A large proportion of microbiome research is focused on the balance of bacterial communities that live in our gut systems, and their effects on health and disease. However, the gastrointestinal microbiome doesn’t just comprise bacteria, it is also home to single-cell eukaryotes and viruses. Laboratory and in vivo studies by a team of scientists at Yong Loo Lin School of Medicine, National University of Singapore (NUS), have now shown how one subtype of the common gut eukaryote Blastocystis directly kills off the good gut bacterium Bifidobacterum, and is linked with reduced numbers of another good microbiome bacterium, Lactobacillus.

“This is the first detailed study to show a causal link between Blastocystis, a common single-cell eukaryote of the human gut, and the host microbiota,” commented research lead Kevin Tan, PhD, from the department of microbiology and immunology at NUS Medicine. “We reveal how it reduces the numbers of beneficial bacteria, which may, in turn, lead to an unbalanced gut microbiome and poorer gut health.”

The NUS team described its studies and findings in Microbiome, in a paper titled, “Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies.”

Blastocystisis a common gut eukaryote in humans and other animal hosts. While many studies have indicated that the single-celled organism is a benign human gut commensal, others have linked Blastocystis with gastrointestinal disease, irritable bowl syndrome, inflammatory bowel disease, and disruption to the gut microbiota.

This discrepancy in research results may be because there are different subtypes of Blastocystis, which might have different influences on the gut microbiota, the NUS authors commented. “Indeed, it has been suggested that microbiota composition in relation to Blastocystis may be dependent on the organism’s subtype identity … One important limiting factor of some of these previous studies was that the subtype of Blastocystis, which has variations in terms of pathogenic potential, was not controlled for or identified.”

With this in mind, the team looked at the interactions between a particular subtype (ST) of Blastocystis, ST7, and different types of bacteria found in the gut microbiome. ST7 Blastocystis is known to feature pathogenic properties not seen in other subtypes, and is resistant to metronidazole, which is the drug of choice for treating eukaryotic single-celled pathogens. Studies have also indicated that ST7 can damage the gut lining. And while Blastocystis ST7 has been reported primarily in Singapore, the subtype has also been identified in Japan and in at least one Danish study, so it is feasible that pathogenic ST7 will also be found in other geographies. Like other Blastocystis subtypes, ST7 is transmitted by eating food that has been contaminated with feces from infected animals, including birds.

Blastocystis could disrupt gut microbiota selectively. In this study, Blastocystis caused reduction of Bifidobacterium longum but an increase in E. coli. This could happen by several mechanisms. There is a direct effect of Blastocystis through oxidative stress, limiting the viability of obligately anaerobic bacteria. Host immune responses as induced by Blastocystis could also limit Bifidobacterium. This bacterium is important to protect the epithelial barrier from Blastocystis-mediated damage. Red and blue arrows signify negative and positive interactions respectively [Dr. Kevin S.W. Tan]

For their reported studies the researchers co-incubated two isolates (ST7-H and ST7-B) of the Blastocystis ST7 organisms with representative commensal gut bacteria, including Escherichia coli, Enterococcus faecalis, Bacteroides fragilis, Bifidobaterium longum, Lactobacillus brevis, and Bacillus subtilis. Findings from their studies indicated that Blastocystis ST7 had a positive effect on some of the gut commensal bacteria and was linked with increased bacterial numbers of multiple bacterial types.

However, when co-cultured with B. longum and E. coli, Blastocystis ST7 negatively affected populations of the “good” bacterium, B. longum, and also reduced numbers of B. longum in the gastrointestinal tracts of live mice. Subsequent studies indicated that ST7 directly caused the death of the Bifidobacterium organisms by inducing oxidative stress mechanisms that released reactive oxygen species. The adverse effects of Blastocystis ST7 on B. longum were even greater in the presence of E. coli. “Indeed, gene expression analysis of B. longum showed that some of the bacterium’s oxidoreductase genes are upregulated, suggesting that it is undergoing oxidative stress in the presence of Blastocystis and E. coli,” the authors stated. “In addition, a greater percentage of B. longum cells exhibited cellular ROS [reactive oxygen species] content when these were incubated with Blastocystis and E. coli.”  Interestingly, Bifidobacterium and E. coli were beneficial to Blastocystis growth.

Separate studies subsequently found that Blastocystis ST7 also reduced numbers of Lactobacillus in vivo, by a mechanism that the researchers have yet to identify. Bifidobacterium and Lactobacillus are generally considered good bacteria because they act to maintain the integrity of the intestinal lining by supporting tight junctions between the lining cells, and Bifidobacterium can also have anti-inflammatory properties. The two bacteria are also commonly used as probiotics to promote gut health.

In addition to its effects on populations of Bifidobacterium and Lactobacillus, Blastocystis ST7 was found to damage the gut lining both directly by disrupting tight junction proteins, and indirectly by triggering an inflammatory response that caused ulcers and disordered structure of the intestinal lining in vivo. The studies reported by Tan confirmed that B. longum acts to protect intestinal epithelial barrier against Blastocystis-induced damage.

Tan is now developing tools to aid studying the mechanisms by which Blastocystis may cause disease. His team has developed a genetic modification system that enables the introduction and expression of foreign genes in Blastocystis. They hope to use this system to illuminate how Blastocystis interacts with its host to cause disease and to explore ways to tackle its detrimental effects.

“To our knowledge, this is the first time wherein in vitro setups complemented by an in vivo system were utilized to investigate the interactions of Blastocystis with the gut microbiota,” the team noted. “While most reports on Blastocystis label it as a commensal and a member of healthy gut microbiota, the findings in this study indicate that different ST of Blastocystis, represented by two pathogenic isolates, may modulate gut microbiota differently from more common STs (e.g., ST1–3). Future work should include other Blastocystis STs with lesser pathogenic potential as well as involving more representatives of gut bacteria. This should provide a clearer picture on where Blastocystis and its STs really stand on gut health and disease.”

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