Molecular characterization of Blastocystis subtypes isolated in the city of Uberaba, Minas Gerais State, Brazil

Abstract INTRODUCTION Blastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates. METHODS Blastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification. RESULTS Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific. CONCLUSIONS The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible.


INTRODUCTION
Blastocystis is a Stramenopile of the Blastocystidae family with a cosmopolitan distribution; they are the most prevalent parasites found in human feces worldwide despite the distinction between their colonization and infection not being well distinguished thus far [1][2][3][4] . Literature suggests that the parasite can interact with the host's microbial flora; however, the consequences of this interaction are not yet well known 4 .
Although underestimated, the prevalence of Blastocystis spp. in human hosts ranges from 17.8% to 86.63% in Brazil, as estimated by the diagnostic methods employed and the technical ability of laboratory technicians for its recognition 1,5,6 .
This study aimed to: (i) genetically characterize the isolates of Blastocystis species obtained from human fecal samples and water supply in the city of Uberaba, MG, Brazil, and (ii) verify the phylogenetic relationship between Blastocystis isolates from humans, pigs, cattle, and water, to define the epidemiological profile of the parasite in this region.

Blastocystis samples
In this study, Blastocystis species present in 26 fecal samples obtained from Uberaba, MG, Brazil, were genetically characterized. Of these, 21 samples were of human origin 6 , four pig feces, and one bovine 26 . In addition, the presence of Blastocystis DNA was tested in 18 water supply samples 27 in the same area.

DNA extraction and PCR
Total DNA was extracted from the fecal samples using the immunomagnetic Magnex DNA Kit (Labtest Diagnóstica S.A., Minas Gerais, Brazil) according to the manufacturer's instructions and protocol described by Moreira et al. 27 . Samples were screened for Blastocystis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described 28 . Briefly, the SSU rDNA gene fragment amplified by PCR with primers 1FB and 1RB ( Table 1) was SpeI-digested to infer parasite classification 28 . PCR-sequence-tagged site (PCR-STS) analysis was performed, and the subtypes were determined 7,29 .
Bovine and pig samples were obtained from another study by our team 26 . The authors classified the S03 pig sample as ST1 using the same methods used in the present study. However, the authors could not determine the subtypes of the pig samples S01, S02, and S06, or bovine sample B01; nonetheless, they were sequenced.

DNA sequencing and analysis
All amplicons from human and supply water samples were partially sequenced. To obtain the sequence of the entire SSU rDNA gene fragment, two pairs of internal primers ( Table 1) were designed, and two additional PCRs were performed, namely PCR-INT 1 and PCR-INT 2, amplifying 907 bp and 850 bp fragments of the SSU rDNA gene, respectively; the amplified products were sequenced. All sequencing reactions were performed using the ABI Prism BigDye Terminator version 3.1, Cycle Sequencing Kits® (Applied Biosystems, Inc., Grand Island, USA) and analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were curated, and the consensus sequence of each sample was generated using the ChromasPro® version 1.7.6.
SSU-rDNA PCR fragments of some Blastocystis isolates from humans (n=7), pigs (n=3), and bovines (n=1) 26 were not classified by PCR-RFLP and PCR-STS; however, they were sequenced. To mitigate this problem, these sequences were aligned using data regarding Blastocystis present in the GenBank database. With sequence alignment greater than 95% and an error less than 5%, the subtypes were defined according to the Genbank classification based on the Stensvold and Clark (2016) statement 4 .
The similarity analysis of the sequence generated with GenBank data was verified, and phylogenetic analysis was performed using the Mega® version 6 software 30 , based on the SSU rRNA dataset of Blastocystis by the 95 Maximum Likelihood method (ML) with 1000 bootstrap replicates. Reference sequences obtained from Blastocystis isolates ST1 (MK801358), ST3 (MK801403), ST5 (MK801414), ST7 (AF408427), and ST10 (MH507326) were used to make comparisons in dendrogram analysis; thus, an outgroup represented for Thecamonas trahens (XR_001290156).

RESULTS
PCR amplicons of 21 isolates from human fecal samples and 18 isolates from water samples were generated; however, these had different sizes (0.9 to 1.3 kb) than expected (1.1 kb) in 18/21 isolates from humans and 17/18 water isolates.
PCR-RFLP showed the restriction profiles of 14 human isolates ( Table 2). After performing the PCR-STS following the generated profile, eight subtypes of Blastocystis were defined: five as ST1, two as ST3, and one as ST2 ( Table 2). Three samples did not have sufficient DNA for PCR-STS analysis, and three were not amplified.
Seven Blastocystis isolates from humans did not show a restriction profile after performing PCR-RFLP, probably because of the small amount of amplified DNA. In this case, PCR-STS was performed with all primer pairs to define each subtype of Blastocystis (ST1-ST7); however, no sample was amplified, and the parasite subtype could not be defined.
Amplicons obtained from Blastocystis isolates from water samples did not show any restriction profile after PCR-RFLP, and the DNA was not amplified by PCR-STS.
In this study, high-quality sequences of 13 isolates from human fecal samples, 1 of an isolate from a bovine sample, 4 of isolates from pig samples ( Table 2), and 4 of isolates from water samples were generated. However, analysis of the water sequences showed that the products amplified by PCR corresponded to the DNA of the algae, Eustigmatophyceae, and not to Blastocystis, thus showing that this PCR is not suitable for environmental samples.

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in GenBank. Comparison of these sequences with GenBank data showed polymorphism of the SSU rDNA gene, showing substitutions in some regions and insertions or deletions (indel events) in others ( Figure S1). Additionally, this analysis allowed the inference of the subtypes of three isolates of Blastocystis from pigs (2 ST5, 1 ST3), one from bovine (ST10), and seven from humans (3 ST1, 4 ST3) not classified by PCR-STS as described in the Methods section (Table 2, Figure 1).
Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by the isolates characterized as ST1, ST5 (S01, S02) and ST10 (B01). Another group was formed with isolates characterized as ST3 and ST7 (Figure 1).

DISCUSSION
In the present study, we confirmed that the SSU rDNA gene is polymorphic, as DNA products of different sizes from the described 1.1 kb fragment 29 were amplified in human feces, indicating a similarity of approximately 99% with the Blastocystis sequences deposited in GenBank. Several indel events were observed in the generated sequences.
These results showed ST1-ST3, ST5, and ST10 in the city of Uberaba, with ST5 and ST10 present only in pigs and cattle, respectively. ST1 and ST3 were isolated from both humans and pigs, demonstrating the zoonotic potential of transmission of Blastocystis species, as reported by other authors [31][32][33][34] .
Few studies have been carried out in Brazil regarding the geographic distribution of Blastocystis subtypes and their hosts 35 . ST1-ST4 have been reported to date, with a predominance of ST1 and ST3 21,26,[35][36] . In the world panorama, the most prevalent subtypes of Blastocystis spp. are ST1-ST4, but ST5-ST9 and ST12 have also been described and have different regional prevalence [37][38][39] . Mixed infections have also been reported 11 , showing that complex genotypes may occur in various regions of the world.
Phylogenetic analysis showed that sequences originating from humans formed two distinct groups, one of which contained the 5/6 sequences of the isolates characterized as ST1 and the other with the isolates characterized as ST3. In recent studies 22,35 , the grouping of subtypes into separate clades was shown by phylogenetic analysis of the different Blastocystis subtypes observed in Brazil in the South, Southeast, and Midwest regions. Pig isolates, although more similar to each other than human isolates, were divided into clades of the dendrogram, either isolated or belonging to one of the groups of human isolates. These data reinforce the potential for zoonotic transmission 22,35,40 .
In our study, 4 of the 18 water samples amplified with Blastocystis-specific primers followed by sequencing did not correspond to sequences of this parasite. Other studies in Brazil using PCR with primers other than those we used showed that amplified DNA fragments of water samples from the Tietê River, State of São Paulo, also did not correspond to the Blastocystis sequences deposited in GenBank 35 . These results show that the primers directed to rDNA targets, which are highly conserved regions among eukaryotes, are inadequate for investigating Blastocystis in environmental samples. Consequently, the use of primers directed to these regions is a limitation for investigating Blastocystis in environmental samples; their use in PCR tests may contribute to false-positive results if other techniques, such as DNA sequencing, are not employed. These observations point to the need for developing new specific primers and/or new techniques to investigate the presence of Blastocystis in environmental samples.
Based on our study, we concluded that the SSU rDNA gene is polymorphic and may define intraspecific variations leading to the grouping of the isolates according to their genetic characteristics. In addition, it was verified that in the studied region, the subtypes of Blastocystis were ST1, ST3, and ST2, and zoonotic transmission is possible, as ST1 was found in both humans and pigs in the region. Finally, it was observed that the PCR of the SSU rDNA gene was not useful for the detection of Blastocystis in environmental samples, as it amplified the non-specific DNA of algae.

ACKNOWLEDGMENTS
We offer our deepest thanks to the institutions that provided technical support for the development and implementation of this study.

FINANCIAL SUPPORT
This work was supported by the Fundação de Amparo À Pesquisa do Estado de Minas Gerais, FAPEMIG (Grant number APQ04094/10).

AUTHORS' CONTRIBUTION
MCS was responsible for conceptualization, formal analysis, funding acquisition, and project administration. MBOS was responsible for conceptualization, and reviewing the manuscript. RGFM performed all the experiments. ALP, and RGFM were responsible for the molecular analysis. DC was responsible for conceptualization, and contributed to reviewing the manuscript.