Previous studies have examined associations of E. histolytica infection with the gut microbiome. Verma et al.,2012 found significant reduction in Bacteroides, two subgroups of Clostridium, Lactobacillus, Campylobacter and increase in Bifidobacterium by absolute quantification using Real time PCR. Gilchrist et al.,2016 showed that expansion of Prevotella copri level was associated with symptomatic diarrhea due to E.histolytica infections in children aged two to three years in an urban slum in Dhaka, Bangladesh. Morton et al. 2015 identified ten most important bacterial taxa which showed changes in abundance during Entamoeba (both histolytica and dispar) infections and reported that colonization by Entamoeba can be predicted by an individual’s gut microbiota composition with 79% accuracy. These taxa included Prevotella copri. A recent study by Yanagawa et al. 2021 looked at the gut microbiome during asymptomatic and symptomatic Entamoeba histolytica infections among patients in Japan, by analysing the V3-V4 region of the bacterial 16 S rRNA. Their analysis showed the asymptomatic cases to have more uniform gut microbiome as evidenced by B diversity analysis. There was increased proportion of Coriobacteriaceae, Ruminococcaceae and Clostridiaceae and Collinsella aerofaciens was significantly higher in the asymptomatic cases. Our study attempts to understand the effect of symptomatic Entamoeba histolytica infections on the gut microbiome by comparing it with that of healthy individuals.
Symptomatic Entamoeba infection perturbs the gut microbiome
Significant decline of (within-sample) alpha diversity in the Entamoeba patients indicated perturbation in the gut microbial ecosystem. While this perturbation was highly significant in terms of reduction in richness, significant decline in evenness was also seen. This suggests that gut microbiome of Entamoeba positive patients not only had relatively fewer microbes, but also showed more variation in abundance across patient samples. On the other hand, the taxonomic profiles of Entamoeba positive patients show a decline in intra-group similarity relative to healthy controls, indicating higher variation in microbiome composition of patients. The absence of grouping of samples into their respective (patients and healthy) groups based on microbiome composition shows that the perturbation however did not affect a major fraction of the taxa, and alterations are possibly directional in only a few of the taxa. The individual taxonomic groups which showed perturbation (in terms of evenness) were few in number, but belonged to all levels of classification. At genus level, Faecalibacterium, Prevotella, Sutterella, Subdoligranulum, Collinsella and Bifidobacterium, significantly reduced in abundance among Entamoeba positive patients.
Reduction in abundance of two species of Prevotella namely, P. Copri and P. stercorea, was reported among rural African samples colonized with Entamoeba (Morton et al. 2015). Although we observed an overall reduction in abundance at genus level, the relative abundance of these two Prevotella species show more variation in Eh positive patients, with a slight increase in the median value, though not significant as shown in Online resource 26. P. Copri can exist as a normal component of the gut microbiome but is also referred to as a pathobiont and is associated with severe inflammation in rheumatoid arthritis (Hofer 2014). Moreover, a study among children with diarrhea in Bangladesh showed an increase in the levels of P. copri in patients with diarrheagenic entamoeba infections (Gilchrist et al. 2016). Therefore, the present study is in consonance with previous studies indicating increased perturbation (rather increase) in the P. copri levels in amoebiasis.
In our study genera Subdoligranulum and Sutterella, also display a lower relative abundance in the Entamoeba positive patients. Several studies have associated Subdoligranulum with a healthy metabolic state. (Leclercq et al. 2014, Qin et al. 2014, Holmstrøm et al. 2004, Vinolo 2011, Van Immerseel et al. 2010, Kaakoush 2012). However Van Hul et al. (2020) in their study, showed that supplementation with S. variabile in diabetic mice did not have any significant beneficial effects and concluded that any correlation between a given bacteria and health as shown by metagenomic data, require further validation with experiments. Our observations suggesting a loss or decline of Subdoligranulum genus during E. histolytica infections, could indicate gut microbiome dysbiosis favouring diarrheal symptoms. However, this needs further validation.
Hiippala et al. 2016 have shown that members of genus Sutterella are widely prevalent commensals with mild pro-inflammatory capacity in the human gastrointestinal tract but their contribution to the dysregulation of epithelial homeostasis connected with the increase of Proteobacteria is insignificant. They suggest that the ability of Sutterella spp. to adhere to intestinal epithelial cells indicate an immunomodulatory role. However, the relationship between disease pathology and loss of abundance of Sutterella in Entamoeba positive patients is not clear and needs to be further investigated.
Collinsella and Bifidobacterium are from the family Coriobacteriaceae. They have been shown as the major lactose utilizers among the human gastrointestinal microbiota with an in vitro model of the human colon. Collinsella species break down carbohydrates to produce acetate, formate and lactate (Kovatcheva-Datchary 2010). Prominent lactate utilizing microbiota Firmicutes and Bacteroides produce Short Chain Fatty acids, butyrate or propionate from lactate (Duncan et al. 2004, Reichardt et al. 2014). Decrease in abundance of Collinsella and Bifidobacterium during amoebiasis may as a consequence reduce the positive effects of butyrate and propionate on the host (Wang et al. 2020). At the species level, our study found Collinsella aerofaciens to be significantly reduced in abundance in the patient samples. C.aerofaciens was also found to be significantly less abundant in symptomatic patients of amoebiasis in the study by Yanagawa et al.2021. Butyrate producing bacteria of genus Butyricoccus and species Butyricoccus Pullicaecorum were significantly less abundant in the patient samples. These have been shown to reduce inflammatory pathogen load in the gut (Eeckhaut et al.2016)
Apart from above mentioned genera, there are a few key genera that were found to be in greater abundance in the Entamoeba positive patients, though not statistically significant. These are Escherichia, Klebsiella, Succinovibrio and Ruminococcus. Both Escherichia and Klebsiella belong to the class Gammaproteobacter, and earlier studies have shown that the abundance of bacteria from the class Gammaproteobacter was also shown to be associated with enterocolitis in new born infants (Toro-Londono et al. 2019). An earlier study from this lab found members of Bifidobacterium to be more abundant during E. histolytica infections (Verma et al. 2012). Members of both Bifidobacterium and Ruminococcus ferment mucin and use it as a source of energy. In an in vitro study where colonization of gastric mucin was examined, using faecal samples, they were found to colonize mucin (Leitch et al. 2007). E. histolytica binds to and breaks down mucus with mucin degrading enzymes, however it also potently increases mucus secretion by intestinal goblet cells during symptomatic E. histolytica infections (Leon-Coria et al. 2020). E. histolytica and the gut microbes mutually benefit from each other. E. histolytica derive nutrient source from breakdown of complex carbohydrate by the gut microbes and also feed on them, while gut microbes benefit from increased mucus secretion.
Gut microbiome response to symptomatic Entamoeba infection was different from that during asymptomatic colonization.
Morton et al. (2015) had earlier examined the effect of Entamoeba infection on the gut microbiome, but unlike the current study where samples were collected from active diarrhoeal cases, their samples primarily had Entamoeba colonization without any symptoms. Interestingly, the response of the gut microbiome to these two infectious stages was largely different. For instance, the diversity within samples was significantly higher in the E. histolytica positive samples, as well as the relative abundance of the majority of the phyla including the dominant Firmicutes. This was in clear contrast to the findings of the current study where a declining trend was observed for both. These findings indicate that the E. histolytica does alter the gut microbiome in both stages, but in the diarrhoeal stage of infection it not only shows larger perturbation but also alters abundance of some of the probiotic bacteria as discussed above and favours the colonization of some pathogenic bacteria as the discussion follows.
Perturbation of pathogenic bacteria and their effects in Entamoeba positive patients:
Pathogenic bacteria Staphylococcus aureus and Shigella dysenteriae were reported to increase the virulence of E. histolytica by altering the host’s immune response, while Escherichia coli did not elicit a similar response (Galván-Moroyoqui et al. 2008). Shaulov et al. 2018 have reported that E.coli encoded malate dehydrogenase and its product oxaloacetate contributes to E. histolytica’s resistance to oxidative stress in the lumen of the large intestine. A study by Mousa et al. 2021 showed genus Klebsiella to be associated with pyogenic liver abscess and was isolated in the pus/blood culture of patients. The prevalence of genera belonging to Klebsiella and Enterobacter was observed to increase in Entamoeba positive patients in our V1-V3-PE analysis while the prevalence of genus Streptococcus was greater in the Entamoeba positive patients in the V1-SE and V3- SE datasets. Yanagawa et al. 2021 also found a greater abundance of two species of Streptococcaceae family in patients with symptomatic Entamoeba histolytica infections. Yamashiro et al. 1996 had found abnormal colonization by Streptococcus and Staphylococcus species in children with Kawasaki syndrome and Esposito et al. 2019, in a review article quoted this and other studies to show that imbalance in Streptococcus during Kawasaki syndrome may interfere with their innate and adaptive immunity. In our study, we see a higher prevalence of the pathogenic bacteria of genus Streptococcus in Entamoeba positive patients which could possibly lead to changes in immunity and contribute to the enhanced invasiveness.