Final Report Summary - BLASTOHIT2012 (Clarifying the role of Blastocystis in human intestinal disease)
Blastocystis is a ubiquitous microbial eukaryote; current estimates suggest that over 1 billion people worldwide are colonised by Blastocystis making it the most common protist in the human gut. It is also found in a diverse array of other vertebrates including pigs, cows, and reptiles, which highlight both its low host specificity and zoonotic potential. Despite its ubiquity, many details of Blastocystis ecology remain unknown or insufficiently characterised. Moreover, Blastocystis has been cited as an emerging human pathogen and has been implicated in the aetiology of numerous gastrointestinal disease. Nonetheless its role in human disease remains poorly understood and controversial and crucially its prevalence in healthy human populations has received little research attention.
The first study we conducted investigated the prevalence and genetic diversity of Blastocystis in a two human groups of interest, namely healthy adults and infants with no gastrointestinal symptoms or disease. Using an optimised PCR assay we detected Blastocystis in 56% of adults surveyed (n = 105). This results shows that Blastocystis is a common component of the healthy adult human gut. In addition a diversity of different subtypes or species were detected in this adult group. We also conducted temporal analysis and found that where present Blastocystis is a stable member of the gut ecosystem. Conversely, Blastocystis is virtually absent in the corresponding infant population; only 3 of the 69 infants (~4%) were positive for Blastocystis.
In collaboration with Prof. Rob Knight we also looked at the prevalence, distribution and diversity of Blastocystis with family units and their pets (dogs) to look at the potential for human-human transmission and zoonotic transmission. Here we surveyed the presence of Blastocystis in healthy individuals (n = 139) that belonged to 50 families units living in Boulder, Colorado, USA as our sample-set. We detected a diversity of Blastocystis subtypes, however, the overall prevalence of Blastocystis was comparatively low (7%) in relation to other Western populations. Although we did not find any evidence of human-human transmission, given the world-wide variation in human living conditions and lifestyles we cannot rule out a role for this mode of transmission in other populations but instead we propose that human-human transmission of Blastocystis is unlikely in human populations living in similar circumstances to those outlined here. Moreover, we did not detect any Blastocystis in dogs, which supports a recent finding that dogs may not be natural hosts for Blastocystis and therefore do not pose any potential disease risk in this regard.
We also developed a novel set of primers to investigate the inter-species or inter-subtype diversity of Blastocystis within individual hosts. To do so we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes (STs) of Blastocystis, (ST1—ST4), to a healthy human cohort (n = 50). We detected mixed infections in 22% of cases, all of which had been identified as single ST infections in our earlier study of the adult population. Certain STs occur predominantly as either single (ST3 and 4) or mixed infections (ST1), which we speculate may reflect inter alia transient colonisation patterns and/or co-operative or competitive interactions between different STs. One particularly important aspect of this work was the comparative analysis of our novel primer set with other primers that have been used extensively for ST-specific analysis. These previously published primer set were found to be unsuitable for detection of mixed and, in some cases, single ST infections. Our development of these novel PCR assays will facilitate future work into the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.
The final part of our project looked at the link between Blastocystis and the bacterial component of the gut microbiota in healthy adult samples. We used 16S rRNA data generated as part of an earlier study and found that Blastocystis is significantly associated with high microbial diversity. Crucially this data may also explain the absence of Blastocystis in our infant population given that infants have a comparatively lower bacterial diversity. A number of bacterial species were positively correlated with the presence of Blastocystis, namely Robinsonella and Anaerosporobacter. Two bacterial species were negatively correlated with Blastocystis, namely Bilophila and E. coli. These data suggest that Blastocystis may require the presence of specific microbial species or the by-products of their metabolism to colonise the gut environment or conversely the presence of specific bacterial species may inhibit their colonisation.
Collectively, our studies shed new light on the prevalence, diversity and transmission potential of Blastocystis within and between healthy human hosts including adults and infants. We also provide novel data on the links between Blastocystis and other members of the gut ecosystem. Although Blastocystis is implicated in human disease our data shows that it is also a highly prevalent member of the healthy gut microbiota. We therefore suggest that the role of Blastocystis in human disease is complex and is perhaps similar to what is known about other microbial eukaryotes that infect the gut, such as Entamoeba spp. As such, the potential pathogenicity of Blastocystis may be linked to specific genotypes that are virulent, or opportunistic genotypes that are only virulent in specific host background or if there is an environmental change in the host such as antibiotic administration or immunocompromisation.
The first study we conducted investigated the prevalence and genetic diversity of Blastocystis in a two human groups of interest, namely healthy adults and infants with no gastrointestinal symptoms or disease. Using an optimised PCR assay we detected Blastocystis in 56% of adults surveyed (n = 105). This results shows that Blastocystis is a common component of the healthy adult human gut. In addition a diversity of different subtypes or species were detected in this adult group. We also conducted temporal analysis and found that where present Blastocystis is a stable member of the gut ecosystem. Conversely, Blastocystis is virtually absent in the corresponding infant population; only 3 of the 69 infants (~4%) were positive for Blastocystis.
In collaboration with Prof. Rob Knight we also looked at the prevalence, distribution and diversity of Blastocystis with family units and their pets (dogs) to look at the potential for human-human transmission and zoonotic transmission. Here we surveyed the presence of Blastocystis in healthy individuals (n = 139) that belonged to 50 families units living in Boulder, Colorado, USA as our sample-set. We detected a diversity of Blastocystis subtypes, however, the overall prevalence of Blastocystis was comparatively low (7%) in relation to other Western populations. Although we did not find any evidence of human-human transmission, given the world-wide variation in human living conditions and lifestyles we cannot rule out a role for this mode of transmission in other populations but instead we propose that human-human transmission of Blastocystis is unlikely in human populations living in similar circumstances to those outlined here. Moreover, we did not detect any Blastocystis in dogs, which supports a recent finding that dogs may not be natural hosts for Blastocystis and therefore do not pose any potential disease risk in this regard.
We also developed a novel set of primers to investigate the inter-species or inter-subtype diversity of Blastocystis within individual hosts. To do so we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes (STs) of Blastocystis, (ST1—ST4), to a healthy human cohort (n = 50). We detected mixed infections in 22% of cases, all of which had been identified as single ST infections in our earlier study of the adult population. Certain STs occur predominantly as either single (ST3 and 4) or mixed infections (ST1), which we speculate may reflect inter alia transient colonisation patterns and/or co-operative or competitive interactions between different STs. One particularly important aspect of this work was the comparative analysis of our novel primer set with other primers that have been used extensively for ST-specific analysis. These previously published primer set were found to be unsuitable for detection of mixed and, in some cases, single ST infections. Our development of these novel PCR assays will facilitate future work into the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.
The final part of our project looked at the link between Blastocystis and the bacterial component of the gut microbiota in healthy adult samples. We used 16S rRNA data generated as part of an earlier study and found that Blastocystis is significantly associated with high microbial diversity. Crucially this data may also explain the absence of Blastocystis in our infant population given that infants have a comparatively lower bacterial diversity. A number of bacterial species were positively correlated with the presence of Blastocystis, namely Robinsonella and Anaerosporobacter. Two bacterial species were negatively correlated with Blastocystis, namely Bilophila and E. coli. These data suggest that Blastocystis may require the presence of specific microbial species or the by-products of their metabolism to colonise the gut environment or conversely the presence of specific bacterial species may inhibit their colonisation.
Collectively, our studies shed new light on the prevalence, diversity and transmission potential of Blastocystis within and between healthy human hosts including adults and infants. We also provide novel data on the links between Blastocystis and other members of the gut ecosystem. Although Blastocystis is implicated in human disease our data shows that it is also a highly prevalent member of the healthy gut microbiota. We therefore suggest that the role of Blastocystis in human disease is complex and is perhaps similar to what is known about other microbial eukaryotes that infect the gut, such as Entamoeba spp. As such, the potential pathogenicity of Blastocystis may be linked to specific genotypes that are virulent, or opportunistic genotypes that are only virulent in specific host background or if there is an environmental change in the host such as antibiotic administration or immunocompromisation.