Harnessing the Gut Microbiome in the Fight against Anthelminthic Drug Resistance

Abstract

Intestinal helminth parasites present major challenges to the welfare of humans and threaten the global food supply. While the discovery of anthelminthic drugs empowered our ability to offset these harms to society, the alarming rise of anthelminthic drug resistance mitigates contemporary efforts to treat and control intestinal helminthic infections. Fortunately, emerging research points to potential opportunities to combat anthelminthic drug resistance by harnessing the gut microbiome as a resource for discovering novel therapeutics and informing responsible drug administration. In this review, we highlight research that demonstrates this potential and provide rationale to support increased investment in efforts to uncover and translationally utilize knowledge about how the gut microbiome mediates intestinal helminthic infection and its outcomes.

Introduction

Intestinal helminth parasites (IHPs) plague humanity. A quarter of the world’s population lives with IHPs [1–5], including several nematodes, such as hookworms (e.g., Ancylostoma and Necator), Ascaris lumbricoides, Strongyloides stercoralis, whipworm (Trichuris trichiura), and pinworm (Enterobius vermicularis), as well as cestodes, such as tapeworms (e.g. Taenia spp.). These IHPs predominately infect people from impoverished nations and account for three of the WHO’s 20 Neglected Tropical Diseases. IHP infections often protract [6] and yield stunting [7], anemia [7], and decreased cognitive ability [8] that collectively contribute to humanity’s five million year loss of “healthy” life span (disability-adjusted life years) due to these infections [9].

Of course, IHP infections are not limited to humans. All major lineages of vertebrates carry IHPs. Among wildlife, IHPs are relatively ubiquitous [10], but increasingly play a role in wildlife population declines [11], in part due to the human influence of the transmission dynamics of wildlife nematodes [12]. IHPs also present a major cost to the global livestock industry in the form of billions of dollars of loss per annum worldwide due to infections of grazing livestock [13,14]. Unfortunately, these costs are expected to increase as recent evidence links climate change to on-going livestock helminth range shift expansions in temperate regions [15].

Against this backdrop of IHP prevalence and harm, anthelminthic drug resistance emerges as a critical social danger. Drug-resistant IHPs, especially nematodes, commonly infect livestock [16] and increasingly infect humans [17]. Resistance has been observed across the three major antiparasitic drug classes (Table 1)[16,18,19], including avermectins (macrocyclic lactones), benzimidazoles, and cholinergic receptor anthelminthics (e.g., levamisole). In some regions, multi-drug resistant IHPs have even risen to the level that livestock industry cannot be sustained [20,21] and thus, overall, the problem of drug resistance threatens global food security [22]. Resistance in veterinary species serves as a sentinel for how anthelminthic resistance will increase within the human population [18], possibly because resistance may spread from livestock to soil and ultimately arrive in human infectious helminths [9,20].

Table 1.

Major Anthelminthic Drug Classes and Reported IHP Resistance

Drug Class	Drug Examples	Mechanism Of Action	Activity Spectrum	Hosts with high levels of reported resistance [16]	Recent Resistance xamples
Benzimidazoles	• Fenbendazle • Albendazole • Mebendazole • Thiabendazole	Inhibits nematode egg production by binding to beta tublin and preventing microtubule assembly.	Antinematodal +/− anticestodal activity	• Sheep • Goat • Equine • +/− Cattle	[111–113]
Macrocyclic Lactones	Avermectins • Abamectin • Ivermectin • Eprinomectin • Doramectin • Selamectin Milibemycins • Moxidectin • Milbemycin • Nemadectin	Activation of glutamate-gated chloride channels, inhibiting neuromuscular transmission in nematodes.	Antinematodal Anti-arthropodal	• Sheep • Goats • Cattle • +/− Horses	[111–114]
Imidothiazoles-tetrahydropyrimidines	• Pyrantel • Levamisole	Interacts with nematode nicotinic acetylcholine receptors leading to paralysis.	Antinematodal +/− anticestodal activity	• Equines • Sheep • Goats • Cattle	[112,113]

While drug resistance has been most prevalently reported in nematodes [18], it is less common but possibly emerging in cestodes and trematodes. This difference may in part be due to the fact that while certain cestodes (tapeworms) and trematodes are important pathogens in both humans and in veterinary medicine, the most severe diseases occur with infections in extraintestinal locations (e.g., blood flukes Schistomsoma spp. (Digenea), larval tapeworms (Echinococcus spp., Taenia solium)). That said, schistosomasis, which is caused by a trematode, is one of the most important parasite infections in humans and has manifested resistance, particularly to praziquantel, which is the most widely administered treatment for the infection [18,23]. Moreover, while resistance in adult tapeworms that infect the intestinal lumen is not widely recognized, the dog tape worm Dipylidium caninum, which is zoonotic, appears to resist praziquantel as well [24]As a result of this growing drug resistance, the limited repertoire of anthelminthics begets concern about the future of IHP chemotherapy [17]. While responsible use can slow the spread of resistance, it is increasingly evident that we also need new therapeutics [25,26].

Cryptic IHP Etiology Drives Drug Administration Strategies that Yield Resistance

Our limited understanding of the etiology of IHPs has in part fueled the rise of anthelminthic drug resistance. For example, though research points to risk factors such as immune status and environmental mediators of exposure [27,28], we still struggle to explain why some individuals are especially sensitive to IHP infection [29]. Our inability to explain and ultimately predict infection outcomes has contributed to the overuse of anthelminthic drugs, which in turn accelerates the evolution of drug resistance. IHP infection burden is overdispersed in a population, meaning that some individuals are infected with multiple worms. In fact, 80% of all worms are found in less than 20% of infected individuals [30]. Myriad parasite and host taxa present this overdispersion [31–34], including animal lines bred to resist IHP infection [35]. This pattern even manifests in controlled conditions with animal models, such as zebrafish Danio rerio [36,37], wherein all individuals from the same population receive the same parasite exposure, and is irrespective of whether exposure occurs passively through the environment or actively through the diet.

In the past, in part because we cannot easily predict burden outcomes, livestock management often adopted shotgun antiparasitic treatment strategies [38], which disregard individual IHP burden or infection likelihood and involve administering drugs to all members of the population to prevent infection or suppress transmission. Unfortunately, these strategies yield the prolific use of anthelminthic drugs, which creates ample opportunity for IHPs to evolve resistance [39]. Contemporary treatment strategies often include maintaining a refugia of unexposed, and hence sensitive, worms [39,40]. To achieve this, only heavily infected animals are treated. This practice is now often employed among small ruminants, such as sheep and goats, and is increasingly employed among cattle and horses in developed countries in an attempt to slow the inevitable emergence of resistant alleles in parasite populations [41]. However, shotgun treatment strategies remain common in developing countries and it remains to be seen to what extent the contemporary strategies will slow the rise of anthelminthic drug resistance. By determining which factors modulate the success of infection upon exposure to a helminth, we may ultimately be able to develop targeted intervention strategies that either prevent infection from establishing or mitigate its overall burden on the individual. This, in turn, will promote selective treatment strategies that minimize the amount of a drug introduced into IHP populations.

The Gut Microbiome as a Potential Modulator of IHP Infection

Recent research suggests that the gut microbiome may be a cryptic promoter or detractor of IHP infection. The diverse community of microbes that occupy the gastrointestinal tract collectively express a variety of biological functions that influence the ecology of the gut that an IHP seeks to infect [42]. Indeed, the gut microbiome impacts the success of a variety of enteric microbial pathogens, such as through the production of antimicrobial compounds that directly attack bacterial pathogens [43–45], biotransformation of bile acids and other biochemical cues that induce the growth of pathobionts [45,46], and modulation of the host immune system to influence infection success [47–49]. These or related functions may also impact IHP infection outcomes.

Consistent with this expectation, the interindividual variation of the relative abundance of gut microbiota is often overdispersed [50,51], which mirrors the overdispersion observed in IHP infection burden. These linked patterns may result from processes, like host immunity, that similarly impact microbiota and IHP success in the gut. Alternatively, these analogous distributions could indicate that the assemblage of microbes carried in the gut influences the success of infection. Indeed, IHPs are exposed to gut microbes and their metabolites upon entering the gut, frequently before parasite eggs hatch or larvae interface with host tissue. Moreover, the gut microbiota heavily influences the chemical ecology of the gut [52,53]. Hence, these microbes and their metabolites may play an especially critical role during the sensitive period of IHP egg hatching and gut colonization in such that different functional assemblages of microbiota carried in the gut may yield gut ecosystems that are more tolerant of IHPs than others.

Recent investigations link the gut microbiome to IHP infection, burden, and even pathology. These linkages appear across disparate vertebrate species despite aspects of gut specialization that hold potential to impact the interaction between the microbiota and parasites (i.e. anatomical differences, diet, transit time, pH, water content, intestinal mucosal characteristics and immune surveillance). In humans, cross-sectional analyses of cohorts naturally exposed to IHPs identified strong associations between IHP infection and the composition of the fecal microbiome [54–57]. While metagenomic investigations are only beginning to emerge, Rosa et al. [56] found that microbiome gene family variation also links to infection. That said, Easton et al. [58] found that infection by the small intestinal IHPs Necator americanus or Ascaris lumbricoides do not appear to manifest a strong association with the fecal microbiome, possibly indicating that fecal communities are not necessarily always informative for understanding small intestinal microbiome-IHP dynamics.

Livestock and wildlife also manifest associations between gut microbes and infection status, with recent work showing that the composition of the fecal microbiome differs between horses that carry either low or high burdens of IHPs [59–61]. However, abomasal infections in some livestock show mixed associations with fecal microbiota across studies [62–64], again pointing to the potential importance of considering host species and location effects when studying microbiome-IHP interactions. While few studies have compared whether different IHP infections yield distinct associations with the gut microbiome, Kreisinger et al. [65] evaluated how the gut microbiome links to infection in wild mice (Apodemus flavicollis) naturally infected with multiple IHP species [65]. Their study showed that each helminth species could be associated with specific changes in the taxonomic composition and abundance of the gut microbiome.

Animal model studies of controlled exposure demonstrate similar linkages between gut microbes and IHP infection [66–68]. Most of these studies currently consider how exposure to a parasite impacts the microbiome; few assess whether the success of infection (e.g., infection burden) relates to gut microbiome composition. In a recent study using zebrafish, we found that the composition of the gut microbiome explains, in part ,the interindividual variation in the success of whipworm infection of the gut, as well as the variation in the pathological outcomes of infection [69]. While zebrafish do not carry the same microbes in their guts as mammals, the utilitarian features of this model system, including high levels of experimental control, rapid growth, and large sample sizes available with relatively minor costs, affords opportunities to inexpensively and rapidly resolve sensitive and otherwise obscure processes that can subsequently be validated in mammalian systems.

The cause and effect relationships that drive the associations between IHPs and gut microbes are beginning to be uncovered. Most of this research has identified myriad mechanisms through which IHPs can impact gut microbiota through alteration of the intestinal environment upon exposure and infection (which are excellently reviewed by Peachey et al.[70] and Leung et al.[42]). That said, a growing body of work points to how gut microbes can both enhance and interfere with IHP infection [71,72]. For example, in vitro investigations demonstrate that both cecum contents and specific microbial taxa influence the rate of egg hatching by the whipworm Trichurs murius [73]. Studies in mice also find that T. murius requires bacteria for its eggs to hatch in the gut, and that microbiome abundance associates with Heligmosomoides polygyrus bakeri burden [6]. Other studies find that the presence of the gut microbiome reduces infection, and that administering specific microbes reduces Trichinella spiralis and Strongyloides venezuelensis worm burdens [6]. Research in invertebrate hosts also point to microbiome mediated control of parasites. For example, a single gut bacterium protects bumble bees from IHPs [74]. Additionally, the presence of the maternally transmitted bacterium Spiroplasma protects Drosophilia neotestacea from the sterilizing effects of a parasitic nematode[75].

Some of the association between IHP infection and the gut microbiome could occur by way of the immune system, to which both IHPs and the gut microbiome are sensitive. Extensive research has demonstrated that the immune status of an individual impacts the success of an IHP infection [76,77] and that successful IHP infections can modulate the host’s immune state [3,78–80]. For example, IHP infection can alter immune function against co-infection by microbial pathogens [81]. Building on observations that gut microbes can also modulate and, in some instances, favorably calibrate host immune state [82–86], recent research has begun to explore the potential multidirectional interactions between immunity, the gut microbiome, and IHP infection. For more extensive discussions on this subject, we recommend recent reviews that have covered this research area at length, namely Reynolds et al. [66] and Leung et al. [42].

A New Frontier in Treating IHP Infection

This prior research suggests that studying the interaction between the gut microbiome and IHPs holds potential to transform our ability to combat IHP infection and the emergence of drug resistance (Figure 1). We in particular posit that gut microbes may serve as a useful source for the discovery of novel anthelminthic drugs, including compounds that induce improved host resistance to IHP infection. Microorganisms have served as a well-spring in our endeavors to discover bioactive natural products [87]. Efforts to harness the biochemical diversity of microorganisms have transformed our ability to manage various infections [88] , including IHP infections. In fact, phylogenetically diverse microbes (as well as plants) have been shown to produce anthelminthic compounds, several of which have yielded major IHP therapeutics [89]. For example, the widely used drug avermectin (Ivermectin) was derived from soil-borne Streptomyces avermitilis [90]. Aquatic sponge-associated Streptomyces produce similar anthelminthics [91]. A Pseudomonas fluorescens protease reduces egg hatching in the plant pathogenic nematode Meloidogyne incognita [92]. Members of the genus Bacillus secrete potent anthelminthics in the form of crystal proteins [93,94]. Fungi also produce a diverse array of benzenediol lactones, such as the curvularin macrolides and caryospomycins that elicit killing activities against plant parasitic nematodes [95]. Fungal cyclooctadepsipeptides elicit strong anthelminthic properties [96] and appear to elicit these properties against otherwise drug-resistant helminths [26].

Figure 1. Investigation of microbiome-parasite interactions holds potential to transform IHP infection prevention and treatment strategies.

Gut microbes may elicit probiotic effects that impact the success of infection, for example by affecting the environmental cues that induce egg hatching as well as by modulating host immunity to suppress IHP growth. Additionally, microbes may produce anthelminthic compounds that directly inhibit the growth of or kill IHPs, especially upon initial exposure of the worm in the gut. Microbial metabolites may also suppress the growth of other microorganisms in the gut that promote parasite infection. Relatedly, these microbes may influence a host’s sensitivity to IHPs with such effect that microbiomic information may be useful for predicting infection likelihoods and facilitating preventative infection strategies that ultimately decrease the frequency, duration, and dose of drug administration.

The gut microbiome may be especially ripe for discovering novel anthelminthic compounds given the putative evolutionary interplay between gut microbes and IHPs. Indeed, it seems that ecosystems that manifest frequent interactions between helminths and bacteria, such as soil and plant surfaces, tend to harbor microbes that produce anthelminthic compounds, presumably as a means for microbes to compete against worms [72,97,98]. The vertebrate gut also likely witnessed recurrent interactions between IHPs and gut microbiota over evolutionary timescales, given that IHP infections are frequently obligate, prevalent (both in nature and in the laboratory, such as zebrafish facilities), and influences gut ecology [72]. Consequently, gut bacteria may serve as a presently untapped reservoir of anthelminthic compounds.

Recent advances in metagenomics indicate that host-associated microbiota may offer a rich menu of novel bioactive compounds [99,100]. In particular, metagenomic analyses of the mammalian microbiome, including the gut microbiome, have revealed a tremendous suite of biosynthetic gene clusters (BGCs) that appear distinct from previously defined BGCs [101,102]. Using cultured isolates of microbiota that carry BGCs of interest, researchers have discovered and defined new classes of antibiotics, including lactocillin [101] and lugdunin [103]. Because most microbes are not yet in culture collections, recent efforts have begun to zero in on the function of specific gut microbiome encoded BGCs by coupling metagenomic analyses with synthetic biology and chemistry. For example, this approach discovered human gut microbes that produce dipeptide aldehydes, which mediate cathepsin inhibition and consequently, may impact immune recognition [104]. Recent efforts to expand access to available cultured isolates of the vertebrate microbiome [105,106] should empower future discovery of related types of novel bioactive compounds. Regardless, the diversity of natural products encoded in the gut microbiome offer an opportunity to discover new anthelminthic drug leads, if we are willing to look for them.

Future Directions

The gut microbiome intertwines with IHP infection and offers potential resources for combatting anthelminthic drug resistance. Future research investments should seek to define the specific mechanisms, especially at a biochemical level, through which gut microbes mediate infection and harness this knowledge to innovate new therapeutic strategies and resources. For example, the development of high-throughput screens that identify specific gut microbes or their metabolites that impact helminth survival, would expand the collection of available anthelminthic drug candidates. Such therapeutic candidates may include microbial metabolites that directly target IHPs, as well as metabolites that (1) modulate host physiology or immunology in ways that impact IHP infection, (2) selectively kill gut bacteria that promote IHP success in the gut, or (3) eliminate microbiota carried by the worm that may contribute to the IHP’s ability to thrive in the gut [107]. Along these lines, technologies that improve access to cultured isolates of gut microbes or the functional characterization of genes encoded in the gut metagenome would ultimately empower gut microbiome encoded drug discovery [105,106,108]. When coupled with studies that define how the temporal change in the microbiome links to the temporal dynamics of IHP infection, these technologies can provide researchers with methods that validate potential features of the microbiome that predict infection outcomes. We also need to know more about how the microbiota that associate with IHPs, including endosymbionts such as Wolbachia spp. in filarial worms, impact the emergence of drug resistance.

Relatedly, recent work creatively explores manipulation of the IHP microbiome by engineered bacteria, as a potentially targeted and specific drug delivery mechanism [109]. Moving the field of parasitology in the direction of investigating the value of microbiome-based treatment strategies will not only help to address global health issues [110], but with the emerging knowledge offered through studies of gut microbiome-parasite interactions, will potentially mitigate the current challenges posed by anthelminthic drug resistance.

Highlights.

• We need new tools to mitigate the harm posed by anthelminthic drug resistance.

• Evidence links the gut microbiome to helminthic infection and its outcomes.

• The gut microbiome may mediate infection and define infection sensitivity.

• Future research should determine if the gut microbiome encodes novel therapeutics or whether it can guide infection prevention strategies.