where human contact is high by using a qPCR assay in Izmir, Turkey. A total of 45.71% (n = 16) of Acanthamoeba spp., 20% (n = 7) of B. mandrillaris, and 17.4% (n = 6) of N. fowleri were detected in five different soil sources by the qPCR assay. The quantitative concentration of Acanthamoeba spp., B. mandrillaris, and N. fowleri in various soil sources was calculated at 10 × 105 - 6 × 102, 47 × 104 to 39 × 103, and 9 × 103 – 8 × 102 plasmid copies/gr, respectively. While the highest quantitative concentration of Acanthamoeba spp. and B. mandrillaris was determined in garden soil samples, N. fowleri was detected in potting soil samples. Three different genotypes T2 (18.75%), T4 (56.25%), and T5 (25%) were identified from Acanthamoeba-positive soil samples. Acanthamoeba T4 genotype was the most frequently detected genotype from soil samples and is also the most common genotype to cause infection in humans and animals. To the best of our knowledge, the present study is the first study to identify genotype T5 in soil samples from Turkey. In conclusion, people and especially children should be aware of the hidden danger in the garden and potting soil samples that come into contact most frequently. Public health awareness should be raised about human infections that may be encountered due to contact with the soil. Public health specialists should raise awareness about this hidden danger in soil.

Molecular evidence for a new lineage within the Acanthamoeba T4 genotype.

Acanthamoeba is a widespread free-living amoeba capable of causing serious infections in humans and other animals, such as amoebic keratitis, disseminated infections, and fatal encephalitis. Strain identification is usually based on 18S rDNA sequencing, which allows the distinction of over twenty genotypes. Most sequences from environmental and clinical samples belong to the T4 genotype, which can be divided into seven subtypes, T4A to T4G, and by a nearly similar grouping of mitochondrial sequences into T4a to T4j subtypes. The co-clustering of nuclear and mitochondrial groups can be very useful for a better identification of lineages within the very rich T4 genotype. In this study, we provided molecular phylogenetic evidence for the delineation of a new nuclear subtype, hereafter labelled T4H, and its co-clustering with the mitochondrial T4j subtype. At least three cases of amoebic keratitis are due to strains belonging to this new group, present mainly in fresh water and detected in various countries (France, Iran, India and China).

Detection of Acanthamoeba spp. in groundwater sources in a rural area in the Philippines.

Research on free-living amoebae (FLA) and its public health implication as an etiologic agent of parasitic infection in humans has recently gained traction in the Philippines. This study aimed to identify potential FLAs in collected groundwater samples from Masinloc, Zambales, Philippines. Fifty-four (54) water samples were collected in 250-mL sterile polyethylene containers by purposive sampling from selected groundwater sources in six (6) barangays of Masinloc. The samples were vacuum filtered through a 1.2-μm pore glass microfiber filter, cultured onto non-nutrient agar (NNA) lawned with Escherichia coli, and observed microscopically for amoebic growth for 14 days using light microscopy. Amoebic growth was observed in 11.1% (6/54) of water samples. DNAs from positive samples were extracted and were made to react with polymerase chain reaction using Acanthamoeba-specific JDP1 (5'-GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'-TCTCACAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). The presence of Acanthamoeba genotypes T4, T7, and T11 was confirmed using molecular and phylogenetic analysis. Our results confirmed that groundwater sources from two of six sampling sites (33.3%) in Masinloc, Zambales, were contaminated with potentially pathogenic FLAs. Proper identification of risk factors that may cause contamination consequently leads to the implementation of programs that will prevent future infections.

Acanthamoeba and a bacterial endocytobiont isolated from recreational freshwaters.

The quality of many freshwater environments is impacted by human activities, so that many rivers may represent a vehicle for the transmission of health-related microorganisms. This work aimed to isolate and identify genetically free-living amoeba (FLA) of the genus Acanthamoeba from a recreational river in Salta, Argentina, and isolate, if possible, an endocytobiont. Sampling took place at four points (P1-P4) throughout the river in the winter and the summer seasons. Free-living amoebae and Acanthamoeba were recovered from 20-L water concentrated through an ultrafiltration system. Isolation was performed in agar plates, confirmation of Acanthamoeba genus by PCR, and fellow identification and classification based on their sequence analyses. High concentrations of indicator bacteria were found especially in P2, which is intensively used for recreation. Out of a total of 29 FLA isolations, 9 were identified as Acanthamoeba genotype T4 subtype A, the most frequent genotype found in nature and associated with causing human disease. From an axenic culture of Acanthamoeba spp. (KY751412), a bacterial endocytobiont was isolated, and identified as Stenotrophomonas maltophilia. The endocytobiont showed resistance and intermediate resistance to a wide range of widely used antibiotics. Results were in concordance with the cosmopolitan behavior of Acanthamoeba, and showed the importance of studying this group of amoebae and related microorganisms in recreational environments.

Genotyping and Molecular Identification of Acanthamoeba Genotype T4 and Naegleria fowleri from Cerebrospinal Fluid Samples of Patients in Turkey: Is it the Pathogens of Unknown Causes of Death?

This study was aimed to investigate the presence of pathogenic free-living amoebae (FLA) in suspected cases of meningoencephalitis with unknown causes of death in Turkey. A total of 92 patients, who were diagnosed as meningoencephalitis, were enrolled. All cerebrospinal fluid (CSF) samples were directly microscopically examined and cultured. Acanthamoeba, N. fowleri and B. mandrillaris were further investigated using molecular diagnostic tools including real-time PCR, sequencing, and phylogenetic analyses. The examined CSF samples were not found positive for the presence of FLA by microscopic examination and culture method. However, two CSF samples were detected positive by real-time PCR assay. Of the positive CSF samples, one was identified as Acanthamoeba genotype T4 and the second positive sample was identified as N. fowleri belonging to genotype II. Furthermore, the pathogens diagnoses was verified through Sanger sequencing. This study was significant to report the presence of Acanthamoeba genotype T4 and N. fowleri genotype II in CSF samples by real-time PCR assay. The present study shows the significance of primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) as one of the differential diagnoses to be considered by clinicians during the evaluation of suspected meningoencephalitis or cases of unknown cause in Turkey. Using real-time PCR, this has made the rapid detection, in a short time-frame, of Acanthamoeba and N. fowleri in CSF samples from patients. The problems with qPCR is that it is not available in every laboratory, reagents are expensive, and it requires skilled and expert personnel to set up these assays.

Real-Time PCR for the Diagnosis of Acanthamoeba Genotype T4.

Acanthamoeba spp. are ubiquitous and opportunistic free-living amoebae (FLA) that can cause Acanthamoeba keratitis and other infections in the human host. A quick and efficient diagnosis is often challenging. Our study aimed to establish a qPCR assay to detect and, at the same time, quantify the predominant Acanthamoeba genotype T4. DNA from clinical corneal scrapings and Acanthamoeba reference strains, including genotypes T3, T4, T5, T6, T10, T11, and T12, were used to develop the new T4 assay and it was compared to published protocols and one commercial kit for evaluation. The T4 assay showed no amplification with Acanthamoeba genotypes T3, T5, T6, T10, T11, and T12. The efficiencies ranged from 92.01 to 97.59% (R2 of 0.9768 to 0.9951). The calculated LOD range was 3.63 to 33.27 cells/µL. The protocol published by Qvarnstrom and colleagues was more sensitive compared to the other assays, and an overall good agreement was observed between the new T4 and the Qvarnstrom assays. We successfully developed and validated a genotype T4 assay that could be run in duplex with the Qvarnstrom assay to reliably and simultaneously diagnose Acanthamoeba genotype T4 and other genotypes from clinical samples.

Survey and first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea, Turkey.

Infection with Acanthamoeba spp. may result in granulomatous amoebic encephalitis and Acanthamoeba keratitis. Water is an important habitat where Acanthamoeba species thrive. Therefore, studying the occurrence of this free-living amoeba in water sources will help understand the infection dynamics. The aim of the study was to survey and report on the presence of Acanthamoeba spp. in water resources from the Ordu and Giresun provinces in Black Sea. Acanthamoeba spp. was found in 1/17 natural spring water samples from Ordu and in 2/18 from Giresun. Acanthamoeba species were not detected in any of the investigated tap water samples. Sequencing of the (SSU) rDNA gene resulted in the identification of haplotype I (Acanthamoeba genotype: KJ094684). T4 (8.6%) was the only isolated genotype in both Ordu and Giresun provinces. This is the first report of Acanthamoeba T4 genotype in natural spring water resources in the Black Sea. The occurrence of Acanthamoeba species in natural spring water sources should be considered as a potential risk for human infection, especially to high-risk populations.

Co-Existence of Free-Living Amoebae and Potential Human Pathogenic Bacteria Isolated from Rural Household Water Storage Containers.

Simple SummaryIn many households in rural communities, water needed for drinking and cooking is fetched from rivers, fountains, or boreholes shared by the community. The water is then stored in various storage containers for several days without treatment and exposed to several conditions that could potentially contaminate the water and cause diseases. If the storage containers are not regularly and properly cleaned, biofilms can form inside the containers. Several microorganisms can be found inside the biofilm that can potentially cause diseases in humans. One such group of organisms is called free-living amoebae, which graze on the bacteria found inside the biofilm. Several of these potentially harmful bacteria have adapted and can survive inside these free-living amoebae and potentially cause diseases when ingested by humans.This study investigated the co-existence of potential human pathogenic bacteria and free-living amoebae in samples collected from stored water in rural households in South Africa using borehole water as a primary water source. Over a period of 5 months, a total of 398 stored water and 392 biofilm samples were collected and assessed. Free-living amoebae were identified microscopically in 92.0% of the water samples and 89.8% of the biofilm samples. A further molecular identification using 18S rRNA sequencing identified Vermamoeba vermiformis, Entamoeba spp., Stenamoeba spp., Flamella spp., and Acanthamoeba spp. including Acanthamoeba genotype T4, which is known to be potentially harmful to humans. Targeted potential pathogenic bacteria were isolated from the water samples using standard culture methods and identified using 16S rRNA sequencing. Mycobacterium spp., Pseudomonas spp., Enterobacter spp., and other emerging opportunistic pathogens such as Stenotrophomonas maltophilia were identified. The results showed the importance of further studies to assess the health risk of free-living amoebae and potential human pathogenic bacteria to people living in rural communities who have no other option than to store water in their homes due to water shortages.

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.

The effectiveness of different culture media on the growth and proliferation of Acanthamoeba T4 genotype

The effectiveness of different culture media on the growth and proliferation of Acanthamoeba T4 genotype

In vitro anti-Acanthamoeba activity of the commercial chitosan and nano-chitosan against pathogenic Acanthamoeba genotype T4.

Acanthamoeba keratitis (AK) is a rare but serious infection of the eye and can lead to blindness. The effective and safe medical therapy remains unclear for AK until present. Antimicrobial activity and biological characteristic of chitosan encourage screening of it against Acanthamoeba. Thus, in vitro anti-amoebic activities of commercial chitosan and nano-chitosan were tested on pathogenic Acanthamoeba genotype T4, a causative agent of human AK. The Acanthamoeba spp. was isolated from the keratitis patient. The Acanthamoeba genotype T4 was approved using PCR method followed by sequencing technique. Chitosan nanoparticles was prepared using ionic gelation method and characterized by their physicochemical properties. In the present study, the in vitro activity of serial dilutions (12.5, 25, 50, 100, and 200 µL/mL) of commercial chitosan and nano-chitosan were evaluated against Acanthamoeba trophozoites and cysts. The finding of nano-chitosan particle size by DLS was 118nm with a PDI of about 0.134. Zeta potential value was found to be 42.7mV. The obtained results showed that the tested chitosan and nano-chitosan presented anti-amoebic activities dependent to time and concentration. The inhibitory effect of the chitosan and nano-chitosan is enhanced by increasing the concentration and incubation time. The inhibitory effect of nano-chitosan on both trophozoites and cyst was more than chitosan. According to the results, nano-chitosan shows the potent activity against Acanthamoeba T4 and could be used for the development of novel and safe therapeutic approaches in the future.

First Evidence for Colonizing of Acanthamoeba T4 Genotype in Urinary Tracts of Patients with Recurrent Urinary Tract Infections.

Limited evidence about the presence of Acanthamoeba spp. in urine specimens collected from urinary catheters of the patients in the intensive care units persuaded our study. No evidence has been found about colonizing of Acanthamoeba spp., in urinary tracts of patients with recurrent urinary tract infections (UTIs) yet. In this study, 50 urine samples were collected from patients presenting with recurrent UTI. The type of bacteria causing UTI was determined by using bacteriological tests. To cultivate Acanthamoeba spp., in a sterile condition, 10mL of urine was centrifuged and the sediment was cultivated on non-nutrient agar. Genotypes were determined by sequencing the DF3 region of the 18S rRNA gene. The bacteriological test findings on the urine samples of the UTI patients (n = 30) demonstrated that those were found to be positive for Escherichia coli (n = 17), Staphylococcus aureus (n = 6), Pseudomonas aeruginosa (n = 4) and Klebsiella spp. (n = 3) respectively. Moreover, a total of 50 urine samples was examined; 6 (6/50; 12%) and 11 (11/50; 22%) were positive by using culture and the PCR test for Acanthamoeba spp., respectively. Sequencing analysis showed all isolates were T4 genotype. Our data showed that the high relative prevalence of Acanthamoeba T4 genotype spp., in the urine of recurrent UTI patients. As well as, providing the first evidence for colonizing of the Acanthamoeba in the urinary tracts of patients with recurrent UTIs. These findings, warrant further investigation among those patients to fully appraise the role of Acanthamoeba spp., as possible latent carriers for resistant bacteria and biofilm formation in the future.