Free-living amoebae (FLA) are amphizoic protozoans with a cosmopolitan distribution. Some strains of species are associated with infections in humans. They feed on microorganisms by phagocytosis; however, some of these can become endocytobionts by resisting this process and taking shelter inside the amoeba. The whole world is experiencing increasing shortage of water, and sewage is being reused, so the study of this environment is important in public health context. The objective of this work was to identify FLA present in sewage treatment plants in Porto Alegre, Brazil. About 1 L samples were collected from eight stations (raw and treated sewage) in January, February, July, and August 2022. The samples were sown in monoxenic culture, and the isolated amoebae were subjected to morphological and molecular identification. Polymerase chain reaction results indicated the presence of the genus Acanthamoeba in 100% of the samples. Gene sequencing showed the presence of Acanthamoeba lenticulata and Acanthamoeba polyphaga - T5 and T4 genotypes - respectively, which are related to pathogenicity. The environment where the sewage is released can be used in recreational activities, exposing individuals to potential interactions with these amoebae and their potential endocytobionts, which may pose risks to public health.

  • Free-living amoebae were isolated for the first time in sewage treatment plants (STPs) in Brazil.

  • In the STPs of Porto Alegre, RS, Brazil, only the genus Acanthamoeba was isolated, and genotypes associated with pathogenicity (T4 and T5) were identified.

  • The Guaíba lake and the Gravataí river receive the city's sewage and, consequently, can disperse Acanthamoeba spp.

  • Further research on the prevalence and identification of free-living amoebae in sewage is still needed in 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.

Study area and sample collection

The study was carried out in eight STPs, which were located in the city of Porto Alegre, southern Rio Grande do Sul, Brazil. The STPs in the study were STP Belém Novo, STP Navegantes, STP Serraria, STP Sarandi, STP Rubem Berta, STP Lami, STP do Bosque, and STP Parque do Arvoredo, which were identified on the map as shown in Figure 1.
Figure 1

Map of Porto Alegre with STPs.

Figure 1

Map of Porto Alegre with STPs.

Close modal

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.

Sample processing

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.

Morphological study of 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).

DNA extraction

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).

Molecular identification and sequencing

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.

Table 1

Specific oligonucleotides used

OrganismGeneSequence 5′–3′AnelamentAmplicon size (pb)Bibliography
Acanthamoeba Região ASA.S1 JDP1: GGCCCAGATCGTTTACCGTGAA
JDP2: TCTCACAAGCTGCTAGGGGATA 
60 °C for 40 s 450–500 Booton et al. (2005)  
Balamuthia 16S rRNA 5′Balspec16S: CGCATGTATGAAGAAGACCA
3′Balspec16S: TTACCTATATAATTGTCGATACCA 
48 °C for 40 s 1,075 Booton et al. (2003)  
Bacterial domain 16S rDNA 27F: AGAGTTTGATCCTGGCTCAG
1492R: GGTTACCTTGTTACGACTT 
55 °C for 60 s 1,200–1,600 Silva et al. (2015)  
Naegleria Region ITS ITS1: GAACCRGCGTAGGGATCATTT
ITS2:
TTTCTTTTCCTCCCCTTATTA 
55 °C for 40 s 400–453 Pélandakis et al. (2000)  
Vermamoeba 18S rDNA Hv1227F: TTACGAGGTCAGGACACTGT
Hv1728R: GACCATCCGGAGTTCTCG 
58 °C for 40 s 505 Kuiper et al. (2006)  
OrganismGeneSequence 5′–3′AnelamentAmplicon size (pb)Bibliography
Acanthamoeba Região ASA.S1 JDP1: GGCCCAGATCGTTTACCGTGAA
JDP2: TCTCACAAGCTGCTAGGGGATA 
60 °C for 40 s 450–500 Booton et al. (2005)  
Balamuthia 16S rRNA 5′Balspec16S: CGCATGTATGAAGAAGACCA
3′Balspec16S: TTACCTATATAATTGTCGATACCA 
48 °C for 40 s 1,075 Booton et al. (2003)  
Bacterial domain 16S rDNA 27F: AGAGTTTGATCCTGGCTCAG
1492R: GGTTACCTTGTTACGACTT 
55 °C for 60 s 1,200–1,600 Silva et al. (2015)  
Naegleria Region ITS ITS1: GAACCRGCGTAGGGATCATTT
ITS2:
TTTCTTTTCCTCCCCTTATTA 
55 °C for 40 s 400–453 Pélandakis et al. (2000)  
Vermamoeba 18S rDNA Hv1227F: TTACGAGGTCAGGACACTGT
Hv1728R: GACCATCCGGAGTTCTCG 
58 °C for 40 s 505 Kuiper et al. (2006)  

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.

Construction of the phylogenetic tree of the samples

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.

Morphological identification

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.

From the samples collected in the summer (Figure 2), a total of 19 clones were obtained, one clone from the affluent samples and one clone from the effluent, in the summer period for the STPs Belém Novo, Navegantes, Sarandi, Rubem Berta, do Bosque, and Parque do Arvoredo. In the STPs Serraria and Lami, one clone of the affluent and two clones of the effluent were obtained from each STP. It is noteworthy that additional clones were derived; however, the selection process was based on their morphology. While it is acknowledged that various other amoebae could potentially exist within this environment, employing this methodology allows us to deduce that the most dominant strains were isolated.
Figure 2

Free-living amoeba from summer samples. (a) STP Sarandi effluent cysts, bar = 37 μm; (b) trophozoites from the STP Lami affluent, bar = 36 μm; (c) cysts from the Parque do Arvoredo STP affluent, bar = 18.5 μm. 40× magnification (images a and b) and 400× magnification (image c).

Figure 2

Free-living amoeba from summer samples. (a) STP Sarandi effluent cysts, bar = 37 μm; (b) trophozoites from the STP Lami affluent, bar = 36 μm; (c) cysts from the Parque do Arvoredo STP affluent, bar = 18.5 μm. 40× magnification (images a and b) and 400× magnification (image c).

Close modal
As for the winter samples (Figure 3), a total of 18 clones were obtained, one clone from the affluent sample and one clone from the effluent, in the summer period for the STPs Belém Novo, Navegantes, Sarandi, Rubem Berta, Lami, do Bosque, and Parque do Arvoredo. From STP Serraria, one clone of the affluent and two clones of the effluent were obtained.
Figure 3

Free-living amoeba from winter samples. (a) Trophozoites from the tributary of STP Belém Novo, bar = 36 μm; (b) cysts from the STP Rubem Berta tributary, bar = 18.5 μm; (c) cyst from the STP do Bosque effluent, bar = 18.5 μm. 40× magnification (image a) and 400× magnification (images b and c).

Figure 3

Free-living amoeba from winter samples. (a) Trophozoites from the tributary of STP Belém Novo, bar = 36 μm; (b) cysts from the STP Rubem Berta tributary, bar = 18.5 μm; (c) cyst from the STP do Bosque effluent, bar = 18.5 μm. 40× magnification (image a) and 400× magnification (images b and c).

Close modal

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.

Cysts from 35 samples were compatible with Group II (Figure 4), which present rounded, polygonal, quadrangular, and triangular endocysts, which were characteristics of Group II in the classification (Visvesvara & Schuster 2008). Cysts from two samples were compatible with Group III (Figure 4), which presents a rounded or slightly angular endocyst (Visvesvara & Schuster 2008).
Figure 4

Summer sample of the effluent from the Belém Novo STP. (a) Trophozoite with acanthopodium shown by the arrow, bar = 18 μm and (b) polygonal cyst, bar = 18.5 μm. Winter sample of the STP Navegantes effluent. (c) Trophozoite with acanthopodium shown by the arrow, bar = 18 μm, and (d) polygonal cyst, bar = 18.5 μm. 400× magnification.

Figure 4

Summer sample of the effluent from the Belém Novo STP. (a) Trophozoite with acanthopodium shown by the arrow, bar = 18 μm and (b) polygonal cyst, bar = 18.5 μm. Winter sample of the STP Navegantes effluent. (c) Trophozoite with acanthopodium shown by the arrow, bar = 18 μm, and (d) polygonal cyst, bar = 18.5 μm. 400× magnification.

Close modal

Molecular identification of FLA

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).

Table 2

Analysis of the identity of summer clones

CloneScientific name/genotypeSequence compared in GenBank and identity (%)Identity GenBank record
BN_AFLU_B_F Acanthamoeba lenticulata/T5 MF076633.1 (95.40%) OR243669 
BN_EFLU_B_F Acanthamoeba lenticulata/T5 MF076633.1 (95.40%) OR233173 
NA_AFLU_C_R Acanthamoeba polyphaga/T4 KT892910.1 (99.27%) OR288588 
NA_EFLU_C_R Acanthamoeba polyphaga/T4 KY934458.1 (99.30%) OR288650 
SE_AFLU_C_F Acanthamoeba polyphaga/T4 KY934458.1 (99.30%) OR289670 
SE_EFLU_A Acanthamoeba polyphaga/T4 KR780551.1 (99.06%) OR364831 
SE_EFLU_C_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.49%) OR241303 
SA_AFLU_B_F Acanthamoeba polyphaga/T4 KT892910.1 (99.76%) OR288579 
SA_EFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.50%) OR241300 
RB_AFLU_A_F Acanthamoeba polyphaga/T4 KT892910.1 (99.53%) OR288523 
RB_EFLU1_B_F Acanthamoeba polyphaga/T4 KT892907.1 (97.52%) OR241297 
RB_EFLU2_A_R Acanthamoeba polyphaga/T4 MF576062.1 (99.49%) OR241299 
LA_AFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.25%) OR241295 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KT894164.1 (97.86%) OR241292 
LA_AFLU_B_F Acanthamoeba polyphaga/T4 KY934458.1 (99.28%) OR241289 
BO_AFLU_C_F Acanthamoeba polyphaga/T4 KT892907.1 (99.28%) OR241293 
BO_EFLU_C_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.49%) OR241286 
AR_AFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.51%) OR241283 
AR_EFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.75%) OR241284 
CloneScientific name/genotypeSequence compared in GenBank and identity (%)Identity GenBank record
BN_AFLU_B_F Acanthamoeba lenticulata/T5 MF076633.1 (95.40%) OR243669 
BN_EFLU_B_F Acanthamoeba lenticulata/T5 MF076633.1 (95.40%) OR233173 
NA_AFLU_C_R Acanthamoeba polyphaga/T4 KT892910.1 (99.27%) OR288588 
NA_EFLU_C_R Acanthamoeba polyphaga/T4 KY934458.1 (99.30%) OR288650 
SE_AFLU_C_F Acanthamoeba polyphaga/T4 KY934458.1 (99.30%) OR289670 
SE_EFLU_A Acanthamoeba polyphaga/T4 KR780551.1 (99.06%) OR364831 
SE_EFLU_C_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.49%) OR241303 
SA_AFLU_B_F Acanthamoeba polyphaga/T4 KT892910.1 (99.76%) OR288579 
SA_EFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.50%) OR241300 
RB_AFLU_A_F Acanthamoeba polyphaga/T4 KT892910.1 (99.53%) OR288523 
RB_EFLU1_B_F Acanthamoeba polyphaga/T4 KT892907.1 (97.52%) OR241297 
RB_EFLU2_A_R Acanthamoeba polyphaga/T4 MF576062.1 (99.49%) OR241299 
LA_AFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.25%) OR241295 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KT894164.1 (97.86%) OR241292 
LA_AFLU_B_F Acanthamoeba polyphaga/T4 KY934458.1 (99.28%) OR241289 
BO_AFLU_C_F Acanthamoeba polyphaga/T4 KT892907.1 (99.28%) OR241293 
BO_EFLU_C_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.49%) OR241286 
AR_AFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.51%) OR241283 
AR_EFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.75%) OR241284 

AFLU, affluent; EFLU, effluent; F, forward; R, reverse.

Table 3

Analysis of the identity of winter clones

CloneScientific name/genotypeSequence compared in GenBank and identity (%)Identity GenBank record
BN_AFLU_A_F Acanthamoeba polyphaga/T4 JQ408989.1 (98.51%) OR241290 
BN_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.24%) OR241302 
NA_AFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.75%) OR241282 
NA_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (95.44%) OR241281 
SE_AFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (99.24%) OR241287 
SE_EFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.25%) OR241291 
SE_EFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.28%) OR241285 
SA_AFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.75%) OR241280 
SA_EFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.75%) OR241301 
RB_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (97.75%) OR241294 
RB_EFLU1_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.00%) OR241298 
RB_EFLU2_B_F Acanthamoeba polyphaga/T4 KR780551.1 (98.25%) OR241288 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.73%) OR241296 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (97.75%) OR233179 
BO_AFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (98.49%) OR233178 
BO_EFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.49%) OR233175 
AR_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.00%) OR233176 
AR_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.48%) OR233174 
CloneScientific name/genotypeSequence compared in GenBank and identity (%)Identity GenBank record
BN_AFLU_A_F Acanthamoeba polyphaga/T4 JQ408989.1 (98.51%) OR241290 
BN_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.24%) OR241302 
NA_AFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.75%) OR241282 
NA_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (95.44%) OR241281 
SE_AFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (99.24%) OR241287 
SE_EFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.25%) OR241291 
SE_EFLU_B_F Acanthamoeba polyphaga/T4 KT892907.1 (99.28%) OR241285 
SA_AFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.75%) OR241280 
SA_EFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (99.75%) OR241301 
RB_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (97.75%) OR241294 
RB_EFLU1_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.00%) OR241298 
RB_EFLU2_B_F Acanthamoeba polyphaga/T4 KR780551.1 (98.25%) OR241288 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.73%) OR241296 
LA_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (97.75%) OR233179 
BO_AFLU_A_F Acanthamoeba polyphaga/T4 HQ833414.1 (98.49%) OR233178 
BO_EFLU_A_F Acanthamoeba polyphaga/T4 KT892907.1 (99.49%) OR233175 
AR_AFLU_A_F Acanthamoeba polyphaga/T4 KR780551.1 (98.00%) OR233176 
AR_EFLU_A_F Acanthamoeba polyphaga/T4 KX688025.1 (98.48%) OR233174 

AFLU, affluent; EFLU, effluent; F, forward; R, reverse.

Phylogenetic analysis of Acanthamoeba spp.

The 37 clones were used with five reference sequences (Table 4) for the construction of the phylogenetic tree. The evolutionary history was inferred using the Neighbor-Joining method. 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 tree was drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 42 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 485 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. The resulting tree can be seen in Figure 5.
Table 4

Reference sequences used to build the phylogenetic tree

BLASTn/accessionSourceStrain/isolation sourceSize (pb)
MF076633.1 Acanthamoeba lenticulata T5 Hospital environment 364 
KT892910.1 Acanthamoeba polyphaga T4 Environmental 452 
KT892907.1 Acanthamoeba polyphaga T4 Environmental 451 
KX688025.1 Acanthamoeba polyphaga T4 Hospital environment 405 
HQ833414.1 Acanthamoeba polyphaga T4 Hospital environment 399 
BLASTn/accessionSourceStrain/isolation sourceSize (pb)
MF076633.1 Acanthamoeba lenticulata T5 Hospital environment 364 
KT892910.1 Acanthamoeba polyphaga T4 Environmental 452 
KT892907.1 Acanthamoeba polyphaga T4 Environmental 451 
KX688025.1 Acanthamoeba polyphaga T4 Hospital environment 405 
HQ833414.1 Acanthamoeba polyphaga T4 Hospital environment 399 

Note: The sequences used for the construction of the phylogenetic tree were based on their similarity and/or similar isolation conditions to this work.

Figure 5

The evolutionary relationship of Acanthamoeba spp. was based on the sequence of Region ASA.S1. Summer clones are identified with yellow circles and winter clones are identified with blue circles.

Figure 5

The evolutionary relationship of Acanthamoeba spp. was based on the sequence of Region ASA.S1. Summer clones are identified with yellow circles and winter clones are identified with blue circles.

Close modal

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.

Al-Lahham
O.
,
El Assi
N. M.
&
Fayyad
M.
2003
Impact of treated wastewater irrigation on quality attributes and contamination of tomato fruit
.
Agricultural Water Management
61
(
1
),
51
62
.
doi:10.1016/S0378-3774(02)00173-7
.
ANA – National Water Agency (Agência Nacional de Águas) Brazil)
.
2017
Atlas Sewers: Depollution of River Basins (Atlas esgotos: despoluição de bacias hidrográficas)
.
ANA
,
Brasília
, p.
88
.
Bastos
R. K. X.
1999
Agricultural use of Sewage (Utilização agrícola de esgotos Sanitários)
.
Apostila de Curso, ABES-Recife, Brazil
, p.
116
.
Bellini
N. K.
,
Thiemann
O. H.
,
Reyes-Batlle
M.
,
Lorenzo-Morales
J.
&
Costa
A. O.
2022
A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil – a systematic review
.
Memórias do Instituto Oswaldo Cruz
117
,
e210373
.
doi:10.1590/0074-02760210373
.
Booton, G. C., Carmichael, J. R., Visvesvara, G. S., Byers, T. J. & Fuerst, P. A. 2003 Identification of Balamuthia mandrillaris by PCR assay using the mitochondrial 16S rRNA gene as a target. Journal of Clinical Microbiology 41 (1), 453–5. doi: 10.1128/JCM.41.1.453-455.2003.
Booton
G. C.
,
Visvesvara
G. S.
,
Byers
T. J.
,
Kelly
D. J.
&
Fuerst
P. A.
2005
Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections
.
Journal of Clinical Microbiology
43
(
4
),
1689
1693
.
doi:10.1128/JCM.43.4.1689-1693.2005
.
Cabello-Vílchez
A. M.
2015
Acanthamoeba spp. an opportunistic agent in human infections (Acanthamoeba spp. um agente oportunista em infecciones humanas)
.
Revista de Investigación de la Universidad Norbert Wiener
4
(
1
).
doi:10.37768/unw.rinv.04.01.002
.
Carlesso
A. M.
,
Simonetti
A. B.
,
Artuso
G. L.
&
Rott
M. B.
2007
Isolation and identification of potentially pathogenic free-living amoebae in samples
.
Revista da Sociedade Brasileira de Medicina Tropical
40
(
3
),
316
320
.
doi:10.1590/S0037-86822007000300013
.
Caumo
K.
,
Frasson
A. P.
,
Pens
C. J.
,
Panatieri
L. F.
,
Frazzon
A. P.
&
Rott
M. B.
2009
Potentially pathogenic Acanthamoeba in swimming pools: a survey in the southern Brazilian city of Porto Alegre
.
Annals of Tropical Medicine and Parasitology
103
(
6
),
477
485
.
doi:10.1179/136485909X451825
.
Delafont
V.
,
Rodier
M.-H.
,
Maisonneuve
E.
&
Cateau
E.
2018
Vermamoeba vermiformis: a free-living amoeba of interest
.
Microbial Ecology
76
,
991
1001
.
doi:10.1007/s00248-018-1199-8
.
De Souza
T. K.
,
Soares
S. S.
,
Benitez
L. B.
&
Rott
M. B.
2017
Interaction between Methicillin-resistant Staphylococcus aureus (MRSA) and Acanthamoeba polyphaga
.
Current Microbiology
74
(
5
),
541
549
.
doi:10.1007/s00284-017-1196-z
.
Diehl, M. L. N., Paes, J. & Rott, M. B. 2021 Single cell cloning of free-living amoebae with isolated square agar. Parasitology Research 120 (9), 3331–3333. doi: 10.1007/s00436-021-07275-9.
Dos Santos
D. L.
,
Virginio
V. G.
,
Berté
F. K.
,
Lorenzatto
K. R.
,
Marinho
D. R.
,
Kwitko
S.
,
Locatelli
C. L.
,
Freitas
E. C.
&
Rott
M. B.
2022
Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont
.
Parasitology Research
121
,
1447
1454
.
doi:10.1007/s00436-022-07474-y
.
Fabres
L. F.
,
Rosa Dos Santos
S. P.
,
Benitez
L. B.
&
Rott
M. B.
2016
Isolation and identification of Acanthamoeba spp. from thermal swimming pools and spas in Southern Brazil
.
Acta Parasitology
61
(
2
),
221
227
.
doi:10.1515/ap-2016-0031
.
Garner
E.
,
McLain
J.
,
Bowers
J.
,
Engelthaler
D. M.
,
Edwards
M. A.
&
Pruden
A.
2017
Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale reclaimed water distribution systems
.
Environmental Science and Technology
52
(
16
),
9056
9068
.
doi:10.1021/acs.est.8b02818
.
Gaze
W. H.
,
Morgan
G.
,
Zhang
L.
&
Wellington
E. M. H.
2011
Mimivirus-like particles in Acanthamoebae from sewage sludge
.
Emerging Infectious Diseases
17
(
6
),
1127
1129
.
doi:10.3201/eid1706.101282
.
Gomes
T. S.
,
Vaccaro
L.
,
Magnet
A.
,
Izquierdo
F.
,
Ollero
D.
,
Martínez-Fernández
C.
,
Mayo
L.
,
Moran
M.
,
Pozuelo
M. J.
,
Fenoy
S.
,
Hurtado
C.
&
Del Águila
C.
2020
Presence and interaction of free-living amoebae and amoeba-resisting bacteria in water from drinking water treatment plants
.
The Science of the Total Environmental
719
,
137080
.
doi:10.1016/j.scitotenv.2020.137080
.
Hajialilo
E.
,
Behnia
M.
,
Tarighi
F.
,
Niyyati
M.
&
Rezaeian
M.
2016
Isolation and genotyping of Acanthamoeba strains (T4, T9, and T11) from amoebic keratitis patients in Iran
.
Parasitology Research
115
(
8
),
3147
3151
.
doi:10.1007/s00436-016-5072-8
.
Henker
L. C.
,
Lorenzett
M. P.
,
Dos Santos
D. L.
,
Virginio
V. G.
,
Driemeier
D.
,
Rott
M. B.
&
Pavarini
S. P.
2021
Naegleria fowleri-associated meningoencephalitis in a cow in Southern Brazil – first molecular detection of N. fowleri in Brazil
.
Parasitology Research
120
,
2873
2879
.
doi.org/10.1007/s00436-021-07209-5
.
Hsu
B.-M.
,
Lin
C.-L.
&
Shis
F.-C.
2009
Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area
.
Water Research
43
(
11
),
2817
2828
.
doi:10.1016/j.watres.2009.04.002
.
INCT Sustentáveis – UFMG
.
2019
Main Sewage Treatment Methods (Principais métodos de tratamento de esgoto)
.
Available from: https://etes-sustentaveis.org/metodos-tratamento-de-esgoto/. (accessed 15 August 2019)
.
Jacobi
P. R.
,
Empinotti
V. L.
&
Schmidt
L.
2016
Water scarcity and human rights (Escassez Hídrica e Direitos Humanos)
.
Ambiente e Sociedade
19
(
1
).
doi:10.1590/1809-4422ASOCeditorialV1912016
.
José Maschio
V.
,
Corção
G.
&
Rott
M. B.
2015
Identification of Pseudomonas spp. as amoeba-resistant microorganisms in isolates of Acanthamoeba
.
Revista do Instituto de Medicina Tropical de São Paulo
57
(
1
),
81
83
.
doi:10.1590/S0036-46652015000100012
.
Khan, N. A. 2006 Acanthamoeba: biology and increasing importance in human health. FEMS Microbiology Reviews 30 (4), 564–595. doi: 10.1111/j.1574-6976.2006.00023.x.
Kuiper
M. W.
,
Valster
R. M.
,
Wullings
B. A.
,
Boonstra
H.
,
Smidt
H.
&
van der Kooij
D.
2006
Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR
.
Applied and Environmental Microbiology
72
(
9
),
5750
5756
.
doi:10.1128/AEM.00085-06
.
Lass
A.
,
Guerrero
M.
,
Li
X.
,
Karanis
G.
,
Ma
L.
&
Karanis
P.
2017
Detection of Acanthamoeba spp. in water samples collected from natural water reservoirs, sewages, and pharmaceutical factory drains using LAMP and PCR in China
.
Science of the Total Environment
584–585
,
489
494
.
doi:10.1016/j.scitotenv.2017.01.046
.
Lorenzo-Morales
J.
,
Martínez-Carretero
E.
,
Batista
N.
,
Alvarez-Marin
J.
,
Bahaya
Y.
,
Walochnik
J.
&
Valladares
B.
2007
Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella
.
Parasitology Research
102
(
1
),
167
169
.
doi:10.1007/s00436-007-0754-x
.
Manaia
C. M.
,
Rocha
J.
,
Scaccia
N.
,
Marano
R.
,
Radu
E.
,
Biancullo
F.
,
Cerqueira
F.
,
Fortunato
G.
,
Iakovides
I. C.
,
Zammit
I.
,
Kampouris
I.
,
Vaz-Moreira
I.
&
Nunes
O. C.
2018
Antibiotic resistance in wastewater treatment plants: tackling the black box
.
Environmental International
115
,
312
324
.
doi:10.1016/j.envint.2018.03.044
.
Marciano-Cabral
F.
&
Cabral
G.
2003
Acanthamoeba spp. as agent of disease in humans
.
Clinical Microbiology Reviews
16
(
2
),
273
307
.
doi:10.1128/CMR.16.2.273-307.2003
.
Maritz
J. M.
,
Eyck
T. A. T.
,
Alter
S. E.
&
Carlton
J. M.
2019
Patterns of protist diversity associated with raw sewage in New York City
.
The ISME Journal
13
,
2750
2763
.
doi:10.1038/s41396-019-0467-z
.
Milanez
G. D.
,
Carlos
K. B.
,
Adao
M. E.
,
Ayson
B. B.
,
Dicon
A. V.
,
Gahol
R. A. M.
,
Lacre
S. K. S.
,
Marquez
F. P. E.
,
Perez
A. J. M.
&
Karanis
P.
2023
Epidemiology of free-living amoebae infections in Africa: a review
.
Pathogens and Global Health
117
(
6
),
527
534
.
doi:10.1080/20477724.2022.2160890
.
Monteiro
M. A.
,
Spisso
B. F.
,
Santos
J. R. P. d.
,
Costa
R. P. d.
,
Ferreira
R. G.
,
Pereira
U. M.
,
Miranda
T. d. S.
,
Andrarde
B. R. G. d.
&
D'Avila
L. A.
2016
Occurrence of antimicrobials in river water samples from rural region of the State of Rio de Janeiro, Brazil
.
Journal of Environmental Protection
7
,
230
241
.
doi:10.4236/jep.2016.72020
.
Moreno-Mesonero
L.
,
Moreno
Y.
,
Alonso
J. L.
&
Ferrús
M. A.
2017
Detection of viable Helicobacter pylori inside free-living amoebae in wastewater and drinking water samples from Eastern Spain
.
Environmental Microbiology
19
(
10
),
4103
4112
.
doi:10.1111/1462-2920.13856
.
Muchesa
P.
,
Mwamba
O.
,
Barnard
T. G.
&
Bartie
C.
2014
Detection of free-living amoebae using amoebal enrichment in a wastewater treatment plant of Gauteng Province, South Africa
.
Biomedical Research International
,
575297
.
doi:10.1155/2014/575297
.
Municipality of Porto Alegre – Municipal Department of Water and Sewage
.
2013
Municipal Basic Sanitation Plan
.
Oliveira
T. F. d.
&
Buss
D. F.
2018
A protocol for evaluating the probability of the recreational use of rivers and its application in risk analysis
.
Ciência e saúde coletiva
23
(
11
),
3705
3717
.
doi:10.1590/1413-812320182311.22232016
.
Oliveira
M. C. d. S.
,
Regitano
L. C. d. A.
,
Roese
A. D.
,
Anthonisen
D. G.
,
Patrocínio
E. d.
,
Parma
M. M.
,
Scagliusi
S. M. M.
,
Timóteo
W. H. B.
&
Jardim
S. N.
2007
Theoretical-Practical Foundations and DNA Extraction and Amplification Protocols Through Polymerase Chain Reaction (Fundamentos teórico-práticos e protocolos de extração e de amplificação de DNA por meio de reação em cadeia da polimerase)
.
Embrapa Pecuária Sudeste
,
São Carlos
.
Page
F. C.
1988
A New Key to Freshwater and Soil Amoebae
.
Freshwater Biological Association Scientific Publications
,
Cumbria
,
UK
, p.
122
.
Pélandakis
M.
,
Serre
S.
&
Pernin
P.
2000
Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri
.
Journal of Eukaryotic Microbiology
47
(
2
),
116
121
.
doi:10.1111/j.1550-7408.2000.tb00020.x
.
Pussard, M. & Pons, R. 1977 Morphologie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 13, 557–598.
Pickup
Z. L.
,
Pickup
R.
&
Parry
J. D.
2007
Growth of Acanthamoeba and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey
.
FEMS Microbiology Ecology
61
(
2
),
264
272
.
doi:10.1111/j.1574-6941.2007.00346.x
.
Ramirez
E.
,
Robles
E.
,
Martinez
B.
,
Ayala
R.
,
Sainz
G.
,
Martinez
M. E.
&
Gonzalez
M. E.
2014
Distribution of free-living amoebae in a treatment system of textile industrial wastewater
.
Experimental Parasitology
145
(
Suppl
),
S34
S38
.
doi:10.1016/j.exppara.2014.07.006
.
Rodríguez-Zaragoza
S.
1994
Ecology of free-living amoebae
.
Critical Reviews in Microbiology
20
(
3
),
225
241
.
doi:10.3109/10408419409114556
.
Saeid
A.
,
Rahimi
H. M.
,
Niyuati
M.
,
Shalilieh
F.
,
Nemati
S.
,
Rouhani
S.
,
Zali
M. R.
,
Mirjalali
H.
&
Karanis
P.
2022
Free-living amoebae in an oil refinery wastewater treatment facility
.
Science of the Total Environment
839
,
156301
.
doi:10.1016/j.scitotenv.2022.156301
.
Salahuldeen
A.
,
El-Aziz
A. A.
,
Gad
M. A.
,
El-Nour
M. F. A.
&
Al-Herrawy
A. Z.
2021
An investigation on potentially pathogenic Acanthamoeba species from a wastewater treatment plant in Alexandria, Egypt
.
Egyptian Journal of Aquatic Biology and Fisheries
25
(
2
),
1067
1079
.
doi:10.21608/EJABF.2021.170934
.
Scheid
P.
2018
Free-living amoebae as human parasites and hosts for pathogenic microorganisms
.
Proceedings
2
(
11
),
692
.
doi:10.3390/proceedings2110692
.
Scheid
P.
2019
Free-living amoebae in rivers and ponds and their multiple role in environmental health
.
Parasitology Research Monographs
12
,
431
444
.
doi:10.1007/978-3-030-29061-0_20
.
Schuster
F. L.
2002
Cultivation of pathogenic and opportunistic free-living amebas
.
Clinical Microbiology Review
15
(
3
),
342
354
.
doi:10.1128/CMR.15.3.342-354.2002
.
Schuster
F. L.
&
Visvesvara
G. S.
2004
Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals
.
International Journal for Parasitology
34
(
9
),
1001
1027
.
doi:10.1016/j.ijpara.2004.06.004
.
Siddiqui
R.
&
Khan
N. A.
2012
Biology and pathogenesis of Acanthamoeba
.
Parasites and Vectors
5
,
6
.
doi:10.1186/1756-3305-5-6
.
Silva, T. N. L., Chaves, M. A., Granjeiro, P. A., Valadares, H. M., Lopes, D. de O. & Magalhães, J. T. de. 2015 Molecular identification of lamic bacteria with probiotic potential present in artisan standard minas cheese (Identificação molecular de bactérias lácticas com potencial probiótico presentes em queijo minas padrão artesanal). 87. In: Anais da V Jornada Acadêmica Internacional de Bioquímica [Blucher Biochemistry Proceedings, vol.1, n.1]. São Paulo, Brazil: Blucher. doi: 10.5151/biochem-jaibqi-0093
Simões
K. d. S.
,
Peixoto
M. d. F. d. S. P.
,
Almeida
A. T.
,
Ledo
C. A. d. S.
,
Peixoto
C. P.
&
Pereira
F. A. d. C.
2013
Wastewater from treated domestic sewage on soil microbial activity and castor bean growth (Água residuária de esgoto doméstico tratado na atividade microbiana do solo e crescimento da mamoneira)
.
Revista Brasileira de Engenharia Agrícola e Ambiental
17
(
5
),
310
316
.
doi:10.1590/S1415-43662013000500008
.
Soares
S. S.
,
Souza
T. K.
,
Berté
F. K.
,
Cantarelli
V. V.
&
Rott
M. B.
2017
Occurrence of infected free-living amoebae in cooling towers of Southern Brazil
.
Current Microbiology
74
,
1461
1468
.
doi:10.1007/s00284-017-1341-8
.
Soller
J. A.
,
Schoen
M. E.
,
Bartrand
T.
,
Ravenscroft
J. E.
&
Ashbolt
N. J.
2010
Estimated human health risks from exposure to recreational waters impacted by human and non-human sources of faecal contamination
.
Water Research
44
(
16
),
4674
4691
.
doi:10.1016/j.watres.2010.06.049
.
Trabelsi
H.
,
Dendana
F.
,
Sellami
A.
,
Sellami
H.
,
Cheikhrouhou
F.
,
Neji
S.
,
Makni
F.
&
Ayadi
A.
2012
Pathogenic free-living amoebae: epidemiology and clinical review
.
Pathologie Biologie (Paris)
60
(
6
),
399
405
.
doi:10.1016/j.patbio.2012.03.002
.
Visvesvara
G. S.
&
Schuster
F. L.
2008
Opportunistic free-living amebae, part II
.
Clinical Microbiology Newsletter
30
(
21
),
159
166
.
doi:10.1016/j.clinmicnews.2008.10.001
.
Visvesvara
G. S.
,
Moura
H.
&
Schuster
F. L.
2007
Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea
.
FEMS Immunology & Medical Microbiology
50
(
1
),
1
26
.
doi:10.1111/j.1574-695X.2007.00232.x
.
Visvesvara
G. S.
,
Sriram
R.
,
Qvarnstrom
Y.
,
Bandyopadhyay
K.
,
Da Silva
A. J.
,
Pieniazek
N. J.
&
Cabral
G. A.
2009
Paravahlkampfia francinae n. sp. masquerading as an agent of primary amoebic meningoencephalitis
.
The Journal of Eukaryotic Microbiology
56
(
4
),
357
366
.
doi:10.1111/j.1550-7408.2009.00410.x
.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

Supplementary data