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Prevalence of Blastocystis and its association with Firmicutes/Bacteroidetes ratio in clinically healthy and metabolically ill subjects

BMC Microbiology volume 21, Article number: 339 (2021) Cite this article

Abstract

Background

Blastocystis is a typical anaerobic colon protist in humans with controversial pathogenicity and has relation with alterations in the intestinal microbiota composition (dysbiosis), whose eventual indicator is the Firmicutes/Bacteroidetes ratio (F/B ratio); this indicator is also linked to complications such as diabetes, obesity, or inflammatory bowel disease. The present study investigated the prevalence of Blastocystis and its association with Firmicutes/Bacteroidetes ratio in healthy and metabolic diseased subjects.

Methods

Fecal and blood samples were collected consecutively from 200 healthy subjects and 84 subjects with metabolic disease; Blastocystis and its most frequent subtypes were identified by end-point PCR and the two most representative phyla of the intestinal microbiota Firmicutes and Bacteroidetes by real-time PCR.

Results

The prevalence of Blastocystis in healthy subjects was 47.0, and 65.48% in subjects with metabolic disease; the most prevalent subtype in the total population was ST3 (28.38%), followed by ST1 (14.86%), ST4, ST5, and ST7 (each one of them with 14.19% respectively), and finally ST2 (8.78%). The low F/B ratio was associated with the prevalence of Blastocystis in the two cohorts FACSA (OR = 3.78 p < 0.05) and UNEME (OR = 4.29 p < 0.05). Regarding the subtype level, an association between the FACSA cohort ST1 and ST7 with low Firmicutes/Bacteroidetes ratio was found (OR = 3.99 and 5.44 p < 0.05, respectively).

Conclusions

The evident predatory role of Blastocystis over Firmicutes phylum was observed in both cohorts since the abundance of bacterial group’s Bacteroidetes increases in the groups colonized by this eukaryote and, therefore, may have a beneficial effect.

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Background

The population of microorganisms living in the human body, especially in the gut and its communication, is termed microbiota [1]. About 1014 bacterial cells live in the colon besides viruses and eukaryotic microorganisms [1, 2], which participate in human metabolism interrelatedly [3]. The gut microbiota plays a crucial role in maintaining the host’s physiological functions; disrupting the fragile host-microbiota interaction equilibrium could affect the onset of several metabolic diseases [4]. Recently, this disruption has been implicated in some chronic diseases ranging from inflammatory bowel disease (IBD), type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD) to colorectal cancer [5].

The two predominant phyla Firmicutes and Bacteroidetes, are determined mainly by the characteristics of the diet and by some genetic and environmental factors that influence the predominance of some organisms over others [6]. In case of an imbalance in the microbiota, dysbiosis will occur [7]. The modification in Firmicutes/Bacteroidetes ratio is an eventual [8] indicator of changes in the microbiota’s composition, which leads to the development of long-term complications such as obesity, diabetes, or inflammatory bowel disease [9, 10]. Noteworthy, the measure of the Firmicutes/Bacteroidetes ratio is a rough method to characterize the microbiota and should note that it is an analysis at the phylum level, which does not have a high resolution from the taxonomic point of view [11]. More standardized and accurate procedures are needed to compare studies from different laboratories and a more taxonomically detailed description than phylum-level changes [8]. Several mechanisms have been proposed to describe the intestine’s changes and energy metabolism from diets rich in carbohydrates and fats [12] that condition the gut microbiota’s dysbiosis, showing a Firmicutes phylum predominance in individuals with obesity and type 2 diabetes mellitus [13, 14].

Conversely, some parasites can colonize the human intestine causing changes in the typical microbiota composition. Blastocystis is the most frequent enteric protozoa found in the human intestine and various animals; however, its clinical significance remains controversial [15]. Infections by Blastocystis range from asymptomatic carriage to non-specific gastrointestinal symptoms; besides, it has been linked to irritable bowel syndrome and urticaria in some populations [16]. This protozoan can inhabit the human intestine for long periods without causing symptoms, so it can probably be part of the normal intestinal microbiota, perhaps its controversial role is mainly due to the predominant subtype or the association with viruses and bacteria [17, 18]. Currently, 22 subtypes (STs) have been identified, 17 have been recognized, and the rest are still under investigation; in humans, only ten have been identified (ST1-ST9 and ST12) [19].

The presence of Blastocystis increases the diversity of gut bacteria [20, 21], and healthier individuals often harbour greater gut microbiological diversity [22]. A negative correlation between Blastocystis and body mass index (BMI) has been suggested [23]; however, it is unknown whether the bacterial microbiota of these individuals induces colonization by Blastocystis or the presence of this protozoan promotes specific bacterial microbiota in individuals with low BMI [21, 24]. Blastocystis may cause a low Firmicutes/Bacteroidetes ratio and gastrointestinal symptoms, and even has been extensively studied in populations with irritable bowel syndrome and disease, the associations found are not strong enough to be attributed to the parasite [25, 26]. Currently, it is unknown whether Blastocystis participates in changes in the microbiota or changes in microbiota and metabolic dysfunctions that cause high colonization by this protozoan [27]. However, a higher relative abundance of Bacteroidetes and a decrease in Firmicutes does not necessarily respond to beneficial effects. Although, it has been demonstrated an increase in Firmicutes (Faecalibacterium and Clostridia family) in individuals with Blastocystis [20, 26] and a decrease in Bacteroidetes related to healthy individuals [28], or in patients with Clostridium difficile colonized by Blastocystis [29]; the association with alterations of the microbiota in metabolically ill subjects is unknown. This study aimed to evaluate the prevalence of Blastocystis, subtypes and its association with low Firmicutes/Bacteroidetes ratio in clinically healthy and metabolically ill subjects.

Results

Two hundred two young university students (FACSA cohort) were included (two were eliminated due to insufficient samples). Of 95 metabolically ill adults (UNEME cohort), 11 were eliminated due to bad samples and incomplete questionnaires. The subjects of the FACSA cohort were younger than the UNEME cohort; the median age in the groups was 20 (19–21) vs. 55 (46–63) years, respectively (Mann-Whitney-Wilcoxon test p = 0.0001) respectively. The subjects of the UNEME cohort have a higher body mass index than the FACSA cohort. The percentage of subjects with normal BMI is higher in the FACSA cohort, and the urban area is the most represented in the total sample. Still, in sick adults, the rural area is more representative (Table 1).

Table 1 Characterization of FACSA and UNEME cohort
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Prevalence of Blastocystis and subtypes

Blastocystis was more prevalent in the UNEME cohort 55 (65.48%) vs. 94 (47.0%) (x2 test, p = 0.004). For subtypes, ST5 was the most prevalent in the UNEME cohort 16 (29.63%) (x2 test, p = 0.001) while ST4 was more prevalent in the FACSA cohort, although the data are not statistically significant (Table 2).

Table 2 Prevalence of Blastocystis and subtypes, gut intestinal in FACSA and UNEME cohort
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Relative abundance units Firmicutes (RAUF), Relative abundance units Bacteroidetes (RAUB), and Firmicutes/Bacteroidetes ratio (F/B ratio).

The RAUF was higher in UNEME cohort 1.92 (0.71–3.53) vs. FACSA cohort 0.80 (0.05–2.08), similar results were obtained with the RAUB 1.6 (0.63–3.29) vs. 0.82 (0.44–1.65), and F/B ratio 1.40 (0.32–4.03) vs. 0.83 (0.07–3.50) (Mann-Whitney-Wilcoxon test, p = 0.0001, p = 0.005, and p = 0.031, respectively) (Table 2).

Prevalence of Blastocystis and their association con Firmicutes/Bacteroidetes ratio

The prevalence of Blastocystis was associated with a lower F/B ratio both in the FACSA cohort and in the UNEME cohort, 0.23 (0.02–1.6) carriers vs. 1.3 (0.5–8.2) no carriers, and 0.88 (0.31–2.5) carriers vs. 2.4 (1.2–6.1) no carriers, (Mann-Whitney-Wilcoxon test, p = 0.0001, and p = 0.015, respectively) (Fig. 1). This association was also observed with subtypes 1,2,4 and 7 in the FACSA cohort, (Mann-Whitney-Wilcoxon test, p = 0.003, p = 0.027, p = 0.006 and p = 0.0007, respectively) (Fig. S1: B, C, E and G). The prevalence of Blastocystis was not associated with age, sex, or obesity in either two cohorts, Table 3. Regarding the ST2 and ST3 subtypes, were associated with age in the UNEME cohort, the oldest patients are those colonized by these subtypes (Mann Whitney-Wilcoxon test p = 0.043 and p = 0.039, respectively) (Table S7).

Fig. 1
figure 1

Relative Abundance Units of Firmicutes (F), Bacteroidetes (B) and Firmicutes / Bacteroidetes Ratio (F / B) in non-carriers and carriers by Blastocytsis of the FACSA and UNEME cohort. The F/B Ratio was decreased in subjects colonized by Blastocystis in both cohorts. (Mann-Whitney-Wilcoxon test, p = 0.0001, and p = 0.015, respectively)

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Table 3 Prevalence of Blastocystis and its association with age, sex, hematologic parameters and gut microbiota in FACSA and UNEME cohort
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We considered for the logistic regression analysis a high F/B ratio > 0.83 and low F/B ratio < 0.83 in the FACSA cohort; and a high F/B ratio > 1.40 and low F/B ratio < 1.40 in the UNEME cohort according to the results of Table 2. The association was corroborated with respect to Blastocystis OR = 3.78 (95% CI 2.10–6.81 p = 0.0002) and OR = 4.24 (95% CI 1.59–11.31 p = 0.002) (Tables 4 and 5, respectively), ST1 and ST7 were associated with low Firmicutes/Bacteroidetes ratio in the FACSA cohort but not in the UNEME cohort, ST1 has OR = 3.99 (CI 95% 1.07–14.79 p = 0.039) and ST7 OR = 5.44 (95% CI 1.16–25.52 p = 0.022) (Table 4).

Table 4 Blastocystis, subtypes and their association with Low Firmicutes/Bacteroidetes ratio in FACSA cohort
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Table 5 Logistic regression between Blastocystis and subtypes and its association with low Firmicutes/Bacteroidetes ratio in a UNEME cohort
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Discussion

Our most important findings are the higher Blastocystis prevalence in the UNEME cohort than in the FACSA cohort (Table 2). The faecal-oral transmission of Blastocystis is due to poor hygiene practices, exposure to animals infected with the parasite, and intake of contaminated water or food [30]. This transmission route is probably most common since the most UNEME cohort subjects live in rural areas (Table 1).

The F/B ratio comparison between cohorts showed an F/B ratio high in UNEME cohort subjects (Table 2) according to a high F/B ratio (Median of 1.40; IR: 0.32–4.03) compared to the subjects of the FACSA cohort (Median of 0.830; IR: 0.07–3.50, p < 0.05). A high F/B ratio agrees with previous reports, in which it is found in metabolically ill subjects [4]; this relationship implies a predisposition to disease states [31]. Likewise, a low Firmicutes/Bacteroidetes ratio is related to weight loss [32], which corresponds with FACSA cohort individuals with lower BMI in this study (Table 1).

Obese and diabetic individuals, compared to healthy individuals, have a higher relative abundance of Firmicutes and a reduced abundance of Bacteroidetes [13], as well as a low microbial gene count and a dominance in the genera Bacteroides and Ruminococcus, all this is associated with a more remarkable ability to obtain energy from the diet, systemic inflammation, adiposity, insulin resistance, and dyslipidemia [22, 33]. Bacteroidetes are known to produce mainly acetate and propionate, while Firmicutes produce more butyrate, attributed to anti-inflammatory activities, regulation of energy metabolism, and increases leptin [34, 35].

The increase in Firmicutes in metabolically ill patients could cause an increase in butyrate production leading to antiobesogenic effects, which is contradictory. It has been speculated that in obese subjects, the butyrate-producing bacteria decrease and are replaced by other bacteria belonging to the same phylum resulting in lower butyrate production in the colonic lumen [8]. For example, increased abundances of Staphylococcus spp. and Lactobacillus reuteri (both from the phylum Firmicutes) have been reported in obese people and positively correlated with energy intake and plasma C-reactive protein (CRP), respectively [36, 37]. On the contrary, the decreased abundance of the butyrate-producing Faecalibacterium prausnitzii (phylum Firmicutes) correlated negatively with the intensity of low-grade inflammation in obese subjects and type 2 diabetes patients [38, 39].

Blastocystis is a widely distributed organism with great adaptability that could colonize healthy and diseased subjects. We analyzed the Firmicutes/Bacteroidetes relationship and the presence of Blastocystis in these two cohorts, and the results showed an association with a low F/B ratio (Table 3) (Fig. 1).

The relationship of Blastocystis with the gut microbiota is a subject of debate, as it has been linked to a low F/B ratio and irritable bowel disease [26]. However, Audebert et al. [20] found a greater abundance of Clostridiales at the class level and a greater abundance of Rumminococcaceae and Prevotellaaceae at the family level in subjects with Blastocystis, while Enterobacteriaceae increased in patients without Blastocystis. It has been suggested that Blastocystis is not associated with dysbiosis observed in intestinal, metabolic diseases or infections commonly associated with inflammation of the lower gastrointestinal tract; instead, colonization by this protozoan could be associated with a healthy intestinal microbiota [20].

Since previous studies that have found a high Firmicutes / Bacteroidetes ratio in healthy patients when Blastocystis is present, a study in faecal samples from 35 Swedish university students found that the presence of Blastocystis was accompanied by higher abundances of the bacterial generates Sporolactobacillus. Also, Blastocystis carriage was positively associated with high bacterial genus richness and negatively correlated to the Bacteroides-driven enterotype; although the results were not significant, the associations between Blastocystis and the bacterial microbiota found in this study could imply a link between Blastocystis and a healthy microbiota as well as with diets high in vegetables [28].

Another study carried out in stool samples from 57 school-aged children in Colombia found a higher microbial richness in Blastocystis-colonized children, which could be benefit intestinal health. The phylum Firmicutes was the predominant taxonomic unit in both groups analyzed; nevertheless, the composition of the intestinal bacterial community was not significantly different between Blastocystis- free and Blastocystis-colonized children [40]. It is worth mentioning that diet [41, 42], antibiotics [43], age, geographic area [44], inflammation [45] and, to a lesser extent, genetics of the host [46] affect the ecology of the intestinal bacterial community. On the other hand, the heterogeneity of the results in humans, regarding the Firmicutes/ Bacteroidetes ratio, could be due to the insufficient number of subjects included in most of the studies, making a poor statistical power to detect small variations [47].

Regarding experimental studies in a study, reported the decrease in the Firmicutes/Bacteroidetes ratio (p = 0.06) [48] in a model of IBS (Irritable Bowel Syndrome) in rats infected with Blastocystis ST4 from healthy humans. This partially agrees with our findings since in the FACSA cohort ST2 had an OR = 3.12 (0.61–15.88, 95% CI), ST3 OR = 1.97 (0.86–4.52, 95% CI), while ST4 OR = 2.34 (0.78–7.02, 95% CI), p > 0.05 (Table 4) was not associated with low F/B ratio. However, there was an association with low F/B at the gender level OR = 3.78 (2.10–6.81, 95% CI), for the subtypes ST1 OR = 3.99 (1.07–14.79, 95% CI) and ST7 OR = 5.44 (1.16–25.52, 95% CI) p < 0.05, (Table 4). Regarding ST7, Yason et al. [49] reported that the presence of ST7 decreases the Bifidobacterium and Lactobacillus populations while increasing the Escherichia populations.

About ST1, the presence of an alternative oxidase provides a partially dependent metabolism of molecular oxygen to resist the stress that the high oxygen concentration entails, which may occur in the intestine of the FACSA subjects due to the low F/B ratio [50]. Also, in the UNEME cohort, a low F/B ratio was found concerning gender OR = 4.24 p < 0.05 (Table 5) but not for subtypes.

The low F/B ratio found in this study might imply that the subjects of the two cohorts infected by Blastocystis present gastrointestinal symptoms; however, the association of the Blastocystis prevalence with gastrointestinal symptoms showed an inverse association between abdominal pain and ST1. At the same time, ST4 was inversely associated with abdominal distension in the FACSA cohort in previous data published [51] (Tables S2 and S3); however, no association of Blastocystis with gastrointestinal symptoms was found in the UNEME cohort (Tables S4 and S5). Contrary to the results obtained, a study found that Blastocystis negatively correlates with Bacteroidetes [20], while the phylum Firmicutes presents a positive correlation in Blastocystis positive samples [24]. These results could be biased in type 2 diabetes subjects due to the low carbohydrate consumption [52]. Since Firmicutes have more coding genes for enzymes involved in metabolism, a decrease in carbohydrate consumption could lead to Firmicutes decrease and Bacteroidetes increase [13, 53]; however, we did not analyse diet in this study.

Possibly, a high F/B ratio does not always lead to inflammation or disease, but certain conditions that lead to an inflammatory state would have to be present to affect the individual. For example, in obese individuals, the proportion of Firmicutes and Proteobacteria increases, compared to Bacteroidetes; in this case, an inflammatory environment is present [54]. In the UNEME cohort, we observed Firmicutes/Bacteroidetes ratio increase in the UAR of Bacteroidetes, but this does not necessarily imply inflammation. Additionally, there is evidence that Blastocystis modulates the immune system through IL-22 release that stimulates mucus production, alleviates colitis symptoms [55] and induces an immune response with a predominance of the Th2 cell response, favouring an anti-inflammatory environment [56].

Interestingly, ST5 was more prevalent in the UNEME cohort. The detection of this subtype is not common in humans and, although low frequencies of ST5 have been reported in Bolivia, Pakistan, Thailand, the United Kingdom, Colombia, and China [18, 57,58,59,60,61], there are no reports of this subtype in Mexico [62]. Additionally, this subtype is mainly detected in non-human primates, pigs, ostriches, dogs, rats, and some ungulate mammals; and therefore, a zoonotic profile has been attributed to it [63, 64]. The above agrees with our findings since most of the subjects of the UNEME cohort are from rural areas (Table 1). ST5 may survive in the possible inflammation scenario, judging by the high Firmicutes/Bacteroidetes ratio of the UNEME cohort (Tables 1 and 2). ST5 could enrich different groups of bacteria while reducing competition between other bacterial communities of the microbiota.

Regarding ST4, although the prevalence was not high, it was more frequent in the FACSA cohort (clinically healthy subjects) subjects with lower BMI (Tables 1 and 2) and subjects with lower BMI in the total sample (Table S6). These findings agree with Beghini et al. [24] that found a strong negative correlation between BMI and Blastocystis prevalence. Also, consistent with findings from the Danish’s subjects study [23], the difference in Blastocystis prevalence between average weight and obese subjects (p = 5E-03), average weight and overweight (p = 0.01), and between non-overweight and overweight (p = 0.02) was significant. Between specific subtypes, only ST4 reached statistical significance (p = 0.03 between average weight and obese). Besides, Tito et al. [21] found a positive and significant correlation (R = 0.26 p = 0.00028) between ST4 and Akkermansia and Methanobrevibacter; the first is an abundant bacterium in healthy people that degrades intestinal mucin, which is associated with weight loss; the second is a methanogenic archaeon that plays an essential role in carbohydrate digestion and may protect against weight gain [22].

ST3 was the most prevalent in the FACSA cohort, 29.79%, and the second most prevalent, 25.93%, in the UNEME cohort (Table 2). This subtype was not associated with an intestinal low F/B ratio in either of the two cohorts (Tables 4 and 5), agreeing with previous reports [58, 65, 66]. A higher bacterial diversity has been reported in ST3-Blastocystis-carriers (high abundance of Prevotella, Methanobrevibacter, and Ruminococcus), while a high percentage of Bacteroides found in Blastocystis-free subjects [66]. Asnicar F. et al. [67] reported interesting findings of the presence of Prevotella copri and Blastocystis spp. as markers of improved postprandial glucose response; both were strongly linked with favourable glucose homeostasis and a decrease of the estimated visceral adipose tissue mass.

One of the limitations of our study was the analysis at the phylum level since it does not have a high resolution from the taxonomic point of view. However, the advantages of using a small template are the high sensitivity, high-performance processing and affordable cost [11]. And it is used in research that aims to characterize microbial communities [68]; such is the case in our study. In future studies, we will perform ultra-high-throughput sequencing methods. Also in future studies, we will analyse the dietary habits and the microbiota composition, including Blastocystis, in both cohorts. Another limitation of our work was qualitative PCR, which only identifies the presence or absence of ST. The implementation of a more sensitive molecular technique, such as Next-generation amplicon sequencing (Maloney (2019), could give us additional information, such as the most predominant ST or the existence of more than two subtypes in a single sample [69]. Also, this technique could help with the identification of genotypes that were not detected with the primers used. A more accurate assessment of Blastocystis diversity is key to understanding the transmission mechanism and pathogenicity in our population. Another limitation was that the majority of the subjects in the UNEME cohort were obese, and the analysis of the comparison with thin individuals between the two cohorts could not be carried out. Therefore, the objective is to increase the sample size in this cohort concerning this group.

Conclusions

The present study provides an overview of the two most representative phyla behaviour, the intestinal microbiota Firmicutes and Bacteroidetes and the Firmicutes/Bacteroidetes ratio when Blastocystis is present. The modulation caused by the parasite mainly in the Firmicutes phylum is evident, which decreases in two cohorts. Some studies have reported the variability between subtypes but have not focused on the host’s variability. Based on this, it is interesting to analyze the type of diet since this may favour the colonization and the predatory function of Blastocystis in the intestine; additionally, follow-up studies will be carried out in the two cohorts.

Methods

Subjects and sample collection

This study was a cross-sectional design with a nonprobabilistic sampling conducted between March of 2018 to April 2019. Two hundred clinically healthy university young adults (FACSA cohort) and 84 adults with metabolic disease were included (UNEME cohort). Inclusion criteria for UNEME cohort were diabetes, hypertension, dyslipidemia, and overweight/obesity. Exclusion criteria were kidney failure, heart disease, lung disease, amputation, and pregnancy.

Inclusion criteria for the FACSA cohort were Bachelor students of the medicine and nutrition program. In this group, patients with chronic degenerative diseases were excluded.

Exclusion criteria for both groups were patients who received any medication with antibiotic treatment in the last 3 months before the study.

Data of patients with insufficient sample or incomplete information were eliminated.

Questionnaire survey

A digital gastrointestinal symptoms questionnaire (abdominal pain and constipation), consisting of multiple-choice questions, based on Rome III diagnostic criteria [70, 71], was used to collect information about each participant including sex, age, city of origin, and clinical data. All data collected from each subject remained confidential and were fully anonymized through the encryption of the identity of individuals.

A 5 ml blood sample was taken of all individuals to perform hematic biometry and requested a stool sample to identify the intestinal microbiota and the presence of Blastocystis.

Blastocystis identification

Parasitological examination

Samples were collected in containers with 10% formaldehyde for coproparasitological exams in triplicate. Each microscopic identification of Blastocystis sp was carried out on a different day of the deposition. The sample preparation was developed as described previously [51]. Briefly, the modified Ritchie technique was performed for the preparation of the samples. Ten microliters of each stool sample were mixed with 20 μl of Lugol’s iodine solution and covered with a 21 × 26 mm coverslip. Three hundred optical fields were examined for Blastocystis with a magnification of 250× (20× objective and 12.5 eyepieces) and, in case of suspected organisms, 500 × (40 × 12.5×) magnification. The observation of each slide lasted an average of 5 min. The diagnostic criterion for positivity was at least 2 precise vacuolar forms of the parasite in either of the three samples [72].

DNA extraction

To confirm the microscopy diagnosis, molecular biology techniques were used to detect Blastocystis subtypes. A fresh sample was dispensed into a DNase and RNAse-free sterile bottle and kept under refrigeration until transport to the laboratory, stored at − 20 °C until use. The sample extraction was developed as described previously [51]. Briefly, from 200 mg of faeces, the nucleic acid extraction was carried out using the E.Z.N.A.® Stool DNA Kit (USA). DNA concentration and purity were determined using NanoDrop 1000 Thermo Scientific (Saveen Werner ApS®, Denmark).

Genus determination

The extracted DNA samples were used to determine the presence of Blastocystis. Three microlitres of each DNA sample were mixed with Radiant™ Red 2x Taqman Mastermix (Alkali Scientific Inc.) to a final volume of 13 μl for the PCR. The primers used were: F1- 5′-GGA GGT AGT GAC AATAAA TC-3′ and R1- 5′-CGT TCA TGA TGA ACA ATT AC-3′ [73] (T4 Oligo®, Irapuato, México). All samples underwent PCR test.

Subtyping of Blastocystis using sequence-tagged sites (STS) primers

For the genotyping of Blastocystis (ST1-ST5, ST7), a set of sequence-tagged site primers derived from products of randomly amplified polymorphic DNA (RAPD) sequences were used [73, 74]. Four μl of each DNA sample positive for Blastocystis in a Polymerase chain reaction (PCR) was mixed with Radiant™ Red 2x Taqman Mastermix (Alkali Scientific Inc.) with primers (Table S1) in a final volume of 13 μl.

The PCR conditions were an initial denaturation step at 94 °C for 4 min; followed by 35 denaturation cycles at 94 °C for 30 s; annealing at 55 °C for 45 s; extension at 72 °C for 45 s; and a final extension at 72 °C for 10 min (PTC-100 thermocycler, MJ Research Inc) [30]. The ß-globin gene was amplified as an internal extraction control. The samples that were negative for gender but beta-globin positive underwent subtyping. The PCR products were resolved in a 1.5% agarose gel (Ultrapure Agarose, Invitrogen™) stained with RedGel™ Nucleic Acid Gel Stain (Biotium), and a molecular weight marker was used to establish the size of the amplicon (100 bp DNA Ladder. Biobasic Inc.). Additionally, the samples were randomized to the analysis by PCR. Sanger sequencing was used to corroborate both the presence of Blastocystis and genotypes, contrasted them with sequences reported in https://blast.ncbi.nlm.nih.gov/Blast.cgi using a blast. The nucleotide sequences generated in present study have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/) under accession numbers: MZ351752-57.

Identification of the gut microbiota

The analysis of the microbiota profile was performed by real-time PCR (qPCR) using the 16S rRNA taxon-specific to detect the presence of Bacteroidetes/Firmicutes and universal primers to amplify all members of the taxon. The sequences of the primers were: Bacteroidetes: CRAACAGGATTAGATACCCT (Forward) and GGTAAGGTTCCTCGGCTAT (Reverse); Firmicutes: TGAAACTYAAGGAATTGACG (Forward) and ACCATGCACCACCTGTC (Reverse); universal: AAACTCAAAKGAATTGACGG (Forward) and CTCACRRCACGAGCTGAC (Reverse) [11] (T4 Oligo®, Irapuato, México). The specificity of the amplification products and the absence of primer dimers were determined by performing melting curve analyses in all cases. The standard curve for each primer was generated by 5-fold serial dilutions of bacterial DNA. The efficiency of PCR amplification for each gene was calculated using the standard curve method, E = 10(− 1/slope) − 1.

To each PCR reaction, 5 μl of SYBR Green (Maxima SYBR Green qPCR Master Mix, Thermofisher Scientific TM), 1 μl of each primer (concentration of 5 pmol for Reverse and 10 pmol for Forward), 1 μl of DNA, and 2 μl of DNase/RNase-free water were added to a final volume of 10 μl. Each reaction was performed in duplicate.

The analysis of the qPCR amplification was performed with the Rotor-Gene Q equipment (QIAGEN®, Germany). The samples were processed under the following amplification conditions: an initial thermal denaturation cycle of 5 min at 95 °C, alignment with 30 cycles at 59 °C for 15 s and elongation for 20 s at 72 °C. The conditions were the same for the three pairs of primers used (Universal, Bacteroidetes and Firmicutes). The expression analysis was carried out by quantifying the relative abundance units (RAU) of Firmicutes and Bacteroidetes with the formula RAU = 2-∆Ct where: RAU = Relative Abundance Units and ∆Ct = Ct specific primers-Ct universal primers [75].

Hematic biometry

The quality control was performed through 3 controls: low, normal, and high (KX-21 N SYSMEX LOT: 2R0301) of whole blood to determine haemoglobin, erythrocytes and leukocytes (lymphocytes, MXD and neutrophils) in the automated KX-21 N equipment. Each blood differential of a complete red series and white series grouping (Lymphocytes, neutrophils, and the sum of basophils and eosinophils monocytes (MXD)) were performed.

Statistical analysis

A descriptive analysis of the variables studied of the 284 participants was performed. For all continuous values, normality hypotheses were evaluated using the Kolmogorov-Smirnov test. The quantitative variables were summarized in terms of means and standard deviation or median and interquartile range [25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75], and the qualitative variables were summarized in frequencies and proportions. Non-parametric U-Mann-Whitney tests were used for comparisons between the medians of the two groups. To group the participants into two equal groups the Firmicutes/Bacteroidetes ratio, was operationalized by taking the cut-off point above and below the median of the RAU on each phylum. A chi-square test (×2) or Fisher’s exact test was applied for the bivariate analysis of qualitative variables. The odds ratio (OR) and 95% confidence interval (CI) were estimated for measuring the association between Blastocystis and the Firmicutes / Bacteroidetes ratio. A p value < 0.05 was considered significant. The statistical analysis was performed using the Stata® Statistics Package, version 13.0 and Graphpad Prism Software, L.L.C.Version 9.2.0.

Availability of data and materials

The nucleotide sequences generated in the present study have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/) under accession numbers: MZ351752-57.

Abbreviations

RAU:

Relative abundance units

RAUF:

Relative abundance units of Firmicutes

RAUB:

Relative abundance units of Bacteroidetes

ratio  F/B :

Ratio Firmicutes/Bacteroidetes

IBD:

Inflammatory bowel disease

T2D:

Type 2 diabetes

CVD:

Cardiovascular disease

BMI:

Body mass index

FACSA:

Facultad de Ciencias de la Salud

UNEME:

Unidades en especialidades médicas

UNEME-EC:

Unidades en especialidades médicas en enfermedades crónicas

mm3 :

Cubic milimetre

mg/dL:

Milligrams per deciliter

mt2 :

Square meter

DNase:

Deoxyribonuclease

RNase:

Ribonuclease

USA:

United States of America

RAPD:

Random Amplified Polymorphic acid deoxyribonucleic

PCR:

Polymerase Chain Reaction

qPCR:

Quantitative polymerase chain reaction

rRNA:

Ribosomal ribonucleic acid

ST1:

Subtype 1

ST2:

Subtype 2

ST3:

Subtype 3

ST4:

Subtype 4

ST5:

Subtype 5

ST7:

Subtype 7

IR:

Interquartile range

OR:

Odds Ratio

CI 95%:

Confidence interval to 95%

n:

Number

%:

Percentage

CHS:

Colonic Hypersensitivity

SCFAs:

Short-Chain-Fatty-Acids

AOX:

Alternativa oxidasa

MXD:

Sum of Basophils eosinophils and monocytes

References

  1. Rai R, Saraswat VA, Dhiman RK. Gut microbiota: its role in hepatic encephalopathy. J Clin Exp Hepatol. 2015;5(1):S29–36 Available from: https://linkinghub.elsevier.com/retrieve/pii/S0973688314004319.

    PubMed  Google Scholar 

  2. Sekirov I, Russell SL, Antunes LCM, Finlay BB. Gut microbiota in health and disease. Physiol Rev. 2010;90(3):859–904 Available from: www.prv.org.

    CAS  PubMed  Google Scholar 

  3. Bibbò S, Ianiro G, Giorgio V, Scaldaferri F, Masucci L, Gasbarrini A, et al. The role of diet on gut microbiota composition. Eur Rev Med Pharmacol Sci. 2016;20(22):4742–9.

    PubMed  Google Scholar 

  4. Agus A, Clément K, Sokol H. Gut microbiota-derived metabolites as central regulators in metabolic disorders. Gut. 2020;0:1–9.

    Google Scholar 

  5. Pace LA, Crowe SE. Complex relationships between food, diet, and the microbiome. Gastroenterol Clin North Am. 2016;45(2):253–65. https://doi.org/10.1016/j.gtc.2016.02.004.

    Article  PubMed  PubMed Central  Google Scholar 

  6. García-Peña C, Álvarez-Cisneros T, Quiroz-Baez R, Friedland RP. Microbiota and aging. A review and commentary. Arch Med Res. 2017;48(8):681–9.

    PubMed  Google Scholar 

  7. Pascale A, Marchesi N, Govoni S, Coppola A, Gazzaruso C. The role of gut microbiota in obesity, diabetes mellitus, and effect of metformin: new insights into old diseases. Curr Opin Pharmacol. 2019;49:1–5.

    CAS  PubMed  Google Scholar 

  8. Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P, et al. The Firmicutes/Bacteroidetes ratio: a relevant marker of gut dysbiosis in obese patients? Nutrients. 2020;12(5):1–17 Available from: https://www.mdpi.com/2072-6643/12/5/1474.

    Google Scholar 

  9. Mathur R. Obesity and the microbiome. In: Expert review of gastroenterology and hepatology, vol. 9. Taylor and Francis Ltd; 2015. p. 1087–99.

  10. Rodiño-Janeiro BK, Vicario M, Alonso-Cotoner C, Pascua-García R, Santos J. A review of microbiota and irritable bowel syndrome: future in therapies. Adv Ther. 2018;35(3):289–310.

    PubMed  PubMed Central  Google Scholar 

  11. Bacchetti De Gregoris T, Aldred N, Clare AS, Burgess JG. Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa. J Microbiol Methods. 2011;86(3):351–6 Available from: https://linkinghub.elsevier.com/retrieve/pii/S0167701211002247.

    CAS  PubMed  Google Scholar 

  12. John GK. The gut microbiome and obesity. In: Current oncology reports, vol. 18. Current Medicine Group LLC 1; 2016. p. 1–7.

  13. Muñoz A, Diaz-perdigones C, Tinahones FJ. Gut microbiota and type 2 diabetes mellitus. Endocrinol Nutr. 2016;63(12):560–8.

    Google Scholar 

  14. Schoeler M. Dietary lipids, gut microbiota and lipid metabolism. In: Reviews in endocrine and metabolic disorders. New York: Springer LLC; 2019.

    Google Scholar 

  15. Tan TC. Phenotypic and genotypic characterisation of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential. Parasitol Res. 2008;104(1):85–93.

    CAS  PubMed  Google Scholar 

  16. Ajjampur SSR, Tan KSW. Pathogenic mechanisms in Blastocystis spp. — interpreting results from in vitro and in vivo studies. Parasitol Int. 2016;65(6):772–9.

    CAS  PubMed  Google Scholar 

  17. Nieves-Ramírez ME, Partida-Rodríguez O, Laforest-Lapointe I, Reynolds LA, Brown EM, Valdez-Salazar A, et al. Asymptomatic intestinal colonization with protist Blastocystis is strongly associated with distinct microbiome ecological patterns. Lozupone C, editor. mSystems. 2018;3(3):1–18 Available from: https://journals.asm.org/doi/10.1128/mSystems.00007-18.

    Google Scholar 

  18. Yakoob J, Jafri W, Beg MA, Abbas Z, Naz S, Islam M, et al. Blastocystis hominis and Dientamoeba fragilis in patients fulfilling irritable bowel syndrome criteria. Parasitol Res. 2010;107(3):679–84.

    PubMed  Google Scholar 

  19. Stensvold CR, Clark CG. Forum pre-empting Pandora’s box: Blastocystis subtypes revisited trends in parasitology. Trends Parasitol. 2020;36(3):229–32.

    PubMed  Google Scholar 

  20. Audebert C, Even G, Cian A, Blastocystis Investigation Group, Loywick A, Merlin S, et al. Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota. Sci Rep. 2016;6:1–11.

    Google Scholar 

  21. Tito RY, Chaffron S, Caenepeel C, Lima-Mendez G, Wang J, Vieira-Silva S, et al. Population-level analysis of Blastocystis subtype prevalence and variation in the human gut microbiota. Gut. 2019;68(7):1180–9.

    CAS  PubMed  Google Scholar 

  22. Le Chatelier E, Nielsen T, Qin J, Prifti E, Hildebrand F, Falony G, et al. Richness of human gut microbiome correlates with metabolic markers. Nature. 2013;500(7464):541–6.

    PubMed  Google Scholar 

  23. Andersen LO, Ida B, Nielsen HB, Stensvold CR. A retrospective metagenomics approach to studying Blastocystis. FEMS Microbiol Ecol. 2015;91(6):1–9.

    Google Scholar 

  24. Beghini F. Large-scale comparative metagenomics of Blastocystis, a common member of the human gut microbiome. ISME J. 2017;11(12):2848–63 Available from: http://www.nature.com/articles/ismej2017139.

    PubMed  PubMed Central  Google Scholar 

  25. Denoeud F. Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biol. 2011;12(3):1–6.

    Google Scholar 

  26. Nourrisson C, Scanzi J, Pereira B, NkoudMongo C, Wawrzyniak I, Cian A. Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects. PLoS One. 2014;9(11):1–9.

    Google Scholar 

  27. Scanlan PD, Stensvold CR, Rajilić-Stojanović M, Heilig HGHJ, De Vos WM, O’Toole PW, et al. The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiol Ecol. 2014;90(1):326–30.

    CAS  PubMed  Google Scholar 

  28. Forsell J, Bengtsson-Palme J, Angelin M, Johansson A, Evengård B, Granlund M. The relation between Blastocystis and the intestinal microbiota in Swedish travellers. BMC Microbiol. 2017;17(231):1–9.

    Google Scholar 

  29. Vega L, Herrera G, Munoz M, Patarroyo MA, Maloney JG, Santin M, et al. Gut microbiota profiles in diarrheic patients with co-occurrence of Clostridioides difficile and Blastocystis. PLoS One. 2021;16(3 March):1–23.

    Google Scholar 

  30. Kumarasamy V. Blastocystis sp., parasite associated with gastrointestinal disorders: an overview of its pathogenesis, immune modulation and therapeutic strategies. Curr Pharm Des. 2018;24(27):3172–5 Available from: http://www.eurekaselect.com/164390/article.

    CAS  PubMed  Google Scholar 

  31. Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Reddy DN. Role of the normal gut microbiota. World J Gastroenterol. 2015;21(29):8836–47.

    Google Scholar 

  32. Mariat D. The firmicutes/bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol. 2009;9:123.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Krajmalnik-Brown R. Effects of gut microbes on nutrient absorption and energy regulation. Nutr Clin Pract. 2012;27:201–14.

    PubMed  PubMed Central  Google Scholar 

  34. Louis P, Flint HJ. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett. 2009;294(1):1–8.

    CAS  PubMed  Google Scholar 

  35. Everard A, Lazarevic V, Derrien M, Girard M, Muccioli GM, Neyrinck AM, et al. Responses of gut microbiota and glucose and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistant mice. Diabetes. 2011;60(11):2775–86.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Bervoets L, Van Hoorenbeeck K, Kortleven I, Van Noten C, Hens N, Vael C, et al. Differences in gut microbiota composition between obese and lean children: a cross-sectional study. Gut Pathog. 2013;5(1):1–10.

    Google Scholar 

  37. Million M, Angelakis E, Paul M, Armougom F, Leibovici L, Raoult D. Comparative meta-analysis of the effect of Lactobacillus species on weight gain in humans and animals. Microb Pathog. 2012;53(2):100–8.

    PubMed  Google Scholar 

  38. Balamurugan R, George G, Kabeerdoss J, Hepsiba J, Chandragunasekaran AMS, Ramakrishna BS. Quantitative differences in intestinal Faecalibacterium prausnitzii in obese Indian children. Br J Nutr. 2010;103(3):335–8.

    CAS  PubMed  Google Scholar 

  39. Furet JP, Kong LC, Tap J, Poitou C, Basdevant A, Bouillot JL, et al. Differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers. Diabetes. 2010;59(12):3049–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Castañeda S, Muñoz M, Villamizar X, Hernández PC, Vásquez LR, Tito RY, et al. Microbiota characterization in Blastocystis-colonized and Blastocystis-free school-age children from Colombia. Parasit Vectors. 2020;13(1):1–12.

    Google Scholar 

  41. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505(7484):559–63.

    CAS  PubMed  Google Scholar 

  42. Machate DJ, Figueiredo PS, Marcelino G, Guimarães RDCA, Hiane PA, Bogo D, et al. Fatty acid diets: regulation of gut microbiota composition and obesity and its related metabolic dysbiosis. Int J Mol Sci. 2020;21(11):1–22.

    Google Scholar 

  43. Keeney KM, Yurist-Doutsch S, Arrieta M-C, Finlay BB. Effects of antibiotics on human microbiota and subsequent disease. Annu Rev Microbiol. 2014;68:217–35.

    CAS  PubMed  Google Scholar 

  44. Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. Human gut microbiome viewed across age and geography. Nature. 2012;486(7402):222–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Slingerland AE, Schwabkey Z, Wiesnoski DH, Jenq RR. Clinical evidence for the microbiome in inflammatory diseases. Front Immunol. 2017;8(400):1–15.

    Google Scholar 

  46. Goodrich JK, Davenport ER, Beaumont M, Jackson MA, Knight R, Ober C, et al. Genetic determinants of the gut microbiome in UK twins. Cell Host Microbe. 2016;19(5):731–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Schloss PD, Sze MA. Looking for a signal in the noise: revisiting Obesity and the microbiome. Am Soc Microbiol. 2016;7(4):1–9.

    Google Scholar 

  48. Defaye M, Nourrisson C, Baudu E, Lashermes A, Meynier M, Meleine M, et al. Fecal dysbiosis associated with colonic hypersensitivity and behavioral alterations in chronically Blastocystis-infected rats. Sci Rep. 2020;10(1):1–12.

    Google Scholar 

  49. Yason JA, Liang YR, Png CW, Zhang Y, Tan KSW. Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies. Microbiome. 2019;7(1):1–13.

    Google Scholar 

  50. Gentekaki E, Curtis BA, Stairs CW, Klimes V, Elias M. Extreme genome diversity in the hyper- prevalent parasitic eukaryote Blastocystis. PLoS Biol. 2017;15(9):e2003769.

    PubMed  PubMed Central  Google Scholar 

  51. Pérez MR, Yáñez CM, Hernández AM, Sustaita JJD, Jiménez EG, Andrade MR, et al. Blastocystis infection frequency and subtype distribution in university students. Heliyon. 2020;6(12):0–5.

    Google Scholar 

  52. Arora SK, McFarlane SI. The case for low carbohydrate diets in diabetes management. Nutr Metab. 2005;2(7):1–9.

    Google Scholar 

  53. Blandino G, Inturri R, Lazzara F, Di Rosa M, Malaguarnera L. Impact of gut microbiota on diabetes mellitus. Diabetes Metab. 2016;42(5):303–15.

    CAS  PubMed  Google Scholar 

  54. Hand TW, Vujkovic I, Ridaura V, Belkaid Y. Linking the microbiota, chronic disease and the immune system. Trends Endocrinol Metab. 2016;27(12):831–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Leung JM, Davenport M, Wolff MJ, Wiens KE, Abidi WM, Poles MA, et al. IL-22-producing CD4+ cells are depleted in actively inflamed colitis tissue. Mucosal Immunol. 2014;7(1):124–33.

    CAS  PubMed  Google Scholar 

  56. Chan KH, Chandramathi S, Suresh K, Chua KH, Kuppusamy UR. Effects of symptomatic and asymptomatic isolates of Blastocystis hominis on colorectal cancer cell line, HCT116. Parasitol Res. 2012;110(6):2475–80.

    PubMed  Google Scholar 

  57. Zou Y, Yang W-B, Zou F-C, Lin R-Q, Zhu X-Q, Hou J-L. Molecular detection and subtype distribution of Blastocystis in farmed pigs in southern China. Microb Pathog. 2021;151:104751 Available from: https://www.sciencedirect.com/science/article/pii/S0882401021000231.

    CAS  PubMed  Google Scholar 

  58. Ramírez JD, Sánchez A, Hernández C, Flórez C, Bernal MC, Giraldo JC, et al. Geographic distribution of human Blastocystis subtypes in South America. Infect Genet Evol. 2016;41:32–5. https://doi.org/10.1016/j.meegid.2016.03.017.

    Article  PubMed  Google Scholar 

  59. Pintong A, Sunyanusin S, Prasertbun R, Mahittikorn A, Mori H, Changbunjong T, et al. Blastocystis subtype 5: predominant subtype on pig farms, Thailand. Parasitol Int. 2018;67(6):824–8 Available from: https://linkinghub.elsevier.com/retrieve/pii/S1383576917305482.

    CAS  PubMed  Google Scholar 

  60. Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, et al. Genetic diversity of Blastocystis in livestock and zoo animals. Protist. 2013;164(4):497–509 Available from: https://linkinghub.elsevier.com/retrieve/pii/S143446101300045X.

    CAS  PubMed  Google Scholar 

  61. Vega-Romero LC, Ramírez-González JD, Muñoz CM. Cambios en el bacterioma y eucarioma intestinal en pacientes con Blastocystis y Clostridium difficile. Universidad del Rosario; 2020.

  62. Rojas-Velázquez L, Maloney JG, Molokin A, Morán P, Serrano-Vázquez A, González E, et al. Use of next-generation amplicon sequencing to study Blastocystis genetic diversity in a rural human population from Mexico. Parasit Vectors. 2019;12(1):1–9.

    Google Scholar 

  63. Cian A, El Safadi D, Osman M, Moriniere R, Gantois N, Benamrouz-Vanneste S, et al. Molecular epidemiology of Blastocystis sp. in various animal groups from two French zoos and evaluation of potential zoonotic risk. PLoS One. 2017;12(1):1–29.

    Google Scholar 

  64. Zhu W, Tao W, Gong B, Yang H, Li Y, Song M, et al. First report of Blastocystis infections in cattle in China. Vet Parasitol. 2017;246(August):38–42.

    PubMed  Google Scholar 

  65. Stensvold CR, Clark CG. Current status of Blastocystis: a personal view. Parasitol Int. 2016;65(6):763–71 Available from: https://linkinghub.elsevier.com/retrieve/pii/S1383576916301544.

    PubMed  Google Scholar 

  66. Gabrielli S, Furzi F, Fontanelli Sulekova L, Taliani G, Mattiucci S. Occurrence of Blastocystis-subtypes in patients from Italy revealed association of ST3 with a healthy gut microbiota. Parasite Epidemiol Control. 2020;9:1–9 Available from: https://linkinghub.elsevier.com/retrieve/pii/S2405673120300039.

    Google Scholar 

  67. Asnicar F, Berry SE, Valdes AM, et al. Microbiome connections with host metabolism and habitual diet from 1,098 deeply phenotyped individuals. Nat Med. 2021;27(1):321–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 2009;67(1):6–20.

    CAS  PubMed  Google Scholar 

  69. Maloney JG, Molokin A, Santin M. Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections. Infect Genet Evol. 2019;73(March):119–25. https://doi.org/10.1016/j.meegid.2019.04.013.

    CAS  Article  PubMed  Google Scholar 

  70. Bai T, Xia J, Jiang Y, Cao H, Zhao Y, Zhang L, et al. Comparison of the Rome IV and Rome III criteria for IBS diagnosis: a cross-sectional survey. J Gastroenterol Hepatol. 2017;32(5):1018–25.

    PubMed  Google Scholar 

  71. Drossman DA. The functional gastrointestinal disorders and the Rome III process. Gastroenterology. 2006;130(5):1377–90.

    PubMed  Google Scholar 

  72. Bart A, Wentink-Bonnema EM, Gilis H, Verhaar N, Wassenaar CJ, Van Vugt M, et al. Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands. BMC Infect Dis. 2013;13:1.

    Google Scholar 

  73. Mohamed RT, El-bali MA, Mohamed AA, Abdel-fatah MA, El-malky MA, Mowafy NM, et al. Subtyping of Blastocystis sp . isolated from symptomatic and asymptomatic individuals in Makkah, Saudi Arabia. Parasit Vectors. 2017;10(174):1–7.

    CAS  Google Scholar 

  74. Yoshikawa H, Wu Z, Kimata I, Iseki M, Ali IKMD, Hossain MB, et al. Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitol Res. 2004;92(1):22–9.

    PubMed  Google Scholar 

  75. Schmittgen T, Livak K. Analyzing real-time PCR data by comparative CT method. Nat Protoc. 2008;3:1101–8.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Dr. Juan Francisco González Bautista UNEME Gómez Palacio and Dra. María de Lourdes Zermeño González UNEME Lerdo.

Funding

The protocols for the development of this study were approved and partially financed by the Program for Teacher Professional Development for the higher type (PRODEP) UJED-CA-135 and UJED-PTC-116.

Author information

Authors and Affiliations

  1. Laboratorio de Microbiología y Parasitología Molecular, Facultad de Ciencias de Salud, Universidad Juárez del Estado de Durango, Sixto Ugalde y Palmas I S/N, Col Revolución, C.P. 35050, Gómez Palacio, Durango, Mexico

    Claudia Muñoz Yañez, Alondra Martínez Sandoval, María Aurora Maravilla Domínguez, Soraya Amalí Zavaleta Muñiz & Janeth Oliva Guangorena Gómez

  2. Instituto de Ciencia y Medicina Genómica, Av. Juarez #1822, Primero de Cobián Centro, C.P. 27000, Torreón, Coahuila, Mexico

    Alejandra Méndez Hernández

Authors
  1. Claudia Muñoz Yañez
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  2. Alejandra Méndez Hernández
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  3. Alondra Martínez Sandoval
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Contributions

JOGG and CMY conceived and designed the study and wrote the paper. Faecal samples were collected and analysed by CMY and ALMH. Sociodemographic and anthropometric data were collected by AMS, MAMD and SAZM. Data were analysed by JOGG, MAMD and SAZM. All authors have read and approved the submitted version of this manuscript.

Corresponding author

Correspondence to Janeth Oliva Guangorena Gómez.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Faculty of Health Sciences of the Juarez University of the State of Durango (PI-01-2018). Written informed consent was obtained from the subjects included in this study. It guaranteed private handling of patient data for research purposes only. It was Researched according to the Declaration of Helsinki. This study corresponds to a study with minimal risk according to article 17 of the second Title: On the Ethical Aspects of Research in Human Beings, of the Regulation of the General Health Law on Research for Health (General Health Law, 2014).

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Figure S1.

A) Relative Abundance Units of Firmicutes (F), Bacteroidetes (B) and Firmicutes / Bacteroidetes ratio (F / B) in non-carriers and carriers by Blastocystis of the FACSA cohort and UNEME cohort. B) Comparison between FACSA and UNEME subjects non-carriers (NC) and carriers (C) of Blastocystis ST-1 using the Mann-Whitney test. Significant difference is shown between ST-1 non-carriers vs carriers FACSA cohort and carriers of both cohorts. C) Comparison between FACSA and UNEME subjects non-carriers (NC) and carriers (C) of Blastocystis ST-2 using the Mann-Whitney test. A significant difference was found in the sample of FACSA carriers and non-carriers. D) Comparison between FACSA and UNEME subjects non-carriers (NC) and carriers (C) of Blastocystis ST-3 using the Mann-Whitney test. No significant differences were found for this subtype in FACSA or UNEME sample. E) Comparison between FACSA and UNEME subjects non-carriers (NC) and carriers (C) of Blastocystis ST-4 using the Mann-Whitney test. Significant differences were found for this subtype in FACSA cohort, and carrier of both cohorts. F) Comparison between FACSA and UNEME subjects non-carriers (NC) and carriers (C) of Blastocystis ST-5 using the Mann-Whitney test. No significant differences were found in the cohorts. G) Comparison between FACSA and UNEME non-carriers (NC) and carriers (C) of Blastocystis ST-7 using the Mann-Whitney test. A significant difference was found in the FACSA cohort in no carriers’ vs carriers and between the carriers of both cohorts. *p < 0.05.

Additional file 2: Table S7.

Prevalence of Blastocystis subtypes and its association with age, sex, and Hematic Biometry in FACSA cohort.

Additional file 3: Table S2.

Prevalence of Blastocystis and subtypes and their asociation with abdominal pain in FACSA cohort.

Additional file 4: Table S3.

Prevalence of Blastocystis and subtypes and their asociation with abdominal constipation in FACSA cohort.

Additional file 5: Table S4.

Prevalence of Blastocystis and subtypes and their association with abdominal pain in a UNEME.

Additional file 6: Table S5.

Prevalence of Blastocystis and subtypes and their su association with abdominal constipation in UNEME cohort.

Additional file 7: Table S6.

Characterization of the subjects by geographical area, normal weight and obesity according to Blastocystis and subtypes.

Additional file 8: Table S1.

Primer sequences for genotyping Blastocystis.

Additional file 9: Table S8.

Prevalence of Blastocystis subtypes and its association with age, sex, Hematic Biometry and gut microbiota in UNEME cohort.

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Yañez, C.M., Hernández, A.M., Sandoval, A.M. et al. Prevalence of Blastocystis and its association with Firmicutes/Bacteroidetes ratio in clinically healthy and metabolically ill subjects. BMC Microbiol 21, 339 (2021). https://doi.org/10.1186/s12866-021-02402-z

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Keywords

  • Blastocystis
  • Firmicutes
  • Bacteroidetes
  • F/B ratio
  • Subtypes

BMC Microbiology

ISSN: 1471-2180

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