First report of free-living amoebae in sewage treatment plants in Porto Alegre, southern Brazil

Free-living amoebae (FLA) are protozoan, which can be found in water for human consumption, water courses, sewage, hot springs, lakes, swimming pools, and rivers (Khan 2006; Carlesso et al. 2007; De Souza et al. 2017; Scheid 2018), which are known as amphizoics, as they can be in the environment or parasitizing humans and animals. Among some strains of potentially pathogenic species and/or genera we have: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, Paravahlkampfia, Sappinia, and Vermamoeba vermiformis, that can cause diseases such as keratitis, granulomatous amoebic encephalitis (GAE), skin lesions, lung lesions and primary amoebic meningoencephalitis (PAM), in association or alone (Rodríguez-Zaragoza 1994; Visvesvara et al. 2009; Trabelsi et al. 2012; José Maschio et al. 2015; Delafont et al. 2018; Scheid 2019).

These amoebae have two morphological stages, the trophozoite (active form) and the cyst (resistance form), except N. fowleri which has an intermediate flagellate stage (Visvesvara et al. 2007; Delafont et al. 2018). Nutrition is carried out by amoebic phagocytosis of algae, bacteria, fungi, or protozoa. However, some microorganisms can survive this process, shelter inside, and protect themselves from chlorination and biocidal processes; these microorganisms are called endocytobionts (Lorenzo-Morales et al. 2007; De Souza et al. 2017; Scheid 2019; Gomes et al. 2020). In this way, some amoebae can serve as a ‘Trojan horse’, carrying and disseminating some microorganisms such as Bacillus anthracis, Cryptococcus neoformans, Escherichia coli O157, Legionella pneumophila, Staphylococcus aureus methicillin-resistant and others (Balczun & Scheid 2017; De Souza et al. 2017; Soares et al. 2017; Scheid 2019).

Data from the World Health Organization (WHO) revealed that about 85% of known diseases are associated with or transmitted by water (Monteiro et al. 2016). Wastewater (sewage) is composed of several microorganisms, which vary depending on the location and seasonality, and the indicator of the quality of the water that is released into the environment can be done with the microbiological analysis of these waters (Maritz et al. 2019).

In Brazil, 55% of sewage is treated, with 43% of the population having sewage collected and treated and 12% using a septic tank (individual solution). However, 45% of the sewage that is generated in the country is still not treated; this problem is associated with deficits in sewage collected and in the treatment capacity of sewage treatment plants (STPs) (ANA 2017). The most used sewage treatments in Brazil are activated sludge, stabilization ponds, and anaerobic sewage treatments (INCT 2019).

The treatment of sewage by activated sludge is considered an aerobic treatment, where the activated sludge is composed of a mass of algae, bacteria, fungi, and protozoa, which make the degradation of the organic matter present in the sewage in the presence of oxygen. The effluent is directed to a tank for aeration, where the organic matter is consumed by the aerobic microorganisms and after that, the effluent is sent to the decanter where the solid part (sludge) is separated from the treated sewage. This is one of the most used treatments in Brazil (Manaia et al. 2018; INCT 2019).

Stabilization ponds are straightforward treatment systems, offering various configurations based on the available conditions. These configurations include aerobic, anaerobic, facultative, maturation and polishing ponds. Within these ponds, the breakdown of organic matter can take place within an aerobic, anaerobic, or combined environment, contingent upon the specific type of pond. Warmer regions accelerate the metabolic rate of microorganisms, although this can be detrimental to the overall efficiency of the ponds (INCT 2019).

The anaerobic sewage treatment takes place using anaerobic microorganisms that break down organic matter. In this process, the affluent passes through a sealed reactor, and the organic matter undergoes degradation. There are two primary types of sealed reactors: the Upflow Anaerobic Reactor (UAR) and the Upflow Anaerobic Sludge Blanket (UASB). The UASB treatment is like the UAR treatment; however, it can capture the biogas generated during the degradation of organic matter, which can then be utilized for energy production. This approach is the most widely employed treatment method in Brazil (INCT 2019).

Sewage is released into the aquatic environment, a medium that can be used for aquatic and recreational activities, such as swimming and diving, but these environments can present a potential risk of the transmission of pathogenic microorganisms to individuals in these places (Soller et al. 2010; Oliveira & Buss 2018). The scarcity of drinking water in the world is increasing (Jacobi et al. 2016), and with this, solutions to reuse water have been used, such as the use of sewage as a source of water for irrigation of plantations, which has been a practice adopted for years (Bastos 1999; Simões et al. 2013). However, one concern is that sewage can contain an abundance of biological contaminants, and these can end up accumulating in soil and plants (Al-Lahham et al. 2003).

The present study aimed to isolate potentially pathogenic FLA in eight STPs in Porto Alegre, RS, Brazil. The treated sewage from these stations is released into Lake Guaíba and/or the Gravataí River, which are easily accessible to the population and commonly utilized for recreational purposes.

The characteristics of the STPs can be obtained from the Municipal Basic Sanitation Plan (2013) of Porto Alegre, where the STPs such as Belém Novo and Lami use treatment by a stabilization pond and release treated sewage into Lake Guaíba; the STPs such as Sarandi and Bosque use treatment by the UASB and release the treated sewage into the Gravataí River; STP Serraria uses treatment by the UASB and release treated sewage into Lake Guaíba, and STPs Navegantes, Rubem Berta and Parque do Arvoredo use activated sludge treatment and release treated sewage into the Gravataí River.

Seventeen samples were collected in the summer period (January and February) and 17 samples in the winter period (July and August), with two samples from each point: affluent (1) and effluent (1) from each station, except for STP Rubem Berta which has two different modules in operation, each one of them in a treatment phase and, therefore, three samples were collected: affluent (1) and effluent (2), totaling 34 samples. Samples were collected in 1 L sterilized glass bottles.

All water samples were sedimented in a sedimentation cup for 24 h. Afterwards, the sediment obtained was centrifuged at 2,500 rpm for 10 min. After centrifugation, 200 μL of the sedimented material was transferred to a Petri dish containing 1.5% non-nutrient agar previously coated with heat-inactivated E. coli (ATCC 10536), and this process was performed in quintuplicate. The plates were inoculated at 30 °C (ideal FLA growth temperature) (Schuster 2002) for up to 15 days, examined daily, and reinoculated to eliminate any contamination by other microorganisms. Cellular cloning was performed by using the methodology of Diehl et al. (2021) to obtain different FLA isolates.

Each selected clone was analyzed for movement, size, and morphological aspects of trophozoites and cysts, presence of acanthopods, and contraction time of contractile vacuoles using the criteria described by Page (1988). The diameter of the trophozoites and cysts was randomly measured by selecting 10 cells from each isolate; and the contraction time of the pulsatile vacuole of five cells randomly selected was also noted. For the identification of groups of the genus Acanthamoeba, the morphology of the cysts was analyzed as proposed by Pussard & Pons (1977) 3 days after the encystment process. For the species N. fowleri identification, which has the flagellar phase, the exflagellation technique was performed (Silva & Rosa 2003).

An Na-adapted protocol from Embrapa (Oliveira et al. 2007) was used to extract DNA. Briefly, the plate containing amoebae was placed in contact with ice, 3 mL of 1× phosphate-buffered saline solution (PBS) was added, and gentle movements were made to detach the amoebae from the agar. The liquid from the plates was placed in a Falcon tube and centrifuged at 1,800 rpm for 10 min. The supernatant was discarded and the pellet was resuspended in 500 μL of 1× PBS and homogenized. About 300 μL of the homogenate was transferred to a 2 mL microtube, and 2.2 μL of proteinase K and 500 μL of 20% sodium dodecyl sulfate were added, homogenized by vortexing, and incubated in a water bath for 1.5 h at 60 °C. Subsequently, 800 μL of chloroform was added and vortexed, and 350 μL of protein precipitation solution was added (3 M potassium acetate with 6.6 M glacial acetic acid) and stirred three times by hand. Afterwards, the microtube was centrifuged for 15 min at 11,000 rpm. Approximately 1 mL of the supernatant was transferred to a new 2 mL tube in which 1 mL of cold absolute ethanol was added and homogenized by inversion for 2 min, after which it was centrifuged at 11,000 rpm for 15 min. Discarding the supernatant, 1 mL of ice-cold 70% ethanol was added and centrifuged at 11,000 rpm for 5 min. After discarding the supernatant, the open microtube was placed on a flat surface for 10 min. After drying, 30 μL of Tris–EDTA buffer and 3 μL of RNAse were added and incubated for 1 h at 37 °C. All extracted DNA was stored at −14 °C. The DNA was quantified with a nanospectrophotometer (Kasvi^® K23-0002, version 01/13).

To perform the polymerase chain reaction (PCR) amplification technique, gene-specific oligonucleotides were used (Table 1). PCRs were performed with 10 pmol of each primer, 2.5 mM deoxynucleoside triphosphate (DNTP), 50 mM MgCl₂, 2.5 μL of 10× buffer, and 1 U of Taq polymerase (Invitrogen^®) to a final volume of 25 μL.

Thermal cycling conditions common to Acanthamoeba spp., Naegleria spp., and Vermamoeba spp. were initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 45 s, extension at 72 °C for 1 min and 15 s for Acanthamoeba spp. and Naegleria spp. and 72 °C for 30 s for Vermamoeba spp., and, for Domain Bacteria, thermal cycling conditions were initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 60 s and extension at 72 °C for 2 min. The annealing conditions and sequence are in Supplementary Table S1.

The negative control was made with DNA-free, and the positive control was made with the clinical isolate of Acanthamoeba spp. (Dos Santos et al. 2022), Domain Bacteria (Dos Santos et al. 2022), and N. fowleri (Henker et al. 2021), and an environmental isolate of V. vermiformis (Soares et al. 2017). After the PCR, the generated amplicons were submitted to electrophoresis and analyzed in 1.2% agarose gel. Sequencing was performed using the same specific primers by the company ACTGene Molecular analyses using an ABI Prism 3500 Genetic Analyzer sequencer (Genetic Analyzer – Applied Biosystems^®). The identified forward and reverse sequences were analyzed 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 results obtained in sequencing. Evolutionary analyses were performed on MEGA11 to build a phylogenetic tree based on the Neighbor-Joining method using forward and reverse sequences. To determine the statistical reliability of each node, 500 bootstrap replicates were performed. This analysis involved 42 nucleotide sequences.

Of the 34 samples that were collected from the STPs, all (100%) were positive for the presence of FLA. Using the cell cloning technique, a total of 37 clones were selected.

The clones had their morphological characteristics analyzed, and the contraction time of the pulsatile vacuoles timed (Supplementary Tables S2 and S3). Some amoebae had their dimensions compatible with species of the genera Acanthamoeba and Naegleria. None of the samples showed dimensions that coincided with species of the genera Balamuthia, Vermamoeba, Paravahlkampfia, or Sappinia.

Pussard & Pons's (1977) classification was used for all samples, since they presented morphological characteristics compatible with Acanthamoeba, such as slow locomotion, cytoplasm with well-defined endoplasm and ectoplasm, presence of acanthopods, and presence of a nucleus with a perinuclear halo.

After DNA extraction, the PCR was used as a molecular tool for identification. The reaction was performed for the 37 clones obtained and the results indicated positivity for the genus Acanthamoeba in 100% of the samples (Supplementary Table S4). The exflagellation test was performed for all samples, and the result was negative.

Sequences obtained were submitted for analysis using the BLAST (Basic Local Alignment Search Tool) program of the U.S. National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST) in order to classify the Acanthamoeba isolates. All isolates obtained identity greater than 95.4% reaching 99.76%, and the sequences were deposited in GenBank (Tables 2 and 3).

FLA feed on microorganisms such as algae, bacteria, fungi, protozoa, and organic matter, which is important in the maintenance of the ecosystem (Pickup et al. 2007). Despite not having been identified, with the oligonucleotides used, endocytobionts in the samples of this study, the capacity of Acanthamoeba spp. harboring endocytobionts is a risk to human health, as the amoeba protects chlorination processes for pathogenic microorganisms, processes that occur in STPs, and protection against biocides, which are used to combat microorganisms. Thus, Acanthamoeba spp. acts as a reservoir of microorganisms and can transmit them to the host (De Souza et al. 2017; Delafont et al. 2018; Scheid 2018, 2019).

In the present study, Acanthamoeba spp. were isolated in 100% of the isolates both in morphological identification and in the PCR technique. This data is similar to those found in the literature since studies carried out in aquatic environments demonstrate the genus Acanthamoeba spp. as the most isolated worldwide, and also the one with the highest number of species and pathogenic genotypes (Carlesso et al. 2007; Bellini et al. 2022; Milanez et al. 2023). Of the 37 isolates, two species with the T5 genotype and 35 with the T4 genotype were identified. These genotypes are known to be related to infections in humans (Siddiqui & Khan 2012; Fabres et al. 2016; Hajialilo et al. 2016).

Marín et al. (2015) collected samples from a domestic STP for 1 year and found Acanthamoeba in both raw and treated sewage, commenting that the treatment that was used in that plant was not effective in eliminating Acanthamoeba. Ramirez et al. (2014), in a textile STP, found species of the genera Acanthamoeba, Naegleria, and Vermamoeba, a worrying fact since the textile effluent is rich in residues of dyes, fixatives, and surfactants, evidencing the resistance of FLA in different environments.

Another study analyzed an STP at an oil refinery, an environment that is known to have pH variations and high loads of toxic components in the water; the study found species of Acanthamoeba with genotypes T4 and T11 (Saeid et al. 2022). These genotypes are known to cause infections in humans (Hajialilo et al. 2016).

Acanthamoeba polyphaga species is known to cause keratitis in humans (Marciano-Cabral & Cabral 2003; Schuster & Visvesvara 2004; Caumo et al. 2009), and the species A. lenticulata is associated with cases of keratitis and granulomatous amoebic encephalitis (Cabello-Vílchez 2015). Therefore, the presence of these species in the treated and untreated sewage in our work is worrying, as they may present a risk to individuals who use Lake Guaíba and River Gravataí for aquatic activities.

The presence of Acanthamoeba spp. in treated and untreated sewage has already been studied in some parts of the world such as South Africa, China, Egypt, Spain, the United States of America (USA), Iran, Mexico, the United Kingdom, and Taiwan (Hsu et al. 2009; Gaze et al. 2011; Muchesa et al. 2014; Ramirez et al. 2014; Garner et al. 2017; Lass et al. 2017; Moreno-Mesonero et al. 2017; Salahuldeen et al. 2021; Saeid et al. 2022). However, so far in Brazil, no study has been carried out investigating potentially pathogenic FLA in STPs, with the present study being the first carried out in our country.

The cultivation of FLA was successful, showing the isolation of amoebae in all samples. Using morphological identification and the PCR technique, it was possible to identify the genus Acanthamoeba in all analyzed samples, being the species A. polyphaga and A. lenticulata with genotypes T4 and T5, respectively, identified both genotypes associated with human diseases.

Our study presents environmental and public health importance, since the FLA genus found is known to cause keratitis, granulomatous amoebic encephalitis, and skin and lung infections in humans, in addition to being known to carry pathogenic or non-pathogenic endocytobionts.

The aquatic environment, where treated sewage is discharged, is used for recreation by immunocompetent individuals or not, presenting a risk to these individuals, either by infections caused by the FLA themselves, or by diseases caused by possible endocytobionts carried by them. As it was the first study carried out on STPs in Brazil, it is suggested that further research be carried out in this environmental field to collect more data on these natural and anthropogenic environments.

This study was the result of Silva's master's thesis, TCB at the Federal University of Rio Grande do Sul, Rio Grande do Sul, Brazil. The authors thank CAPES (Coordination for the Improvement of Personnel and Higher Education) for the scholarship granted to T.C.B.S., and CNPq (National Council for Scientific and Technological Development) for the research scholarship to M.B.R.

No funding was received to carry out this study.

T.C.B.S. conceived and wrote the project, collected and analyzed the data, and wrote the manuscript. D.L.d.S. performed data verification and revised the manuscript. M.B.R. managed the project and revised the manuscript. All authors approved the publication of this version of the manuscript.

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

The authors declare there is no conflict.