Collateral Damage in the Human Gut Microbiome - Blastocystis Is Significantly Less Prevalent in an Antibiotic-Treated Adult Population Compared to Non-Antibiotic Treated Controls.

Antibiotics can drive the rapid loss of non-target, phylogenetically diverse microorganisms that inhabit the human gut. This so-called "collateral damage" has myriad consequences for host health and antibiotic mediated changes to the gut microbiota have been implicated in the aetiology of many chronic diseases. To date, studies have largely focused on how antibiotics affect the bacterial fraction of the gut microbiome and their impact on non-bacterial members, including prevalent eukaryal species, such as Blastocystis, remains largely unknown. Here we assessed the prevalence and diversity of Blastocystis in an elderly adult group that were in receipt of antibiotics (n = 86) and an equivalent non-antibiotic treated group (n = 88) using a PCR-based approach. This analysis revealed that although similar subtypes were present in both groups, Blastocystis was significantly less prevalent in the antibiotic-treated group (16%) compared to non-antibiotic treated controls (55%); Fisher's Exact test, p < 0.0001). Given that antibiotics target structures and molecules of prokaryotic cells to kill or inhibit bacterial populations, the most likely explanation for differences in prevalence between both groups is due to secondary extinctions owing to the potential dependence of Blastocystis on bacteria present in the gut microbiome that were negatively affected by antibiotic treatment. Although further work is required to explore this hypothesis in greater detail, these data clearly show that Blastocystis prevalence in human populations is negatively associated with antibiotic treatment. This finding may be relevant to explaining patterns of variation for this microorganism in different human populations and cohorts of interest.

Microbial evolution and ecological opportunity in the gut environment.

Recent genomic and metagenomic studies have highlighted the presence of rapidly evolving microbial populations in the human gut. However, despite the fundamental implications of this intuitive finding for both basic and applied gut microbiome research, very little is known about the mode, tempo and potential functional consequences of microbial evolution in the guts of individual human hosts over a lifetime. Here I assess the potential relevance of ecological opportunity to bacterial adaptation, colonization and persistence in the neonate and germ-free mammalian gut environment as well as over the course of an individual lifetime using data emerging from mouse models as well as human studies to provide examples where possible. I then briefly outline how the continued development and application of experimental evolution approaches coupled to genomic and metagenomic analysis is essential to disentangling drift from selection and identifying specific drivers of evolution in the gut microbiome within and between individual human hosts and populations.

Impact of bile salts on coevolutionary dynamics between the gut bacterium Escherichia coli and its lytic phage PP01.

Although host and parasites are typically embedded in complex abiotic and biotic environments our understanding of how environmental variation impacts on host-parasite interactions, including antagonistic coevolution (AC) is poorly understood. Nonetheless, previous studies using bacteria and bacteriophages have shown that variation in just one abiotic parameter can have profound effects not only on the type of AC dynamics observed but also the time-frames over which AC interactions can persist. Here, we investigated the effect of an important component of the abiotic human gut environment, bile salts, on AC dynamics between the bacterium Escherichia coli and the lytic phage PP01 in an in vitro model system. In the absence of bile salts E.coli and PP01 coevolved in a manner that is consistent with a directional arms race dynamic (ARD), with bacteria and phages evolving increasing resistance and infectivity ranges through time. However, in the presence of bile salts, evidence of directional coevolution was weaker and more variable across replicate communities. These effects may be explained, in part, by the negative effect of bile salts on both host and parasite population sizes; lower population sizes for both bacteria and phages will reduce encounter rates which in turn could mitigate the benefits of generalism in both host and parasite resistance and infectivity ranges that are observed for ARDs. The negative effect of bile salts on phage population size may also be partially independent of host population size as bile salts was found to negatively impact phage viability in the absence of bacteria, as well as reducing phage adsorption efficiency. Differences in bacterial morphological diversity between treatments were also noted, with the emergence of mucoid colonies in both bile salts and non-bile salts treatments but only in the presence of phages. These data contribute to the growing body of knowledge on how environmental variation can impact on interactions between hosts and parasites. More specifically, these results are particularly relevant to our understanding of how bacteria-phage interactions may be affected by different abiotic factors relevant to the complex environment of the human gut and have clear implications for the development of phage as therapeutics to target members of the gut microbiota and/or intestinal pathogens.

Within-host interference competition can prevent invasion of rare parasites.

Competition between parasite species or genotypes can play an important role in the establishment of parasites in new host populations. Here, we investigate a mechanism by which a rare parasite is unable to establish itself in a host population if a common resident parasite is already present (a 'priority effect'). We develop a simple epidemiological model and show that a rare parasite genotype is unable to invade if coinfecting parasite genotypes inhibit each other's transmission more than expected from simple resource partitioning. This is because a rare parasite is more likely to be in multiply-infected hosts than the common genotype, and hence more likely to pay the cost of reduced transmission. Experiments competing interfering clones of bacteriophage infecting a bacterium support the model prediction that the clones are unable to invade each other from rare. We briefly discuss the implications of these results for host-parasite ecology and (co)evolution.

The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome.

The human gut is host to a diverse range of fungal species, collectively referred to as the gut "mycobiome". The gut mycobiome is emerging as an area of considerable research interest due to the potential roles of these fungi in human health and disease. However, there is no consensus as to what the best or most suitable methodologies available are with respect to characterizing the human gut mycobiome. The aim of this study is to provide a comparative analysis of several previously published mycobiome-specific culture-dependent and -independent methodologies, including choice of culture media, incubation conditions (aerobic versus anaerobic), DNA extraction method, primer set and freezing of fecal samples to assess their relative merits and suitability for gut mycobiome analysis. There was no significant effect of media type or aeration on culture-dependent results. However, freezing was found to have a significant effect on fungal viability, with significantly lower fungal numbers recovered from frozen samples. DNA extraction method had a significant effect on DNA yield and quality. However, freezing and extraction method did not have any impact on either α or ß diversity. There was also considerable variation in the ability of different fungal-specific primer sets to generate PCR products for subsequent sequence analysis. Through this investigation two DNA extraction methods and one primer set was identified which facilitated the analysis of the mycobiome for all samples in this study. Ultimately, a diverse range of fungal species were recovered using both approaches, with Candida and Saccharomyces identified as the most common fungal species recovered using culture-dependent and culture-independent methods, respectively. As has been apparent from ecological surveys of the bacterial fraction of the gut microbiota, the use of different methodologies can also impact on our understanding of gut mycobiome composition and therefore requires careful consideration. Future research into the gut mycobiome needs to adopt a common strategy to minimize potentially confounding effects of methodological choice and to facilitate comparative analysis of datasets.

Forgotten fungi-the gut mycobiome in human health and disease.

The human body is home to a complex and diverse microbial ecosystem that plays a central role in host health. This includes a diversity of fungal species that is collectively referred to as our 'mycobiome'. Although research into the mycobiome is still in its infancy, its potential role in human disease is increasingly recognised. Here we review the existing literature available on the human mycobiota with an emphasis on the gut mycobiome, including how fungi interact with the human host and other microbes. In doing so, we provide a comprehensive critique of the methodologies available to research the human mycobiota as well as highlighting the latest research findings from mycological surveys of different groups of interest including infants, obese and inflammatory bowel disease cohorts. This in turn provides new insights and directions for future studies in this burgeoning research area.

Bacteria-Bacteriophage Coevolution in the Human Gut: Implications for Microbial Diversity and Functionality.

Antagonistic coevolution (AC) between bacteria and bacteriophages plays a key role in driving and maintaining microbial diversity. Consequently, AC is predicted to affect all levels of biological organisation, from the individual to ecosystem scales. Nonetheless, we know nothing about bacteria-bacteriophage AC in perhaps the most important and clinically relevant microbial ecosystem known to humankind - the human gut microbiome. In this opinion piece I review current research on bacteria-phage AC in in vitro and natural populations of microbes. I then examine the evidence and discuss the potential role of AC in driving observed patterns of intra- and interindividual variation in the gut microbiome together with detailing the potential functional consequences of such AC-driven microbial variation for human health and disease.

Modification of Escherichia coli-bacteriophage interactions by surfactants and antibiotics in vitro.

Although experiments indicate that the abiotic environment plays an important role in bacterial interactions with their parasitic viruses (bacteriophages or phages), it is not yet clear how exposure to compounds present in nature alters the impact of phages on bacterial growth and evolution. To address this question, we exposed Escherichia coli K12 MG1655, in combination with three lytic phages, to various substances that natural and clinical microbial populations are likely to encounter: bile salts (present in mammalian gastrointestinal tracts), sodium dodecyl sulfate (SDS, a common surfactant in cleaning and hygiene products) and four antibiotics (present at variable concentrations in natural and clinical environments). Our results show that bile salts and SDS can reduce the detrimental effect of phages on bacterial growth. In some cases these compounds completely mitigated any negative effects of phages on bacterial growth and consequently bacteria did not evolve resistance to phages in these conditions. The proportional effects of phages were unaffected by antibiotics in most combinations, excepting three cases of phage-drug synergy. These results suggest that accounting for interactions between phages and environmental factors such as surfactants and antibiotics will improve understanding of both bacterial growth and resistance evolution to phages in vivo and in nature.

Parasite genetic distance and local adaptation in co-evolving bacteria-bacteriophage populations.

Antagonistic co-evolution between hosts and parasites can lead to local adaptation (LA) such that parasite fitness is greatest in sympatric hosts (or vice versa). The magnitude of LA typically increases with geographical distance, which is assumed to be because genetic (and hence phenotypic) distance increases with geographical distance. Here, we explicitly test the relationships between parasite genetic and phenotypic distance and LA using isolates of co-evolved viral parasites (lytic bacteriophage ϕ2) and the host bacterium Pseudomonas fluorescens SBW25. We find positive relationships between parasite genotype and infectivity phenotype, but the strength of the relationship was greater when infectivity was defined by the identity of hosts that could be infected rather than the actual number of hosts infected (host range), and when measurements were compared within rather than among populations. Crucially, we find a monotonic relationship between LA and genetic distance across phage isolates from different populations, although in contrast to many geographical studies, parasite LA decreased with genetic distance. These results can be explained by the fact that bacteria can rapidly adapt to phage infectivity mutations, but that evolved resistance has a degree of specificity to the local phage population. Our results show that antagonistic co-evolution alone can result in predictable links between genetic distance and host-parasite local adaptation.

Prevalence and genetic diversity of Blastocystis in family units living in the United States.

The human gut is host to a diversity of microorganisms including the single-celled microbial eukaryote Blastocystis. Although Blastocystis has a global distribution, there is dearth of information relating to its prevalence and diversity in many human populations. The mode of Blastocystis transmission to humans is also insufficiently characterised, however, it is speculated to vary between different populations. Here we investigated the incidence and genetic diversity of Blastocystis in a US population and also the possibility of Blastocystis human-human transmission between healthy individuals using family units (N=50) living in Boulder, Colorado as our sample-set. Ten of the 139 (~7%) individuals in our dataset were positive for Blastocystis, nine of whom were adults and one individual belonging to the children/adolescents group. All positive cases were present in different family units. A number of different Blastocystis subtypes (species) were detected with no evidence of mixed infections. The prevalence of Blastocystis in this subset of the US population is comparatively low relative to other industrialised populations investigated to date; however, subtype diversity was largely consistent with that previously reported in studies of European populations. The distribution of Blastocystis within family units indicates that human-human transmission is unlikely to have occurred within families that participated in this study. It is not unexpected that given the world-wide variation in human living conditions and lifestyles between different populations, both the prevalence of Blastocystis and its mode of transmission to humans may vary considerably.

Adaptation to abiotic conditions drives local adaptation in bacteria and viruses coevolving in heterogeneous environments.

Parasite local adaptation, the greater performance of parasites on their local compared with foreign hosts, has important consequences for the maintenance of diversity and epidemiology. While the abiotic environment may significantly affect local adaptation, most studies to date have failed either to incorporate the effects of the abiotic environment, or to separate them from those of the biotic environment. Here, we tease apart biotic and abiotic components of local adaptation using the bacterium Pseudomonas fluorescens and its viral parasite bacteriophage Φ2. We coevolved replicate populations of bacteria and phages at three different temperatures, and determined their performance against coevolutionary partners from the same and different temperatures. Crucially, we measured performance at different assay temperatures, which allowed us to disentangle adaptation to biotic and abiotic habitat components. Our results show that bacteria and phages are more resistant and infectious, respectively, at the temperature at which they previously coevolved, confirming that local adaptation to abiotic conditions can play a crucial role in determining parasite infectivity and host resistance. Our work underlines the need to assess host-parasite interactions across multiple relevant abiotic environments, and suggests that microbial adaption to local temperatures can create ecological barriers to dispersal across temperature gradients.

Experimental evolution and bacterial resistance: (co)evolutionary costs and trade-offs as opportunities in phage therapy research.

Antagonistic coevolution between bacteria and phages (reciprocal selection for resistance and infectivity) has been demonstrated in a wide range of natural ecosystems, as well as experimental populations of microbes, yet exploiting knowledge of coevolution for the prophylactic and therapeutic use of phages is under-explored. In this addendum to our recent paper we discuss how real-time coevolution studies using experimental populations of bacteria and phages can provide novel insight into the changes in bacterial phenotypes that result from resistance evolution against coevolving phages, and how this may ultimately improve our understanding of phage therapy and ability to design effective treatments.

Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population.

The human gut is host to a diversity of microorganisms, including the single-celled microbial eukaryote Blastocystis. Research has shown that most carriers host a single Blastocystis subtype (ST), which is unusual given the considerable within-host species diversity observed for other microbial genera in this ecosystem. However, our limited knowledge of both the incidence and biological significance of Blastocystis diversity within hosts (i.e., so-called mixed infections) is likely due to problems with existing methodologies. Here, we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes of Blastocystis (ST1 to ST4) to a healthy human cohort (n = 50). We detected mixed infections in 22% of the cases, all of which had been identified as single-ST infections in a previous study using state-of-the-art methods. Our results show that certain STs occur predominantly as either single (ST3 and 4) or mixed (ST1) infections, which may reflect inter alia transient colonization patterns and/or cooperative or competitive interactions between different STs. Comparative analyses with other primers that have been used extensively for ST-specific analysis found them unsuitable for detection of mixed- and, in some cases, single-ST infections. Collectively, our data shed new light on the diversity of Blastocystis within and between human hosts. Moreover, the development of these PCR assays will facilitate future work on 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.

Coevolution with bacteriophages drives genome-wide host evolution and constrains the acquisition of abiotic-beneficial mutations.

Studies of antagonistic coevolution between hosts and parasites typically focus on resistance and infectivity traits. However, coevolution could also have genome-wide effects on the hosts due to pleiotropy, epistasis, or selection for evolvability. Here, we investigate these effects in the bacterium Pseudomonas fluorescens SBW25 during approximately 400 generations of evolution in the presence or absence of bacteriophage (coevolution or evolution treatments, respectively). Coevolution resulted in variable phage resistance, lower competitive fitness in the absence of phages, and greater genome-wide divergence both from the ancestor and between replicates, in part due to the evolution of increased mutation rates. Hosts from coevolution and evolution treatments had different suites of mutations. A high proportion of mutations observed in coevolved hosts were associated with a known phage target binding site, the lipopolysaccharide (LPS), and correlated with altered LPS length and phage resistance. Mutations in evolved bacteria were correlated with higher fitness in the absence of phages. However, the benefits of these growth-promoting mutations were completely lost when these bacteria were subsequently coevolved with phages, indicating that they were not beneficial in the presence of resistance mutations (consistent with negative epistasis). Our results show that in addition to affecting genome-wide evolution in loci not obviously linked to parasite resistance, coevolution can also constrain the acquisition of mutations beneficial for growth in the abiotic environment.

The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota.

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.

Loss of social behaviours in populations of Pseudomonas aeruginosa infecting lungs of patients with cystic fibrosis.

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections.

Blastocystis: getting to grips with our guileful guest.

Blastocystis, a common single-celled intestinal parasite of humans and animals, continues to puzzle clinical microbiologists, gastroenterologists, and general practitioners who are still unsure of the clinical significance of the organism. Here we consider some less well-addressed areas of Blastocystis research, which, facilitated by recent technological advances, could potentially turn out to be significant pathways to knowledge. First and foremost we discuss new trends in Blastocystis research, including the 'omics' perspectives, and then highlight some aspects of Blastocystis research in the context of host coevolution, its potential as a biomarker of intestinal functionality, and its relationship to other components of the human intestinal microbiota.

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.

Host-parasite coevolutionary arms races give way to fluctuating selection.

Host-parasite coevolution is a key driver of biological diversity and parasite virulence, but its effects depend on the nature of coevolutionary dynamics over time. We used phenotypic data from coevolving populations of the bacterium Pseudomonas fluorescens SBW25 and parasitic phage SBW25Φ2, and genetic data from the phage tail fibre gene (implicated in infectivity evolution) to show that arms race dynamics, typical of short-term studies, decelerate over time. We attribute this effect to increasing costs of generalism for phages and bacteria with increasing infectivity and resistance. By contrast, fluctuating selection on individual host and parasite genotypes was maintained over time, becoming increasingly important for the phenotypic properties of parasite and host populations. Given that costs of generalism are reported for many other systems, arms races may generally give way to fluctuating selection in antagonistically coevolving populations.