First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp.

Free-living amoebae (FLA) are amphizoic protozoans. Some species of this group are known to cause severe diseases such as fatal encephalitis and cutaneous injuries (Visvesvara et al. 2007; Trabelsi et al. 2012). Because they are free-living organisms, some environments are more likely to be a source of contamination due to the easy exposure of people to the place, such as some water courses intended for recreational purposes, especially on hot days. They have been isolated from different natural and anthropogenic environments, from a wide range of aquatic and land habitats worldwide (Rodríguez-Zaragoza 1994; Visvesvara et al. 2009).

FLA have a cystic structure capable of resisting in unfavourable environmental conditions, which provides viability in chlorine-treated water, food shortages, dissection, extreme pH, and different temperatures. Acanthamoeba, a genus responsible for causing granulomatous amoebic encephalitis in immunosuppressed individuals and amoebic keratitis in healthy people, are easily found in swimming pools and lakes. In addition, some potential pathogens species of the other FLA, such as Vermamoeba vermiformis (famous for causing keratitis when associated with Acanthamoeba), Naegleria fowleri and Naegleria gruberi (which can cause a serious central nervous system infection called primary amoebic meningoencephalitis), are also isolated from various water matrices (Fowler & Carter 1965; Cerva 1969; Jager & Stamm 1972; Aitken et al. 1996; Scheid et al. 2019).

These organisms are considered the ‘Trojan Horse’ of other microorganisms (e.g., bacteria, fungi, and viruses) and can act as promoters or vehicles for the dissemination of pathogens called amoeba-resistant microorganisms, which can survive and multiply within the FLA. In this way, Legionella pneumophila, Pseudomonas spp., Mycobacterium leprae, Candida auris, and adenovirus can survive in unfavourable conditions and continue to present great pathogenic potential (Winiecka-Krusnell et al. 2009; Lovieno et al. 2010).

The study in question aimed to determine the occurrence of pathogenic and opportunistic FLA in two watercourses of great social and economic importance in Porto Alegre, southern Brazil. One of them, Guaíba Lake, is used as a water supply for some metropolitan cities. In addition, the other is a watercourse that runs through the capital city and flows into the lake. Due to the easy access to the population, knowledge about the quality of these courses is essential.

All water samples were sedimented in a sedimentation cup. The sediment obtained after 24 h was centrifuged at 2,500 rpm for 10 min. Then, 200 μL of the sedimented material was transferred to a Petri dish containing 1.5% non-nutrient agar previously inoculated with heat-inactivated Escherichia coli (ATCC 8739). The dishes were inoculated at 30 °C for up to 40 days, while being examined daily and re-inoculated to eliminate any contamination by other microorganisms. Cloning, according to Diehl et al. (2021), was performed to obtain different isolates of FLA.

An extraction method capable of covering species of the Amoebidae family was developed. In this way, using a cell scraper and 3 mL of 1X phosphate-buffered saline (PBS), the amoebae were removed from the culture plates and subjected to 1,800rpm for 10 min. The pellet was resuspended in 500 μL of 1X PBS and homogenized. A total of 300 μL of the contents was transferred to a 2 mL Eppendorf, herewith 2.2 μL of proteinase K and 500 μL of 20% sodium dodecyl sulphate. After being homogenized by vortexing, the material was incubated in a water bath for 1.5 h at 60 °C. After the process, 800 μL of chloroform was added and vortexed. A total of 350 μL of protein precipitation (3M potassium acetate with 6.6 M glacial acetic acid) was added and shaken three times by hand. Then, the Eppendorf was centrifuged for 5 min at 11,000 rpm. The supernatant was transferred to a new 2 mL tube in which 1 mL of ice-cold absolute ethanol was added and homogenized by inversion for 2 min, to be later centrifuged at 11,000 rpm for 2 min. One millilitre of ice-cold 70% ethanol was added to the pellet and subjected to centrifugation again. The supernatant was discarded, and the pellet left to dry upside down for 10 min. After drying, 30 μL of Tris-EDTA buffer and 3 μL of RNAse were added to the microtube and incubated for 1 h at 37 °C. All extracted DNA was stored at −14 °C. The DNA was quantified using a nano spectrophotometer (Kasvi^® K23-0002, version 01/13).

To perform the amplification by polymerase chain reaction (PCR), three gene-specific oligonucleotides were used: JDP1 (5′-GGCCCAGATCGTTTACCGTGAA-3′) and JDP2 (5′- TCTCACAAGCTGCTAGGGGATA-3′) for Acanthamoeba spp.; ITS1 (5′-GAACCTGCGTAGGGATCATTT-3′) and ITS2 (5′-TTTCTTTTCCTCCCCTTATTA-3′) for Naegleria spp.; and Hv1227F (5′-TTACGAGGTCAGGACACTGT-3′) and Hv1728R (5′-GACCATCCGGAGTTCTCG-3′) for Vermamoeba spp. The PCR reactions were performed with 10 pmol of each primer, 2.5 mM of deoxynucleoside triphosphate (DNTP), 50 mM of MgCl₂, 2.5 μL of 10X buffer, and 1 U of Taq polymerase (Invitrogen^®) for a final volume of 25 μL. The PCR conditions were set to initial denaturation at 94 °C for 5 min followed by 30 cycles of 94 °C for 45 s, 60 °C for 40 s, and 72 °C for 1 min and 15 s (Acanthamoeba spp. according to Santos et al. 2022); 94 °C for 45 s, 58 °C for 40 s, and 72 °C for 30 s (Vermamoeba spp.); and 94 °C for 45 s, 55 °C for 40 s, and 72 °C for 1 min and 15 s (Naegleria spp. according to Henker et al. 2021). A final extension at 72 °C for 5 min was promoted in a SimpliAmp™ Thermal Cycler (Applied Biosystems). The negative control was performed with DNA-free, and the positive control was performed using a clinical isolate of N. fowleri (Henker et al. 2021) and Acanthamoeba spp. (Santos et al. 2022), and an environmental isolate of V. vermiformis (Soares et al. 2017). After PCR, the generated amplicons were submitted to electrophoresis and analysed on a 1.2% agarose gel.

The company ACTGene Molecular Analyzes performed sequencing using an ABI Prism 3500 Genetic Analyzer sequencer (Genetic Analyzer – Applied Biosystems^®). The primers used were the same as in the PCR reaction, but, at this stage, they were tested separately for each reaction (only the forward primer or only the reverse primer) using 4.5 pM of each primer for a final volume of 6 μL containing 60 ng. The sequences forward and reverse identified were analysed and submitted to homology analysis in BLAST^®, aligned by Clustal W 2.1, and deposited in the GenBank database.

The phylogenetic analysis was based on the ITS for Naegleria spp. and 18 S rDNA for Vermamoeba spp. The evolutionary analyses were conducted in MEGA11 to build a phylogenetic tree based on neighbour-joining using the forward and reverse sequences. To determine the statistical reliability of each node, 500 bootstrap replicates were performed. This analysis involved 21 nucleotide sequences.

A total of 46 clones were obtained: 21 from Dilúvio Stream and 25 from Guaíba Lake. Some clones had sizes consistent with the genera studied.

The sequences received from ACTGene were analysed in BLASTn to indicate the identity of the clones. Of these clones, only four reverted to a complete sequence that could be aligned together with BLASTn. Table 1 summarizes the results for all the samples with their respective registers in Genbank.

Our findings are demonstrated to be 93–98% identical with GenBank N. gruberi reference sequences, with a query coverage of 96–97% at BLASTn alignment. A total of 98–99% identical with V. vermiformis reference sequences with 95–97% of query coverage.

Despite the molecular identification by PCR for the presence of Acanthamoeba in both study areas, the amplicons sent for sequencing were not reverted to complete sequences that could be aligned and identified by similarity in BLASTn, and therefore, it was not possible to carry out a phylogenetic tree of these.

Table 2 shows the reference sequences used for the construction of the phylogenetic tree with its relevant information.

The neighbor-joining was used as the method for building the phylogenetic tree. The bootstrap consensus tree was inferred from 500 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The icons indicate sequences referring to the same clone.

Of the 46 clones obtained, only 23 were positive for genera known to contain potentially pathogenic species. It is important to highlight that all clones have morphological features of FLA, such as amoeboid shape, presence of contractile vacuole, pseudopods, among others. But none showed dimensions compatible with Sappinia spp. and Paravahlkampfia spp. (in Supplementary Information – SI) and therefore were not tested for both genera. Although the isolation sites were in water, the clones were also tested for the Balamuthia, but all of them were negative.

The results show that the negative clones for the target genera in this study belong to non-potentially pathogenic FLA, though this does not exclude their ability to host other microorganisms with pathogenic characteristics, ensuring their protection in hostile environments and assisting in proliferation.

Almost all points on Guaíba Lake were positive for Vermamoeba spp., with the exception of the second and ninth sampling points. For Acanthamoeba spp., the fourth was the only positive site. Points 1, 7, and 8 of Dilúvio Stream were positive only for Vermamoeba spp., points 5 and 6 were positive for Acanthamoeba spp., and point 4 was positive for Vermamoeba spp. and Naegleria spp. Point 4, the only one where it was possible to isolate two genera, was the first completely urbanized site composed of residences and enterprises, in addition to being a point that precedes a hospital.

Javanmard et al. (2017), in their research that covered different water bodies, demonstrated V. vermiformis as the free-living amoeba most present there, ranking above other genera such as Acanthamoeba and Naegleria. Still, these authors related their results to cases of V. vermiformis keratitis reported in the same region where the study was carried out. Fani et al. (2022) observed the same result. Our results indicate that although there are no cases of infection by V. vermiformis reported yet, users of both watercourses studied are still subject to a possible infection.

The first report of Naegleria philippineneis, Naegleria australiensis, Naegleria dobsoni, and N. gruberi in South American environmental samples was described by Bellini et al. (2020). In this way, this is the first report of V. vermiformis, N. gruberi, and Acanthamoeba spp. in southern Brazil in an environmental sample. Although there are not many studies that point to N. gruberi as an agent of any disease, Cerva (1969) reported a case of an 11-year-old boy who died of acute amoebic meningoencephalitis caused by N. gruberi.

As a result, this study points to the presence of different potentially pathogenic microorganisms, one of them responsible for promoting or intensifying some other pre-existing diseases (V. vermiformis). These results demonstrate that these environments are ideal means for the promotion of diseases caused by FLA agents when in contact with a possible host.

It is important to point out that in Brazil, little environmental research has been carried out so far for a survey and mapping of pathogenic FLA in aquatic and anthropogenic environments. Furthermore, this is the first study on natural sources carried out in Rio Grande do Sul, southern Brazil. The study in question has epidemiological and environmental importance, since the genera found have species responsible for infecting both humans and other animals, in addition to carrying other pathogens. The Dilúvio Stream and Guaíba Lake, which are aquatic and anthropogenic environments often used as recreational facilities, can serve as a means for transmission and contamination by FLA, which gives valuable information on the public health in the city. Thus, we believe that some measures can be taken to mitigate future FLA infections. Using control measures, such as placing signage at the access points to these watercourses, is an effective strategy to promote awareness and safety. The signage can alert people to the risks associated with that specific area, providing important instructions and guidance due to the presence of free-living amoebae known to cause serious infections of the skin, cornea and central nervous system. These measures help prevent FLA infections and ensure that people adopt safe behaviors around watercourses. Additionally, it is crucial that the signs are clear, visible, and understandable to all users.

This study was the result of an MSc thesis by B. T. S. Marinho in Federal University of Rio Grande do Sul, Rio Grande do Sul, Brazil. The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for the scholarship granted to B.T. S. Marinho.

All authors contributed to the study conception and design. The first draft of the manuscript was written by Brenda Teixeira Scardini Marinho and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.