Profiling of the fecal microbiota and circulating microRNA-16 in IBS subjects with Blastocystis infection : a case-control study.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal (GI) tract disorder. Although the main reason for IBS is not clear, the interaction between intestinal microorganisms and the gut barrier seems to play an important role in pathogenesis of IBS. The current study aimed to investigate the effect of Blastocystis on the gut microbiota profile and the circulation levels of microRNA (mir)-16 of IBS patients compared to healthy subjects. Stool and blood samples were collected from 80 participants including 40 samples from each IBS and healthy group. Upon DNA extraction from stool samples, barcoding region and quantitative real-time PCR were analyzed to investigate Blastocystis and the microbiota profile, respectively. RNA was extracted from serum samples of included subjects and the expression of mir-16 was evaluated using stem-loop protocol and qreal-time PCR. Significant changes between IBS patients and healthy controls was observed in Firmicutes, Actinobacteria, Faecalibacterium, and Alistipes. In IBS patients, the relative abundance of Bifidobacteria was directly correlated with the presence of Blastocystis, while Alistipes was decreased with Blastocystis. Lactobacillus was significantly increased in Blastocystis carriers. In healthy subjects, the relative abundance of Bifidobacteria was decreased, but Alistipes was increased in Blastocystis carriers. The changes in the Firmicutes/Bacteroidetes ratio was not significant in different groups. The relative expression of mir-16 in Blastocystis-negative IBS patients and healthy carriers was significantly overexpressed compared to control group. The presence of Blastocystis, decreased the relative expression of mir-16 in IBS patients compared to Blastocystis-negative IBS patients. The present study revealed that Blastocystis has the ability to change the abundance of some phyla/genera of bacteria in IBS and healthy subjects. Moreover, Blastocystis seems to  modulate the relative expression of microRNAs  to control the gut atmosphere, apply its pathogenicity, and provide a favor niche for its colonization.

Microbiota and parasite relationship.

The diversity of microbiota is different in each person. Many health problems such as autoimmune diseases, diabetes, cardiovascular diseases, and depression can be caused by microbiota imbalance. Since the parasite needs a host to survive, it interacts closely with the microbiota elements. Blastocystis acts on the inflammatory state of the intestine and may cause various gastrointestinal symptoms, on the contrary, it is more important for gut health because it causes bacterial diversity and richness. Blastocystis is associated with changes in gut microbiota composition, the ultimate indicator of which is the Firmicutes/Bacteroidetes ratio. The Bifidobacterium genus was significantly reduced in IBS patients and Blastocystis, and there is a significant decrease in Faecalibacterium prausnitzii, which has anti-inflammatory properties in Blastocystis infection without IBS. Lactobacillus species reduce the presence of Giardia, and the produced bacteriocins prevent parasite adhesion. The presence of helminths has been strongly associated with the transition from Bacteroidetes to Firmicutes and Clostridia. Contrary to Ascaris, alpha diversity in the intestinal microbiota decreases in chronic Trichuris muris infection, and growth and nutrient metabolism efficiency can be suppressed. Helminth infections indirectly affect mood and behavior in children through their effects on microbiota change. The main and focus of this review is to address the relationship of parasites with microbiota elements and to review the data about what changes they cause. Microbiota studies have gained importance recently and it is thought that it will contribute to the treatment of many diseases as well as in the fight against parasitic diseases in the future.

Presence of Blastocystis in gut microbiota is associated with cognitive traits and decreased executive function.

Growing evidence implicates the gut microbiome in cognition. Blastocystis is a common gut single-cell eukaryote parasite frequently detected in humans but its potential involvement in human pathophysiology has been poorly characterized. Here we describe how the presence of Blastocystis in the gut microbiome was associated with deficits in executive function and altered gut bacterial composition in a discovery (n = 114) and replication cohorts (n = 942). We also found that Blastocystis was linked to bacterial functions related to aromatic amino acids metabolism and folate-mediated pyrimidine and one-carbon metabolism. Blastocystis-associated shifts in bacterial functionality translated into the circulating metabolome. Finally, we evaluated the effects of microbiota transplantation. Donor's Blastocystis subtypes led to altered recipient's mice cognitive function and prefrontal cortex gene expression. In summary, Blastocystis warrant further consideration as a novel actor in the gut microbiome-brain axis.

Contrasting microbiota profiles observed in children carrying either Blastocystis spp. or the commensal amoebas Entamoeba coli or Endolimax nana.

Recent studies have shown how intestinal parasites can modulate gut microbiota. This observation is not surprising since the human intestinal lumen, like any other niche, is a battlefield of microbial competition, and Eukaryotes can affect bacterial populations. Intestinal pathogenic protist has been associated with reshaping the microbial community structure; however, the interactions between the colonic bacterial communities and parasites like Blastocystis spp., Entamoeba coli, and Endolimax nana have been poorly studied. In this work, we studied the distal intestinal bacterial microbiota of 49 children attending 7 public daycare centers in Medellin, Colombia, and compared the bacterial microbiota structure in the presence or absence of the protists Blastocystis spp., E. coli, and E. nana. Parasite colonization was associated with an increase in bacterial richness. Moreover, Blastocystis spp. presented a positive relationship with Prevotella, since this bacterium was selectively enriched in children carrying it. Remarkably, the E. coli colonized children showed a microbial profile that was closer to uninfected controls, although some bacterial taxa displayed to be enriched. This is the case for Akkermansia, which showed to be favored in E. coli colonized individuals, while notably reduced in the Blastocystis spp. parasitized group.

Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies.

BACKGROUND: Blastocystis is a common gut eukaryote detected in humans and animals. It has been associated with gastrointestinal disease in the past although recent metagenomic studies also suggest that it is a member of normal microbiota. This study investigates interactions between pathogenic human isolates belonging to Blastocystis subtype 7 (ST7) and bacterial representatives of the gut microbiota. RESULTS: Generally, Blastocystis ST7 exerts a positive effect on the viability of representative gut bacteria except on Bifidobacterium longum. Gene expression analysis and flow cytometry indicate that the bacterium may be undergoing oxidative stress in the presence of Blastocystis. In vitro assays demonstrate that Blastocystis-induced host responses are able to decrease Bifidobacterium counts. Mice infected with Blastocystis also reveal a decrease in beneficial bacteria Bifidobacterium and Lactobacillus. CONCLUSIONS: This study shows that particular isolates of Blastocystis ST7 cause changes in microbiota populations and potentially lead to an imbalance of the gut microbiota. This study suggests that certain isolates of Blastocystis exert their pathogenic effects through disruption of the gut microbiota and provides a counterpoint to the increasing reports indicating the commensal nature of this ubiquitous parasite.

Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes.

The microbial parasite Blastocystis colonizes the large intestines of numerous animal species and increasing evidence has linked Blastocystis infection to enteric diseases with signs and symptoms including abdominal pain, constipation, diarrhea, nausea, vomiting, and flatulence. It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of Blastocystis cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for Blastocystis subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5' UTR was identified. Using this promoter combined with the legumain 3' UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in Blastocystis. This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for Blastocystis is a significant advance for Blastocystis research both in vitro and in vivo. This technique will spearhead further research to understand the parasite's biology, its role in health and disease, along with novel ways to combat the parasite.

Efficient and reproducible experimental infections of rats with Blastocystis spp.

Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing "natural" infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.

The relation between Blastocystis and the intestinal microbiota in Swedish travellers.

BACKGROUND: Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. In this study, faecal samples from 35 Swedish university students were examined through shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota. RESULTS: We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly Blastocystis subtypes (STs) found were ST3 and ST4, accounting for 20 of the 31 samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The composition of the gut bacterial community was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was accompanied with higher abundances of the bacterial genera Sporolactobacillus and Candidatus Carsonella. Blastocystis carriage was positively associated with high bacterial genus richness, and negatively correlated to the Bacteroides-driven enterotype. These associations were both largely dependent on ST4 - a subtype commonly described from Europe - while the globally prevalent ST3 did not show such significant relationships. CONCLUSIONS: The high rate of Blastocystis subtype persistence found during travel indicates that long-term carriage of Blastocystis is common. 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. Whether the associations between Blastocystis and the microbiota are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

Comparison of faecal microbiota in Blastocystis-positive and Blastocystis-negative irritable bowel syndrome patients.

BACKGROUND: We investigated whether the carriage of Blastocystis in IBS patients was associated with differences in the faecal microbiota. Forty patients with diarrhoea-predominant IBS (26 Blastocystis-positive and 14 Blastocystis-negative) and 57 healthy controls (HC) (42 Blastocystis-positive and 15 Blastocystis-negative) submitted faecal samples for metataxonomic analysis of the 16S ribosomal RNA gene. Differences in the relative abundance of bacteria in these IBS and HC groups were evaluated from phylum to genus level. RESULTS: Significant changes were observed in two dominant phyla in IBS patients, regardless of Blastocystis infection status, namely a rise in Firmicutes and a statistically significant reduction in relative abundance of Bacteroidetes (with a threefold increase in the Firmicutes to Bacteoridetes ratio). Significant differences at genus level in IBS subjects compared to HC were also observed for many bacterial species. However, further clinical subgroup analysis of Blastocystis-positive and Blastocystis-negative subjects, regardless of symptoms, showed no significant differences at the phylum or genus level in IBS-P compared to IBS-N. CONCLUSIONS: Significant differences in the faecal microbiota between diarrhoea-predominant IBS patients and healthy controls were confirmed, but the carriage of Blastocystis did not significantly alter the faecal microbiota. If Blastocystis-positive patients represent a separate clinical subtype of IBS, this group is not identified by changes in the microbiota.

Associations between common intestinal parasites and bacteria in humans as revealed by qPCR.

Several studies have shown associations between groups of intestinal bacterial or specific ratios between bacterial groups and various disease traits. Meanwhile, little is known about interactions and associations between eukaryotic and prokaryotic microorganisms in the human gut. In this work, we set out to investigate potential associations between common single-celled parasites such as Blastocystis spp. and Dientamoeba fragilis and intestinal bacteria. Stool DNA from patients with intestinal symptoms were selected based on being Blastocystis spp.-positive (B+)/negative (B-) and D. fragilis-positive (D+)/negative (D-), and split into four groups of 21 samples (B+ D+, B+ D-, B- D+, and B- D-). Quantitative PCR targeting the six bacterial taxa Bacteroides, Prevotella, the butyrate-producing clostridial clusters IV and XIVa, the mucin-degrading Akkermansia muciniphila, and the indigenous group of Bifidobacterium was subsequently performed, and the relative abundance of these bacteria across the four groups was compared. The relative abundance of Bacteroides in B- D- samples was significantly higher compared with B+ D- and B+ D+ samples (P < 0.05 and P < 0.01, respectively), and this association was even more significant when comparing all parasite-positive samples with parasite-negative samples (P < 0.001). Additionally, our data revealed that a low abundance of Prevotella and a higher abundance of Clostridial cluster XIVa was associated with parasite-negative samples (P < 0.05 and P < 0.01, respectively). Our data support the theory that Blastocystis alone or combined with D. fragilis is associated with gut microbiota characterized by low relative abundances of Bacteroides and Clostridial cluster XIVa and high levels of Prevotella.

Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects.

Blastocystis is a protistan parasite living in the digestive tract of many animals, including humans. This highly prevalent intestinal parasite is suspected to be linked to Irritable Bowel Syndrome (IBS), a chronic functional bowel disorder. Here, we first compared the prevalence of Blastocystis among 56 IBS patients (40 IBS with constipation (IBS-C), 9 IBS with diarrhea (IBS-D), 4 mixed IBS (IBS-M) and 3 unsubtyped IBS (IBS-U) according to the Rome III criteria) and 56 control (i.e. without any diagnosed chronic or acute gastrointestinal disorder) subjects. The highest prevalence of Blastocystis spp. was observed in the IBS group, but was only statistically significant in men (36.8% in the IBS group versus 4.8% in the control group). We then conducted a meta-analysis including epidemiological studies attempting to determine whether Blastocystis carriage could be linked to IBS, and highlighted that IBS patients had a relative risk of 2.34 to be infected by Blastocystis when compared to non-IBS subjects. We also looked for Dientamoeba fragilis, which is often associated with IBS, and identified this parasite only in some IBS patients (n = 6/56). Several studies provided evidence for a major role of the gut microbiota in the pathophysiology of IBS. Thus, we investigated the possible impact of Blastocystis carriage on the enteric bacterial community through quantification of 8 major bacterial groups from the enteric flora. Our data indicated that men with IBS-C had a significant decrease in Bifidobacterium sp. when infected by Blastocystis. Interestingly, in control subjects (i.e. without any gastrointestinal disorder) positive for Blastocystis, Faecalibacterium prausnitzii, which is known for its anti-inflammatory properties, was significantly decreased in men. Our results support the hypothesis that Blastocystis might be linked to the pathophysiology of IBS-C and intestinal flora imbalance.

Subtype determination of Blastocystis isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

The pathogenic role of the enteric parasite Blastocystis remains controversial. Recent studies have suggested that various subtypes (STs) found in human samples could be correlated to the presence or absence and variability of clinical manifestations, and that STs can differ with respect to drug sensitivity. Polymerase chain reaction (PCR) techniques used to determine these STs are expensive and are usually restricted to research laboratory settings. This study evaluates the potential application of the inexpensive matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) technique to discriminate Blastocystis STs. A database of parasitic protein signatures was constructed for five Blastocystis STs, and the reference spectra were challenged with those from 19 axenic cultures of ST1, ST2, ST3, ST4 and ST8 and those from nine xenic liquid cultures of ST3 and ST4. Samples from axenic cultures were prepared using standard formic acid extraction and direct deposition procedures. The reference spectra revealed five distinct spectral profiles, and the database library allowed for discrimination between all of the cultures with reliability indices ranging from 2.038 to greater than 2.8 when an extraction was performed. The direct deposition procedure resulted in greater variability in the discrimination and direct MALDI-TOF MS identification from xenic liquid cultures was effective in 3 out of 9 samples. MALDI-TOF MS proved to be an effective technology for efficiently discriminating Blastocystis STs in axenic cultures.

Blastocystis: past pitfalls and future perspectives.

Blastocystis is a genetically heterogeneous protist found in the intestinal tract (IT) of many vertebrates, and although it is implicated in a variety of human intestinal disorders, data regarding the clinical relevance of Blastocystis is at best speculative. Several research issues, including a lack of standardization across studies, the potential for intrasubtype variation in pathogenicity, and difficulties associated with diagnostics for many idiopathic disorders of the human IT have led to conflicting reports in support of a role for Blastocystis pathogenicity. Here, several research areas and methodologies are reviewed that if integrated appropriately into a prospective study may prove useful and facilitate a better understanding of the role of Blastocystis in human health and disease.

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts.

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

Varying incidence of Blastocystis hominis in cultures from faeces of patients with diarrhoea and from healthy persons.

A study was performed on the frequency of Blastocystis hominis in the faeces from 100 patients suffering from diarrhoea and from 100 healthy persons. Surprisingly, an increased detection rate was observed in samples from healthy persons after anaerobic cultivation. This increased frequency is obviously not dependent on the kind of serum used as a culture supplement and raises the question whether the protozoa morphologically described as B. hominis represent a homogenous species. When rabbit and horse sera were used instead of human serum for cultivation, in both groups the share of positive cultures increased and more large forms of B. hominis cells were observed. Biological implications are being discussed.