The body of vertebrates is inhabited by trillions of microorganisms, i.e., viruses,
archaea, bacteria, and unicellular eukaryotes, together referred to as the “microbiota.”
Similarly, vertebrates also host a plethora of parasitic worms (the “macrobiota”),
some of which share their environment with the microbiota inhabiting the gastrointestinal
(GI) tract [1]. Complex interactions between the helminths and the gut microbiota
have been associated with establishment of parasite infection, disease manifestations,
and host immune-modulation [2, 3]. Remarkably, not only GI helminths alter the gut
microbiome composition [4], but also the infections with blood flukes of the genus
Schistosoma have been associated with intestinal dysbiosis, that even occurs before
the onset of egg laying [5, 6]. Comparably, over the last decade, evidence has emerged
of the contribution(s) of the resident microbiota to several physiological and reproductive
processes of invertebrate hosts, including insects, arachnids, worms, and snails [7,
8]. These noteworthy discoveries, coupled with the recent expansion of high-throughput
microbiota- and microbiome-profiling approaches (the former referring to the community
of microorganisms themselves and the latter to the microorganisms and their genomes,
within a given ecological niche), are rapidly leading to a much better understanding
of the composition and functions of microbial communities inhabiting parasitic worms
of major public health and socioeconomic significance. This basic knowledge might
expose exploitable vulnerabilities of parasites, thus paving the way to the development
of novel control strategies [9].
In this Viewpoint, we consider the challenges associated with the study of the helminth
microbiota/-me, spanning not only bacteria transiently associated with parasites in
which the life cycle includes free-living and parasitic stages, but also putative
helminth endosymbionts. Indeed, endosymbionts have been described in both roundworms
and flatworms [10, 11]. In nematodes, the most notable example of a mutualistic relationship
between worms and bacteria is represented by filarial parasites [12], i.e., Onchocerca
volvulus, Wuchereria bancrofti, and Brugia malayi, agents of human lymphatic filariasis.
In particular, the fitness, propagation, and survival of these worms depend on endosymbiotic
bacteria of the genus Wolbachia that have thus become the target of intense research
aimed to develop novel filaricidal compounds [11, 13]. On the other hand, bacteria
of the genus Neorickettsia have been identified in the endoparasitic digeneans, i.e.,
trematodes [10]. These intracellular bacteria inhabit the worm reproductive tissues
and are vertically-transmitted to the next generation of parasites via the eggs [10].
In addition, horizontal transmission of Neorickettsia from the fluke to the fluke-infected
vertebrate host, where the bacteria colonize macrophages among other cell types, is
a determinant for the pathogenesis of severe disease in, for example, horses, dogs,
and humans [14]. Recently, we have sequenced and characterized the whole genome of
a Neorickettsia endobacterium in an isolate of adult Fasciola hepatica liver flukes
[15]. The Neorickettsia, related to the etiological agents of human Sennetsu and Potomac
horse fevers, was localized in the gonads of the liver fluke and its DNA detected
by PCR in eggs, thus supporting a germline transmission [15].
To decipher the role of parasite-associated microbiota on the pathophysiology of helminthiases,
the Parasite Microbiome Project (PMP) was launched in January 2019 [9]. Importantly,
the PMP encourages best practices for experimental designs to ensure robust and reliable
comparisons between datasets and promotes the inclusion of appropriate controls to
correctly identify environmental microbial contaminants [9]. These practices are particularly
important in experiments in which microbiota profiling is conducted using next generation
sequencing (NGS) (i.e., high-throughput) technologies that are particularly prone
to exogenous bacterial contamination, such as bacterial 16S rRNA-amplicon sequencing
on low-biomass samples, e.g., helminths [16, 17]. Therefore, given the potential confounders
in helminth-associated microbiome studies, we propose that four elements, outlined
below, must be considered in order to generate reliable and reproducible data (Fig
1).
10.1371/journal.pntd.0008446.g001
Fig 1
Key elements for a reliable and reproducible characterization of the helminth-associated
microbiome.
NGS; next generation sequencing.
1: Appropriate controls
The identification and characterization of the helminth-associated microbiota/-me
includes several experimental steps from sample collection to library preparation
and sequencing, each of which is exposed to different sources of contamination. Therefore,
the inclusion of matching negative controls (“blanks”) in each step of the experiment
is critical. The sample collection should ideally be carried out under clean conditions
by using disposable sterile consumables and autoclaved instruments to minimize the
risk of sample contamination with environmental bacteria. In addition, controls for
each tentative source of environmental contaminants should be included. Following
thorough screening, the sequence data generated from these negative controls from
each experimental step can be subtracted from the datasets under consideration.
2: Microscopical visualisation of helminth-associated bacteria
Following in silico subtraction of putative contaminant sequences, unequivocal identification
and characterization of worm microbiomes can rely on microscopical techniques aimed
to localize bacteria of interest across different helminth tissues and developmental
stages. Widely used approaches to localize specific groups of microorganisms are based
on fluorochromes conjugated to either antibodies or nuclei acid probes that bind to
specific bacterial proteins or nuclei acids, respectively. Neorickettsia bacteria
were identified within the reproductive tissue of the liver fluke F. hepatica via
fluorescent immunohistochemistry [15], whereas a recent report characterized the “core
microbiome” associated with the ovine GI nematode Haemonchus contortus using fluorescence
in situ hybridization and light and transmission electron microscopy [18].
3: “Core microbiome” versus transiently associated bacteria
The localization of microorganisms in helminth tissues is a robust indication of the
occurrence of a worm microbiome, and may provide clues on its function(s); for instance,
Wolbachia localized in the reproductive tissues of filarial parasites have been shown
to be involved in sexual differentiation [19] and worm survival [20]. However, the
distinction between bacteria that might be transiently associated with the parasite,
e.g., coating the surface of free-living larval stages or transported within the alimentary
tract of the parasite among different host niches, and those that might belong to
the worm “core microbiome” is crucial.
Protocols to eliminate bacteria contaminating the external tegument of GI worms have
been implemented. Treatment of Trichuris muris worms with sodium hypochlorite allowed
the identification of a specific parasite intestinal microbiota distinct from that
of its host [2]. Whether transiently associated bacteria have direct effects on parasite
biology still needs to be ascertained; notwithstanding, they might contribute to the
pathophysiology of the infection and comorbidity in the host. The Asian liver fluke
Opisthorchis viverrini provides a signal example in this regard; accumulating evidence
suggests that the juvenile form of the parasite excysted in the duodenum of the human
host might ingest Helicobacter pylori and/or related species of bacteria and transport
the bacillus to the bile duct, where this fluke establishes [21, 22]. Chronic infection
with either H. pylori bacteria or O. viverrini is classified by the International
Agency for Research on Cancer as a Group 1 carcinogen, leading to gastric adenocarcinoma
or cholangiocarcinoma (CCA), respectively [23]. We have previously reported a synergistic
association between the liver fluke and Helicobacter bacteria in the development of
the opisthorchiasis-associated CCA [21], which may result from an eventual horizontal
transmission of bacteria from the parasite to host tissue. On the other hand, a worm
“core microbiome” (particularly, if associated with the parasite gonads) may be vertically-transmitted
to the next generation and, hence, detected across different developmental stages.
Therefore, screening for the presence of bacteria in different developmental stages
of the parasite, either by PCR, qPCR, and/or 16S-rRNA amplicon NGS [15, 18] is recommended
to define the “core microbiome” that might serve as a foundation to explore novel
strategies for transmission control. In addition to bacteria, the “core microbiome”
may comprise microeukaryotes, such as fungi and protozoa, and viruses that can be
detected by shotgun metagenomic approaches [9]. Although these methods are not inexpensive
and generate complex data (which require advanced bioinformatics analyses that include
the identification and in silico subtraction of helminth-derived sequences), their
application is recommended to gain an overall picture of the helminth microbiome and
enable the prediction of bacterial metabolic pathways that might be essential for
worm biology and (patho)physiology associated with the infection [24]. Subsequently,
the use of functional approaches to investigate the roles of this “core microbiome”
in worm biology, helminth infection, establishment, and host–parasite interactions
becomes critical.
4: Functional studies of the helminth-associated microbiome
The follow-on step after the identification of both transiently associated bacteria
and the worm “core microbiome” is to understand the biological relevance of these
interactions. The use of broad- or narrow-spectrum antibiotics to alter the worm microbiome
might assist the determination of the essentiality of these bacteria for worm survival,
fitness, and/or reproduction [25]. In addition, optimization of protocols for in vitro
and ex vivo culturing of parasitic developmental stages [26–28], and the use of organoids
to simulate interactions between parasites, host cells, and selected bacteria [29],
in tandem with functional genomic tools currently under development for helminths
(e.g., genome editing [30–32]) and bacteria [33] will assist the set-up of controlled
experiments to address hypotheses on mechanisms underlying worm-microbes interactions.
Similar approaches have been employed for model organisms such as Caenorhabditis elegans.
The C. elegans–associated microbiome has been analyzed in laboratory settings by culturing
worms on individual bacterial strains and evaluating helminth growth rate and responses
of stress and immune related genes. The majority of the bacterial strains investigated
were found to be beneficial for worm fitness [34]. Finally, where feasible, in vivo
studies using rodent models of helminth infection might provide invaluable functional
insights on transmission of bacteria across parasitic developmental stages, microbial
horizontal translocation to host tissues, and bacteria-mediated pathologies associated
with helminthiases. Germ-free and gnotobiotic mice (i.e., animals exclusively colonised
by known microbes) are extensively used in microbiome studies [35]. However, the dysfunctional
immune response of these animals might add several confounders to the infection model.
On the other hand, the use of antibiotics in well-established murine models of helminthiases
might allow to target specific groups of host- and/or worm-associated bacteria.
To conclude, similarly to the human microbiome, bacteria associated with helminth
parasites likely represent an intrinsic part of these organisms, so much so that parasite
biology might not be completely understood in its absence. However, the characterization
of the genuine helminth microbiome may turn out to be complex, or even daunting, due
to several technical challenges. In our view, rigorous hygiene to exclude or at least
minimize contaminants, together with bacterial localization in helminth tissues and
across developmental stages, and their functional characterization are essential steps
to unequivocally identify bacteria associated with parasitic worms and ascertain their
roles in the dynamic crosstalk among the parasite, the host, and the host microbiome.
Ultimately, this will contribute to the current incomplete understanding of the biology
and pathogenicity of helminths.