Abstract

To date, the majority of research into the human gut microbiota has focused on the bacterial fraction of the community. Inevitably, this has resulted in a poor understanding of the diversity and functionality of other intestinal microorganisms in the human gut. One such nonbacterial member is the microbial eukaryote Blastocystis, which has been implicated in the aetiology of a range of different intestinal and extra-intestinal diseases. However, prevalence data from different studies are conflicting, and crucially, there is limited information on its incidence and diversity in healthy individuals. Here, we survey the prevalence, genetic diversity and temporal stability of Blastocystis in a group of healthy adults (n = 105) using a sensitive PCR assay. Blastocystis was present in 56% of our sample set, which is much higher than previously reported from an industrialised county (Ireland). Moreover, a diversity of different subtypes (species) were detected, and Blastocystis was present in a subset of individuals sampled over a period of time between 6 and 10 years, indicating that it is capable of long-term host colonisation. These results show that Blastocystis is a common and diverse member of the healthy gut microbiota, thereby extending our knowledge of the microbial ecology of the healthy human intestine.

Blastocystis is a prevalent and diverse member of the human gut microbiota.

Blastocystis is a prevalent and diverse member of the human gut microbiota.

The human ‘gut microbiota’ is a collective terms used to refer to the microbial inhabitants of the human intestinal tract (IT). However, a thorough understanding of the composition of the gut microbiota and the microbial ecology of the healthy human gut is lacking due to a research focus on the bacterial fraction of the community. In addition to bacteria, a diverse collection of other microorganisms are present in the human gut (Scanlan & Marchesi, 2008; Hamad et al., 2012) including members of the microbial eukaryotes. Such microorganisms are now the focus of increased interest (Parfrey et al., 2011), especially following greater recognition of their relevance to host health (Iliev et al., 2012). While some of the research that has taken place has focused solely on fungal diversity within the human gut (Chen et al., 2011; Gouba et al., 2013), a number of studies have taken a broader approach using universal primers to explore the diversity of all microbial eukaryotes present (Nam et al., 2008; Scanlan & Marchesi, 2008; Hamad et al., 2012; Pandey et al., 2012). Even though the number of these universal studies is limited and sample sizes are small, one common trend emerging is the widespread prevalence of the intestinal protist Blastocystis (Nam et al., 2008; Scanlan & Marchesi, 2008; Hamad et al., 2012; Pandey et al., 2012; Gouba et al., 2013).

Blastocystis is a unicellular, nonflagellated member of the Stramenopiles (or Heterokonta) (Silberman et al., 1996), which is a branch of the Eukarya that comprises a collection of uni- and multi-cellular organisms including diatoms, algae and oomycetes (Patterson, 1999). To date, seventeen different Blastocystis subtypes (arguably seventeen different species) have been described (Alfellani et al., 2013) and in addition to colonising the human IT, Blastocystis is also found in a range of other hosts including other mammals, birds and reptiles (Ramirez et al., 2013). With respect to its prevalence in human populations, Blastocystis has largely been investigated in the context of disease and some studies have implicated it in a number of different intestinal and extra-intestinal diseases including Inflammatory Bowel Disease, Irritable Bowel Syndrome, autism and urticaria (Boorom, 2007; Tan et al., 2010; Poirier et al., 2012). Blastocystis is also cited as the causative agent of an illness termed Blastocystosis (Tan et al., 2010), which is characterised by a loose collection of nonspecific symptoms (inter alia abdominal pain, diarrhoea and bloating) that could in fact be attributed to and are associated with any number of other infectious microorganisms and/or intestinal disorders. Thus, the question of whether Blastocystis spp. play a role in certain human diseases or not remains both unclear and controversial. Indeed, although in vitro and genomic data that support the role of Blastocystis as an emerging pathogen have been published (Puthia et al., 2006, 2008; Tan et al., 2010; Mirza et al., 2012), no links with human disease have been unequivocally demonstrated and its role as an aetiologic agent is still speculative (Tan et al., 2010; Scanlan, 2012; Scanlan & Stensvold, 2013).

There are a number of factors that have hampered progress in establishing a definitive role for Blastocystis in human disease in the past. Central to this are methodological and experimental design issues (Tan, 2004; Tan et al., 2010; Scanlan, 2012). Fortunately, these problems are now recognised and researchers have begun to switch to molecular-based screening approaches (Poirier et al., 2011; Stensvold et al., 2012a, b; Bart et al., 2013; Clark et al., 2013). Nonetheless, one of the outstanding issues is the lack of reliable epidemiological data (Tan et al., 2010), and in particular, epidemiological data obtained from human populations of healthy, randomly sampled individuals using a standardised sensitive molecular-based approaches are lacking (Scanlan, 2012).

To date, reported prevalence rates are generally higher in developing countries and have been associated with sanitation levels, water source and contact with animals (Tan, 2008; Lee et al., 2012; Wawrzyniak et al., 2013). The highest prevalence reported for Blastocystis is 100% and is from a recent study of 93 children living in the Senegal River Basin (El Safadi et al., 2014). In contrast to this, current estimates of Blastocystis prevalence in developed countries range from 0.5% to 30% depending on the methods used and the population sampled (Bart et al., 2013; Scanlan & Stensvold, 2013; Wawrzyniak et al., 2013). Here, we optimised a PCR assay using the existing Blastocystis primer set RD5 and BhRDr that targets the 18S rRNA gene (Scicluna et al., 2006) to survey the prevalence of Blastocystis in our sample set which consisted of 100 healthy adults living in Cork, Ireland, that had participated in the Eldermet study (Claesson et al., 2012), and five additional healthy adults from Ireland that had participated in an earlier study (Scanlan & Marchesi, 2008). These five additional individuals were also included in the longitudinal analysis, see Table 1; Supporting Information, Table S1.

Table 1

Overview of participants, full details are provided in Table S1

Group (nMean age and range (years) Male to female ratio Ethnicity Blastocystis prevalence (%) Sampling period 
Eldermet Control (< 65), (n = 12) 34.4 (28–46) 1 : 1 Caucasian 58 NA 
Eldermet (> 65), (n = 88) 72.7 (64–93) 1 : 1.3 Caucasian 55 NA 
Irish (prevalence and longitudinal analysis), (n = 5) 45.4 (27–67) 1 : 1.5 Caucasian 80 6–10 years 
the Netherlands (longitudinal analysis), (n = 5) 31 (25–41) 1.5 : 1 Caucasian and Asian 7 years 
Group (nMean age and range (years) Male to female ratio Ethnicity Blastocystis prevalence (%) Sampling period 
Eldermet Control (< 65), (n = 12) 34.4 (28–46) 1 : 1 Caucasian 58 NA 
Eldermet (> 65), (n = 88) 72.7 (64–93) 1 : 1.3 Caucasian 55 NA 
Irish (prevalence and longitudinal analysis), (n = 5) 45.4 (27–67) 1 : 1.5 Caucasian 80 6–10 years 
the Netherlands (longitudinal analysis), (n = 5) 31 (25–41) 1.5 : 1 Caucasian and Asian 7 years 

Please see Claesson et al. (2012) for further information on the Eldermet trial.

For longitudinal analysis, between three and eight samples were analysed per person. Each of the four positive Irish samples were sampled at year 0, 1, 2 and 6, and the remaining Irish individual (Blastocystis negative) was sampled at year 0, 1, 2, 3, 4, 6, 8 and 10 years. Each of the Dutch samples was taken at year 0, 3 and 7. The mean of the averaged age for the time period analysed is given, and the range is the highest and lowest ages for all individuals in that group over the time period analysed. Due to potential biases arising from geographical variation in Blastocystis prevalence, only Irish samples were analysed and reported in the overall prevalence figures (i.e. 59/105 or 56% prevalence).

Our assay had a limit of detection at Blastocystis cell densities of 103/cells per gram of faeces and at DNA concentrations of < 1 ng μL−1 per PCR. Blastocystis was detected in 59 individuals or 56% of the sample population (n = 105). Although this number is lower than an earlier report of 82% prevalence in an Irish population (Scanlan & Marchesi, 2008), the number of individuals assayed previously (n = 17) limited the scope of (statistical) analysis and conclusions. We found no significant difference in the prevalence of Blastocystis between elderly and nonelderly subjects (χ2 = 0.645, d.f. = 1, P=0.422), or between female (61.8%) and male (46.7%) subjects (χ2 = 1.861, d.f. = 1, P=0.173). Blastocystis transmission to humans is thought to occur via contact with other colonised humans or animals or through waterborne transmission (Tan, 2008; Lee et al., 2012). Although it is difficult to trace the potential exposure of an individual to Blastocystis over their lifetime, we looked for a relationship between their current water source (urban or rural) and the incidence of Blastocystis. However, no significant relationship was evident (P=0.754) with equivalent prevalence in urban and rural groups.

We also analysed the temporal stability of Blastocystis in ten individuals to provide some insight into long-term colonisation trends. This sample set consisted of a subset of our Irish population (n = 5) and an additional five individuals from the Netherlands (n = 5) (Rajilic-Stojanovic et al., 2012). These individuals were sampled over different time periods, ranging from 6 to 10 years, see Table 1; Table S1 (Scanlan & Marchesi, 2008; Rajilic-Stojanovic et al., 2012). Four of these individuals were consistently positive for the same Blastocystis strain (determined at allele level) over the time period sampled indicating that, once established, Blastocystis is a stable component of the healthy human gut microbiota. Of the four individuals that were positive, all were Irish; that is, none of the samples from the Netherlands study testing positive. These five Dutch individuals plus one of the Irish subjects tested negative for Blastocystis and, similar to the consistency in the temporal trend observed for positive samples, these individuals were negative at all time-points sampled up to a period of 10 years.

To date, nine different Blastocystis subtypes (arguably separate species) have been found in humans (Clark et al., 2013). All 59 positive PCR products provided single-trace reads indicating the absence of mixed infections. At the subtype (ST) level, all reads could be assigned to one of five STs using the online site http://pubmlst.org/blastocystis/ (Jolley & Maiden, 2010; Stensvold et al., 2012a, b); however, the relative frequency of STs within the dataset was significantly different (χ2 = 27. 02, d.f. = 4, P<0.0001). ST3 was the most common ST accounting for 42% of positive PCR products, followed by ST2 (25%), ST4 (19%), ST1 (12%) and ST8 (2%), see Fig. 1a. Blastocystis ST distribution within the positive subpopulation was independent of host age or gender (P=0.772 and 0.883, respectively). Although there are differences in the geographic distribution of different subtypes (for example, ST4 is much less common in African populations compared with European populations), of the nine STs associated with humans, recent analysis has shown that STs 1–4 are the most common and account for c. 90% of all STs identified in Blastocystis subtyping surveys of human samples (Alfellani et al., 2013b, Alfellani et al., 2013c). Our data are consistent with this pattern of ST1-ST4 predominance in human populations. These subtypes were further delineated to 10 different alleles (intra-subtype variants). Similar to the analysis of ST frequencies, the relative frequencies of different alleles within the sample population differed significantly (χ2 = 76.92, d.f. = 10, P<0.0001). ST3-34 accounted for 37% of alleles in the dataset. The greatest within subtype diversity was observed for ST2 with five different alleles detected, two allele variants were detected for ST3, and only one allele type was identified for ST1, ST4 and ST8, see Fig. 1b.

Fig. 1

Overview of subtype (a) and intra-subtype/allele (b) percentage distributions within the Blastocystis positive subpopulation.

Fig. 1

Overview of subtype (a) and intra-subtype/allele (b) percentage distributions within the Blastocystis positive subpopulation.

In conclusion, the high percentage of individuals positive for Blastocystis and the temporal stability of Blastocystis (when present) show that Blastocystis is a common member of the healthy human intestinal microbial ecosystem and, where present, appears to be a stable feature. Moreover, the range of different Blastocystis subtypes and genotypes present in individuals indicate that a diversity of different Blastocystis spp. can colonise the healthy human gut without resulting in symptomatic carriage. Nonetheless, the question of what role Blastocystis plays in intestinal illness and disease remains outstanding. If a specific Blastocystis sp. is found to be virulent or disease causing, this would be analogous to what is now known about Entamoeba spp. distribution and pathogenicity in human populations. A diversity of different Entamoeba species have been detected in humans to date; however, only some species cause illness and the majority of individuals that are host to Entamoeba spp. are colonised by apparently nonpathogenic species (Stensvold et al., 2011). Similarly, the majority of Blastocystis spp. may be harmless and the virulence of Blastocystis spp. might be limited a subset of genetic variants or intra-subtypes (Tan et al., 2010; Scanlan, 2012). An additional scenario is that different variants might be opportunistic in different host backgrounds (Genotype × Genotype interaction) or where an environmental component(s) that, for example, compromises the immune status of the host facilitates disease initiation and progression (Genotype × Genotype × Environment interaction). As more data emerge from carefully controlled studies that use appropriate methods, this will hopefully facilitate a better understanding of the relationship between Blastocystis and disease, determine what factors govern host colonisation and move towards understanding the ecological role (if any) of this common and diverse resident of the human gut.

Acknowledgements

P.S. is supported by an Intra-European Marie Curie Research Fellowship. The authors are grateful to three anonymous reviewers who provided useful comments on the manuscript.

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Supporting Information

Table S1. Summary data of all study participants.

Author notes

Editor: Julian Marchesi
Intra-European Marie Curie Research Fellowship