Abstract
Free-living amoebae (FLA) are protozoa ubiquitously found in nature. In addition to their natural distribution, some species have been documented as pathogenic to humans. The main aim of the current study was the molecular identification, sequencing and phylogenetic analysis of morphologically detected FLA in water sources in El-Qalyubia, Egypt. A total of 96 water samples were collected from different water sources. Each water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive FLA were subjected to PCR, PCR products were sequenced and the obtained sequences were phylogenetically analysed. FLA were found in 41 water samples examined (42.7%). Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively). Naegleria italica was first identified in Egypt from the waters of the Nile. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. However, other FLA species, including Acanthamoeba spp. and the pathogenic Naegleria fowleri, previously reported in Egypt, were not included in this study. The recent identification of these FLA in the Egyptian waters related to human populations indicates the need for more phylogenetic studies using larger sample sizes to investigate their potential threat to human health.
HIGHLIGHTS
This study documented the presence of FLA in different water sources in El-Qalyubia, Egypt.
Naegleria italica was identified for the first time in Egypt from the waters of the Nile.
Besides, Vahlkampfia spp. and Hartmannella spp. were also detected.
This study presents knowledge of the prior prevalence of FLA that should be considered by the clinicians and the environmental professionals in the region.
INTRODUCTION
Free-living amoebae (FLA) are protozoa that can survive and proliferate in the environment independently or found within a host hence the name amphizoic amoeba. FLA are found all over the world in various natural and man-made aquatic environments such as lakes, ponds, swimming pools and even treated water (Mahmoudi et al. 2021).
Humans are constantly exposed to these amoebae which can cause severe diseases due to infection (Fabros et al. 2021). The causative agents of the disease in humans belong to two super-groups: Amoebozoa, including the genera Acanthamoeba and Hartmannella, and Excavata, including Vahlkampfiidae family with the genera Naegleria and Vahlkampfia as members (Di Filippo et al. 2015; El-Badry et al. 2020).
FLA can induce a wide range of clinical complications. For instance, Naegleria fowleri causes an acute fatal central nervous system infection known as primary amoebic meningoencephalitis (PAM) in healthy young adults and children (Ithoi et al. 2011; El-Badry et al. 2020). Acanthamoeba species cause granulomatous amoebic encephalitis (GAE), and occasionally skin, pulmonary and kidney infections that may affect immunocompromised patients. Besides, amoebic keratitis (AK) can be detected in immunocompetent individuals (Al-Herrawy et al. 2017). Vahlkampfia and Hartmannella species have been detected from the eye surface of humans (Al-Herrawy & Gad 2017).
In Egypt, there is a dearth of available data regarding the prevalence of the FLA in aquatic environments, with a shortage of phylogenetic studies.
The present study aims to identify FLA from water sources used by local populations for drinking, washing, cooking, agriculture and recreational activities in El-Qalyubia, Egypt. Moreover, it aims to characterize isolates at the species/genotypes level to better understand their distribution in the environment and to assess potential risks to human health.
MATERIALS AND METHOD
Study location, water sample collection and filtration
A total of 96 water samples were collected from various districts in El-Qalyubia governorate as follows: 24 from the Nile River (collected from the inlet of Benha Drinking Water Treatment Plants (DWTP)), 24 samples from household potable water (distribution systems), 24 samples from untreated groundwater (pre-chlorinated water from a 128 m deep well of Kaha DWTP) and 24 from treated groundwater (chlorinated water of Kaha DWTP) (Figure 1). The Nile River samples were collected from the inlet of Benha DWTP with a depth of approximately 30 cm, making sure that there are no floating films or organic material, while the untreated groundwater samples were collected from a tape connected to the well pipes. The well had been purged sufficiently (the water is pumped for 5–10 min until the water temperature was stabilized) to ensure that the sample is representative of the groundwater (ISO/FDIS 2006; APHA 2017).
One litre volume of water samples was collected in dry, clean autoclavable polypropylene containers and transferred in iceboxes to the laboratory where they were immediately processed. The collected water samples were concentrated using the membrane filtration technique as per Di Filippo et al. (2015). Each separately collected water sample was concentrated and filtered through cellulose nitrate membranes (0.45 mm pore size and 47 mm in diameter) using a stainless-steel holder attached to a suction pump. When the suction was detached, the holder was separated and the membrane was removed before it dried.
FLA cultivation, sub-cultivation and morphological identification
Cultivation and sub-cultivation of FLA were performed according to Abd El Wahab et al. (2018). Briefly, filter paper for each sample was placed face-down on the surface of plates of a 1.5% non-nutrient agar (NNA) made with Page's amoebae saline, covered with a thin layer of Escherichia coli. NNA plates were incubated at 37 °C and observed daily for 14 days using an inverted microscope for the presence of any amoebic growth. When the amoebic growth was detected, a piece of agar enclosing the amoebic growth was cut out using a sterile scalpel and placed into a fresh NNA-E. coli plate. The sub-cultured plates were incubated in the same way described above and then plates were examined for amoebae growth. Morphological characteristics of trophozoites and cyst stages of FLA were performed according to Page's classification key. The flagellation test was done by incubating the amoebae in a test tube containing 2 ml of distilled water for 1–2 h to differentiate Naegleria spp. from other Vahlkampfiidae (Stockman et al. 2011).
DNA extraction, amplification and sequencing
The growing amoebae were harvested from all positive culture plates, placed in Eppendorf tubes and washed two times with saline buffer prior to the molecular procedure. DNA extraction was performed using G-spin™ Total DNA Extraction Mini Kit following the manufacturer's specifications (iNtRON Bio, South Korea).
For identification of FLA, we used two sets of Naegleria primer pairs, genus-specific primers Naeg1 (forward: 5′GAACCTGCGTAGGGATCATTT3′) and Naeg 2 (reverse: 5′TTTCTTTTCCTCCCC TTATTA3′) and N. fowleri species-specific (forward: 5′ GTGAAAACCTTTTTTCCATTTACA 3′) and (reverse: 5′ AAATAAAAGATTGACCAT TTGAAA 3′) targeting the internal transcribed spacer regions (ITS1 and 2) that contain the 5.8S rDNA gene. PCR reaction conditions and mixtures were performed as previously described (Pélandakis et al. 2000).
For identification of the genus Acanthamoeba, a PCR was carried out to amplify an 18S rDNA region defined as ASA.S1 (Acanthamoeba-specific amplimer) that includes the diagnostic fragment 3 (DF3), using the genus-specific primers JDP1 (forward: 5′GGCCCAGATCGTTTAC CGTGAA3′) and JDP2 (forward: 5′ TCTCACAAGCTGCTAGGGAGTCA3′). PCR reaction conditions and mixtures were performed as per Schroeder et al. (2001).
PCR products were purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen, Germany) according to the manufacturer's instructions. Sequencing was done with the primer pair (Naeg1 and Naeg 2) and using Big-DyeTM Terminator v3.1 with the Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA) on the ABI Prism 310 genetic analyser (Applied Biosystems, USA) according to the manufacturer's instructions.
The sequences of the studied isolates were matched to the reference sequences registered in the Gene Bank database through BLAST-NCBI (https://blast.ncbi.nlm.nih.gov), after which all sequences were aligned using the BioEdit software, which depends on the ClustalW multiple alignment conditions (Hall 1999). Evolutionary analyses were conducted in MEGA X (Version 10.2.4) (Kumar et al. 2018).
RESULTS
Of the 96 water samples, 41 (42.7%) were positive for FLA after cultivation on NNA. Nile water and groundwater were the sources with the highest prevalence rates (83.3 and 62.5%, respectively) (Table 1). Microscopically, vahlkampfiids and Hartmannellidae were identified based on morphological characteristics. Vahlkmapfiids had spherical double-wall cysts and temporarily branched trophozoites (Figure 2(a) and 2(b)). Hartmannella had a small spherical or ovoid cyst, while the trophozoite form was Limax containing a small nucleolus (Figure 2(c) and 2(d)).
Distribution of positive FLA in different water samples with reference to results of sequencing according to sampling site
. | . | . | Results of sequencing . | ||
---|---|---|---|---|---|
. | Examined samples . | Positive FLA samples . | Vahlkampfiidae . | . | |
Water samples . | Total . | n (%) . | Hartmannella . | Naegleria . | Vahlkampfia . |
Nile water | 24 | 20 (83.3%) | 1 | 2 | 1 |
Untreated ground water | 24 | 15 (62.5%) | – | 1 | 1 |
Household tap water | 24 | 1 (4.2%) | – | 1 | – |
Treated ground water | 24 | 5 (20.8%) | – | 1 | – |
Total | 96 | 41 (42.7%) | 1 (N. italica) | 5 | 2 |
. | . | . | Results of sequencing . | ||
---|---|---|---|---|---|
. | Examined samples . | Positive FLA samples . | Vahlkampfiidae . | . | |
Water samples . | Total . | n (%) . | Hartmannella . | Naegleria . | Vahlkampfia . |
Nile water | 24 | 20 (83.3%) | 1 | 2 | 1 |
Untreated ground water | 24 | 15 (62.5%) | – | 1 | 1 |
Household tap water | 24 | 1 (4.2%) | – | 1 | – |
Treated ground water | 24 | 5 (20.8%) | – | 1 | – |
Total | 96 | 41 (42.7%) | 1 (N. italica) | 5 | 2 |
Fresh unstained Naegleria trophozoite (a) and cysts (b) and fresh unstained Hartmannella trophozoite (c) and cysts (d).
Fresh unstained Naegleria trophozoite (a) and cysts (b) and fresh unstained Hartmannella trophozoite (c) and cysts (d).
All the 41 morphologically identified vahlkampfiids and Hartmannellidae were positive by using the Ng.spp_FW and Ng.spp_RV primers. About 500 and 800 bp PCR products were obtained from vahlkampfiids and Hartmannella, respectively (Figure 3). Sequence analysis was successfully performed for only eight PCR products (Figure 4), while the rest of the sequences were non-interpretable possibly due to ineffective and/or insufficient amplified products.
PCR amplification of internal transcribed spacer (ITS) rDNA region. Lane 1: DNA marker of 50 bp molecular weight. Lane 2: negative control sample. Lanes 3 and 6: Hartmannella. Lanes 5, 7 and 8: Vahlkampfiidae samples. Lane 4: negative sample.
PCR amplification of internal transcribed spacer (ITS) rDNA region. Lane 1: DNA marker of 50 bp molecular weight. Lane 2: negative control sample. Lanes 3 and 6: Hartmannella. Lanes 5, 7 and 8: Vahlkampfiidae samples. Lane 4: negative sample.
Optimal tree of ITS1, 5.85, ITS2 and 28S rDNA sequences for the studied FLA samples. Neighbor-joining tree showing the evolutionary history of the studied FLA samples, inferred by the evolutionary distance analysis, was calculated using the maximum composite likelihood method. The percentage of replicate trees in which the associated taxa clustered together in a bootstrap test. The bootstrap value is 5,000 with the sum of the branch length = 0.5.
Optimal tree of ITS1, 5.85, ITS2 and 28S rDNA sequences for the studied FLA samples. Neighbor-joining tree showing the evolutionary history of the studied FLA samples, inferred by the evolutionary distance analysis, was calculated using the maximum composite likelihood method. The percentage of replicate trees in which the associated taxa clustered together in a bootstrap test. The bootstrap value is 5,000 with the sum of the branch length = 0.5.
These sequences were phylogenetically analysed. The phylogenetic tree was generated with sequences of reference species from NCBI-BLAST. The sequencing results are summarized in Table 2 and Figure 4. Isolates 10, 11 and 12 showed 94–98% homological identities with Vahlkampfia Sp. unclassified (MT109103.1) with accession numbers MW843639–MW843641, while isolate 23 showed 96% homological identities with Hartmannella sp. unclassified (JX910447.1) with accession number MW843642 and isolate 29 showed 100% homological identities with Hartmannella sp. unclassified (HE617189.1) with accession number MW876241. Furthermore, isolate 27 showed 95% homological identities with N. italica (Gu597047.1) with accession number MW857141 (Table 2; Figure 4).
Sequences retrieved from the GenBank used for phylogenetic analysis
Organism . | Source . | Location . | Accession number . | References . |
---|---|---|---|---|
Naegleria sp. | Raw water | Malaysia | KT356270 | Richard et al. (2016) |
Vermamoeba vermiformis | Culture media | USA | KT185625 | Fučíková & Lahr (2016) |
Marine sediment sample | United Kingdom | GU001158 | Glücksman et al. (2011) | |
Vahlkampfia sp. Unclassified | Tap water | Japan | AB330062 | Edagawa et al. (2009) |
Hospital ward | Iran | JN608805 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/JN608805) | |
Geothermal waters | Italy | MT109103 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT109103) | |
Lake water | Pakistan | KF153929 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/KF153929) | |
Hartmannella sp. | Biofilm | Ghana | HE617189 | De Jonckheere et al. (2012) |
Geothermal waters | France | JX910447 | Moussa et al. (2013) | |
Naegleria pagei | Aquatic environment | Iran | MT648397 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT648397) |
Aquatic environment | Iran | MT648417 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT648417) | |
N. italica | Biofilm in hot spring water | Taiwan | GU597047 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/GU597047) |
Geothermal water | Italy | MF503261 | Di Filippo et al. (2017) |
Organism . | Source . | Location . | Accession number . | References . |
---|---|---|---|---|
Naegleria sp. | Raw water | Malaysia | KT356270 | Richard et al. (2016) |
Vermamoeba vermiformis | Culture media | USA | KT185625 | Fučíková & Lahr (2016) |
Marine sediment sample | United Kingdom | GU001158 | Glücksman et al. (2011) | |
Vahlkampfia sp. Unclassified | Tap water | Japan | AB330062 | Edagawa et al. (2009) |
Hospital ward | Iran | JN608805 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/JN608805) | |
Geothermal waters | Italy | MT109103 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT109103) | |
Lake water | Pakistan | KF153929 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/KF153929) | |
Hartmannella sp. | Biofilm | Ghana | HE617189 | De Jonckheere et al. (2012) |
Geothermal waters | France | JX910447 | Moussa et al. (2013) | |
Naegleria pagei | Aquatic environment | Iran | MT648397 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT648397) |
Aquatic environment | Iran | MT648417 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/MT648417) | |
N. italica | Biofilm in hot spring water | Taiwan | GU597047 | Unpublished (https://www.ncbi.nlm.nih.gov/nuccore/GU597047) |
Geothermal water | Italy | MF503261 | Di Filippo et al. (2017) |
In our study, no representative of other pathogenic types of FLA (Acanthamoeba spp. and N. fowleri) was detected in any sample, neither by morphology nor by PCR.
DISCUSSION
Humans are constantly exposed to FLA due to their ubiquitous occurrence in the environment (Blair et al. 2008). According to Abdul Majid et al. (2017), the occurrence of these pathogens must be monitored due to their important role within ecosystems and their potential to cause serious infections in humans.
In addition to their pathogenicity, FLA act efficient carriers for certain antibiotic-resistant bacteria and viruses rendering their detection a priority (Khurana et al. 2015).
In our research, FLA were detected in 41 (42.7%) out of 96 water samples examined in El-Qalyubia governorate, Egypt for identification of new species of Naegleria (N. italica), Vahlkampfia spp. and Hartmannella spp.
In the present study, FLA were isolated from 83.3% of the examined Nile water samples. Other studies in Egypt revealed a higher percentage of FLA in the examined Nile water samples with 100% in Minofeya governorate (Al-Herrawy et al. 2017), 91.7% in Fayuom governorate (Al-Herrawy et al. 2015) and 87.5% in Cairo (Hilali et al. 1994), while Hamadto et al. (2003) detected the presence of FLA in a lower percentage (20%) in surface water and canal water samples from different governorates in Egypt.
Other studies in other countries detected FLA at high frequency like Japan (Edagawa et al. 2009) and the USA (Corsaro et al. 2009) found that the percentage of occurrence of FLA in surface water reached 94 and 100%, respectively. In contrast, other researchers recorded a lower incidence of FLA in freshwater samples. 28.7, 61.1 and 69% were recorded from Italy (Di Filippo et al. 2015), Bulgaria (Tsvetkova et al. 2004) and Central Mexico (Bonilla-Lemus et al. 2014), respectively.
In the present study, FLA were isolated from 62.5% of untreated groundwater samples. In Egypt, Al-Herrawy et al. (2017) reported a higher incidence (100%) of FLA in untreated groundwater samples from Minofeya governorate, while a lower incidence (58.3%) was documented by Gad & Al-Herrawy (2016).
In other countries such as Italy (Di Filippo et al. 2015) and Iran (Ghadar-Ghadr et al. 2012), FLA were found in 85.7 and 44.4% of groundwater samples, respectively.
In the present work, FLA were isolated from 4.2 and 20.8% of the examined household tap water and treated groundwater, respectively.
In Egypt, a nearly similar percentage (4%) of FLA was detected by Hamadto et al. (2003) in the examined tap water samples from different governorates. Other workers in Egypt recorded FLA in tap water in a higher percentage of 45.8% (Al-Herrawy et al. 2017), 41.7% (Al-Herrawy et al. 2015) and 50% (Gad & Al-Herrawy 2016) compared to the present work.
Other researchers in other countries such as Leońska-Duniec et al. (2015) in Poland, Shoff et al. (2008) in the UK and Jeong & Yu (2005) in Korea have reported FLA in tap water samples as 44, 46.9 and 48%, respectively.
Significant variation in the prevalence values of FLA was observed worldwide. According to De Jonckheere (2011), Stockman et al. (2011) and Di Filippo et al. (2015), the variation in the occurrence of FLA may be affected by many factors such as differences in geographical location, environmental conditions, water sources and different applied methodologies.
Concerning the phylogenetic analysis, this is the first phylogenetic study revealing the presence of Naegleria italica, Vahlkampfia spp. and Hartmannella spp. in Egypt. Nevertheless, other FLA species, including Acanthamoeba spp. and the pathogenic N. fowleri, previously reported in Egypt, were not investigated in this study.
In Egypt, there is a scarcity of phylogenetic studies. A study done by El-Badry et al. (2020) reported the presence of Vahlkampfia ciguana and the Naegleria species N. australiensis, N. philippinensis and N. neojejuensis in the Nile water. In agreement with our results, this study also documented the absence of the pathogenic N. fowleri.
Although N. fowleri is the only species of the genus Naegleria causing human pathology, N. italica detected in this study has previously proven to be pathogenic in experimental animals (De Jonckheere 2014).
The most common cause of AK is the genera Acanthamoeba, Vahlkampfia and Hartmannella species; nonetheless, it has been isolated from the cornea, alone (Alexandrakis et al. 1998; Al-Herrawy & Gad 2017) or in combination with Acanthamoeba (Arnalich-Montiel et al. 2013) as a cause of keratitis.
CONCLUSION
N. italica was first identified in Egypt from the water of the Nile River. In addition, Vahlkampfia spp. and Hartmannella spp. were also detected. These newly discovered FLA in Egyptian aquatic environments need further phylogenetic studies, using bigger sample sizes from a greater variety of water sources. These studies are needed in order to evaluate the potential pathogenicity of these species to humans.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
FUNDING
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.