Antibiotic resistance genes (ARGs) occur naturally and are evolutionarily ancient (Aminov and Mackie, 2007; D’Costa et al., 2011), but the pervasive use of antibiotics has accelerated their global propagation and precipitated a resistance crisis (Ventola, 2015; Van Puyvelde et al., 2018). The ARGs within a microbial community, collectively known as the resistome, influence the function of native bacteria (Kim et al., 2020), increase pathogen morbidity and mortality rates (Howard et al., 2003; Lin et al., 2015), and diminish the efficacy of antibiotics (Rossolini et al., 2014; Martens and Demain, 2017). ARGs are thus well-studied in clinical populations or agricultural systems that receive antibiotic treatment (French, 2005; Ghosh and LaPara, 2007; Lerminiaux and Cameron, 2019). Although ARGs are also components of anthropogenic disturbance, such that contact with infected hosts or contaminated environments facilitates the transfer and incorporation of ARGs into natural microbial consortia (Hiltunen et al., 2017; Manaia, 2017), the presence and abundance of ARGs in wildlife and non-model animals remains relatively understudied (Dolejska and Literak, 2019; Ramey and Ahlstrom, 2020). Here, we use a site-comparative approach within a single host species, the endangered ring-tailed lemur (Lemur catta), to (a) characterize ARGs in the gut microbiota of multiple wild and captive populations, (b) determine if identified ARGs correlate with components of anthropogenic disturbance faced by these populations, and (c) test for covariation between ARGs in wild lemurs and their natural environments, as proxied by ARGs in soil microbes.

Antibiotic resistance genes evolved as defense mechanisms, so natural ARGs are diverse, present in low abundances, and persist in microbial communities with no known human contamination (Segawa et al., 2013; Jardine et al., 2019). Their propensity to be exchanged via horizontal gene transfer has allowed ARGs to pass between and persist within host-associated and environmental microbial communities (Baquero et al., 2009; Forsberg et al., 2012; Martínez, 2012). The associated arms race between the evolution of protective ARGs and antibiotic production has resulted in myriad, naturally occurring resistance mechanisms (Sengupta et al., 2013; Versluis et al., 2015; Van Goethem et al., 2018). The increased selective pressure from human antibiotic use, coupled with widespread human encroachment into natural environments, is thus expanding ARG reservoirs in wildlife and their environments (Gatica and Cytryn, 2013; Berglund, 2015; Anthony et al., 2018).

The most notable and well-studied route of ARG enrichment within a microbial community stems from treatment with man-made antibiotics (Alanis, 2005). This direct route of enrichment has greatly contributed to the spread of clinically relevant ARGs – those that confer resistance to multiple drug types or to last-resort antibiotics (Koch et al., 2014; Mühlberg et al., 2020). Once resistant microbes are present in a microbial community, they also can indirectly enrich the community resistome by persisting as community members and/or by transferring ARGs to native microbes (Chee-Sanford et al., 2009; Gao et al., 2018). Resistant microbes can be shared between humans and companion animals (Pomba et al., 2017), acquired through consumption of contaminated dietary items (Wang et al., 2006; Nawaz et al., 2011), or acquired via contact with human or agricultural waste that includes antibiotic residues or ARGs (Addison, 1984), the latter prompting investigation of soil microbiomes (Esiobu et al., 2002; D’Costa et al., 2007).

Antibiotic resistance genes are thus a newly recognized component of the Anthropocene, expanding the traditional definition of human disturbance (e.g., habitat degradation) to include perturbations of microbial communities (Tripathi and Cytryn, 2017; Zhu et al., 2017; Trevelline et al., 2019) that can influence host-associated microbiota, transfer ARGs to pathogens, and alter the host’s immune function (Bengtsson-Palme et al., 2018; Dafale et al., 2020). ARG spillover into wild animals also has the potential to subsequently re-infect exposed humans or other animals (Pomba et al., 2017). The risk of ARG enrichment in wildlife is thus linked to the potential for both direct (e.g., antibiotic administration) and indirect (e.g., human and agriculture presence) routes of transmission. As a first step to determining the respective roles of these enrichment routes, we will characterize ARGs in systems that portray varying types and severities of anthropogenic disturbance.

Madagascar is a prime study site. Its ongoing population and agricultural boom has increased the demand for antibiotics to treat human and agricultural diseases, thereby enriching ARGs in human and domestic animal populations (Gay et al., 2017; Padget et al., 2017; Randrianirina et al., 2010). Furthermore, antibiotics are broadly used to treat Madagascar’s semi-regular plague outbreaks (caused by the bacterium Yersinia pestis) (Boisier et al., 2002; Andrianaivoarimanana et al., 2013; Salam et al., 2020), which has fueled the demand for antibiotics (Rasamiravaka, 2020). Coupled with a lack of infrastructure to contain human and agricultural waste, ubiquitous antibiotic use in Madagascar poses a significant risk of widespread ARG propagation to its unique and endangered wildlife.

Unlike many ecologically specialized lemurs endemic to Madagascar, the ecologically flexible ring-tailed lemur is a semi-terrestrial omnivore that performs geophagy or earth-eating (Ganzhorn, 1987; Gould, 2006); it survives and reproduces successfully in greatly disturbed habitats and in a wide range of captivity settings (Jolly et al., 2006; Mason, 2010). Across their natural range, ring-tailed lemurs live along a gradient of disturbance from near-pristine forests, that have minimal human activity, to degraded habitats with heavy human encroachment, including logging, agricultural land use, and hunting. Similarly, captive ring-tailed lemurs live under conditions that range from naturalized settings (e.g., social housing in natural habitat enclosures) to artificial settings, some even living solitarily, as pets, in human dwellings, where they experience consistent human contact and substandard conditions (Reuter et al., 2015; Reuter and Schaefer, 2016; LaFleur et al., 2019).

Regarding direct routes of ARG enrichment, lemurs in natural and captivity settings have differential exposure to antibiotic treatment. Whereas most wild lemurs and lemurs kept as illegal pets in Madagascar will have never experienced antibiotic treatment (the latter owing to lack of veterinary care), populations at well-established field sites may have, on occasion, experienced antibiotic treatment during capture sessions or as part of a research protocol. At the other extreme, lemurs at research or rescue facilities will have experienced routine veterinary care, including potentially frequent antibiotic treatment. Regarding indirect routes of ARG enrichment, wild and captive lemurs differ in their exposure to humans and domestic animals, in their potential for social transmission between conspecifics, and in their potential to acquire ARGs from their environment.

We first characterize ARGs in the gut microbiota of wild and captive ring-tailed lemurs, by population. We predict that the abundance and diversity of ARGs across populations will positively correlate with the degree of overall anthropogenic disturbance, and that any clinically relevant ARGs in wild lemurs will reflect antibiotic use in human and agricultural settings in Madagascar. For captive lemurs, we expect overall ARG abundance to be greater than in wild conspecifics; however, the diversity and type of ARGs could reflect different routes of enrichment. In captive lemurs at facilities that provide naturalized settings and routine veterinary care, we expect ARGs to reflect treatment with common antibiotics. By contrast, in pet lemurs that are unlikely to receive antibiotic treatment, but have consistent and sustained direct contact with humans and domestic animals, we expect to observe ARGs that confer resistance to antibiotics commonly used in Madagascar (such as those used to treat the plague).

Similar to the patterns predicted for lemur ARGs, we expect ARGs in soil microbiota to also correlate with the degree of environmental disturbance and to also reflect the types of antibiotics used in human and agricultural settings in Madagascar. As regards ARG covariation between wild lemur resistomes and soil resistomes, we expect soil from the more disturbed habitats to have greater abundance and diversity of ARGs than would soil from the more pristine habitats. Consistent with evidence that hosts acquire microbes from their environments (Smith et al., 2015; Adair and Douglas, 2017; Selway et al., 2020), we expect wild lemur resistomes to be more similar to soil resistomes from their local habitat than to soil resistomes from outside their home ranges.

By assessing ARGs in multiple ring-tailed lemur populations living under variably disturbed conditions, we (a) shed light on the breadth of ARG presence and diversity outside of traditionally studied settings, (b) highlight the potentially different routes by which ARGs may be acquired by wild versus captive animals, and (c) demonstrate covariance between lemur and soil resistomes in a subset of wild populations. Consistent with previous findings on natural resistomes in wildlife microbiota (Vittecoq et al., 2016; Kipkorir et al., 2019; Marcelino et al., 2019) and environmental microbiota (Esiobu et al., 2002), ARGs were present in nearly all lemur and soil samples collected, including from animals that had potentially minimal exposure to introduced antibiotics (Pallecchi et al., 2008; McCann et al., 2019). As one of the broadest examinations of ARGs in an endangered species and its habitat, our study adds to the growing recognition that wildlife-associated and environmental consortia may act as inextricable reservoirs of ARGs, exemplifying both the ecological and conservation concerns associated with the resistance crisis.

At the lowest end of the anthropogenic disturbance gradient, relatively undisturbed, wild lemur populations (e.g., at IVO and AMO) nonetheless harbored diverse, low-abundance ARGs that likely reflect natural resistomes. Even in these low-risk populations, the composition of lemur resistomes was site-specific, indicating that naturally occurring ARGs are neither randomly present across individuals nor are they homogenized across populations. Thus, under conditions of minimal anthropogenic impact, selective pressures other than human influence may shape animal resistomes.

Although not directly addressed in the present study, one such pressure may involve host sociality. Notably, the site-specificity we observed in lemur resistomes mirrors the site- or group-specificity observed for microbiome composition in highly social species (Theis et al., 2012; Leclaire et al., 2014; Tung et al., 2015; Moeller et al., 2016), including the ring-tailed lemur (Bornbusch et al., 2021). The widely accepted mechanism underlying these patterns is the sharing of microbes between hosts, from many possible bodily sources, particularly between conspecific social partners (reviewed in Archie and Tung, 2015). If social vectors for microbial transmission have a homogenizing effect on the microbiomes of group members, the same pattern could be expected for specific microbial genes, such as ARGs. Our observation of site-specific resistomes in group-living lemurs is thus consistent with the potentially homogenizing impact of ARG social transmission.

Likewise, another pressure may involve components of the host’s diet. Omnivory and geophagy in the ring-tailed lemur (Canington, 2021) promotes ingestion of naturally occurring biomolecules and chemical elements, such as plant secondary compounds (e.g., tannins) and heavy metals, that can modify or co-occur with bacterial resistance mechanisms (Hatano et al., 2005; Compean and Ynalvez, 2014; Pal et al., 2015). The presence and abundance of these compounds could vary with food availability and dietary preferences across the wide range of habitats occupied by wild ring-tailed lemurs. The social transmission of microbes and the diets available within microhabitats are plausible mechanisms for shaping natural resistomes in wild populations and warrant further research to better characterize the potential for even ‘unperturbed’ wildlife to serve as reservoirs of ARGs.

Unexpectedly, we observed ‘outliers’ in most of our wild lemur populations, including those in the most undisturbed habitats, according to our criteria. These outliers appeared to be more ARG-enriched than were their site-mates. To the extent that they represent ‘sentinel’ animals – a concept widely used in epidemiology to describe individuals or species that manifest a specific risk and thus provide advance warning (Van der Schalie et al., 1999) – their resistomes may reflect increased potential of ARG exposure at given sites (Blanco and Bautista, 2020). Sentinel individuals or species may be differentially exposed to ARGs via their different behavioral patterns or biogeographical ranges (Rabinowitz et al., 2005; Vittecoq et al., 2016). Given the probability of host–host transmission of ARGs, sentinels may act as propagators of ARG within their population or community (Sacristán et al., 2020; Plaza-Rodríguez et al., 2021). Alternatively, whereas healthy hosts may be able to combat colonization by resistant bacteria, sentinels may have been those most susceptible to infection (Aguirre, 2009; Taur and Pamer, 2013). In this case, ARG enrichment might reflect vulnerability. Although we could not pinpoint the cause of this variation in our study populations, both scenarios are plausible. Longitudinal data on ARG prevalence in wildlife would help disentangle these two possibilities.

Although degree of anthropogenic disturbance has been suggested as a driver of ARG enrichment in other wildlife species (Tripathi and Cytryn, 2017; Sacristán et al., 2020), most ARG studies have been limited to single or few host populations. Here, across seven populations of wild ring-tailed lemurs, we show that ARG abundance in host gut microbiota positively correlated with our assessments of four anthropogenic components. Compared to subjects from other wilderness sites, those from BEZ and BER (i.e., the sites with the greatest disturbance rankings) had significantly greater abundances of overall and clinically relevant ARGs. Notably, because both of these sites have well-established, long-term research stations (Sauther et al., 1999; Jolly et al., 2006; Jolly, 2012), certain groups had been habituated, captured, and handled for research purposes, and, at BEZ, on rare occasions in the recent past, treated with antibiotics (personal communication with M. Sauther). Because routine captures ceased almost a decade ago, it is improbable that the lemurs sampled for this study received direct antibiotic treatment. Nonetheless, given the persistence and propagation of ARGs, earlier research practices may have left a signal in the lemur resistomes that could have been perpetuated, albeit dampened, over time. Lemurs at TSI have also been handled for research purposes, yet they showed no signal of ARG enrichment. Whereas researcher presence and animal handling at TSI began relatively recently (i.e., in the 10 years before sampling), researcher activity has been occurring for ∼47 and ∼60 years, respectively, at BEZ and BER. Perhaps our findings suggest that, when human contact is infrequent and sporadic, long-term exposure may be required for ARGs to accumulate and disseminate within wildlife populations.

Compared to the ARGs found in wild lemurs, those in captive lemurs were greatly enriched, in some cases by multiple orders of magnitude, likely owing to direct antibiotic treatment received by captive animals at the LRC and DLC. Moreover, the types of ARGs present in these two populations reflected the differences in antibiotic availability and use between the two countries. The resistomes of LRC lemurs, for example, included phenicol ARGs, which most commonly confer resistance to chloramphenicol, an antibiotic that is rarely used in developed countries (Wareham and Wilson, 2002; Fernández et al., 2012), but available in Madagascar for treating the plague (Chanteau et al., 2000; Godfred-Cato et al., 2020). In addition, LRC lemurs harbored notable proportions of ARGs conferring resistance to vancomycin, which, until recently, was considered a last-resort antibiotic for severe bacterial infections, including Staphylococcus aureus (i.e., MRSA: Gardete and Tomasz, 2014; Koch et al., 2014). A recent increase in vancomycin use in Madagascar has led to increased vancomycin resistance and decreased efficacy (Dhanda et al., 2018), including in isolates from humans and other animals (Gay et al., 2017).

By contrast, for DLC lemurs, tetracycline, aminoglycoside, and beta-lactam ARGs were the first, second, and third most prevalent, respectively, potentially reflecting the common use of all three antibiotic families in United States veterinary care (Schwarz and Chaslus-Dancla, 2001; Sarmah et al., 2006; personal communication with veterinarians at the DLC). Nonetheless, not all lemurs in these captivity settings had received equivalent antibiotic treatment. Moreover, DLC lemurs without any history of antibiotic treatment still harbored substantial ARGs that rivaled those seen in conspecifics with numerous previous antibiotics treatments (Bornbusch et al., 2020). In combination, these results indicate that antibiotic treatment produced predictable patterns in ARG enrichment, but those patterns are also present in untreated lemurs. The presence of specific types of ARG enrichment in treatment-naïve individuals perhaps again suggests an independent mechanism of social or environmental transmission.

Another line of evidence in favor of social or environmental transmission is the finding that, despite a lack of antibiotic treatment, pet lemurs harbored some of the greatest abundances of ARGs. Similar to the resistomes of wild and LRC lemurs, those of pet lemurs included ARGs that reflected human use of antibiotics in Madagascar, but at significantly higher abundances than that shown by their wild conspecifics. In a recent report, researchers indicated that ARGs associated with virtually every class of antibiotics have increased in Madagascar, both in humans and in domestic animals (Gay et al., 2017). Whereas for wild lemurs, intraspecific social contact likely facilitates the transmission of ARGs, for pet lemurs isolated from conspecifics, interspecific social contact with humans and domestic animals likely serves as a vector for ARG transmission. Pet lemurs live under unsuitable conditions (Reuter et al., 2015; Reuter and Schaefer, 2016; LaFleur et al., 2021); their poor health may present a particular vulnerability to ARG enrichment. Moreover, because the transmission of ARGs is not host-specific or even route (e.g., fecal-oral) specific, ARG enrichment in pet lemurs poses a significant risk of transmission to the humans who come into contact with these lemurs.

With growing recognition that animal hosts represent only one component of microbial landscapes, researchers are increasingly probing the relationships between host-associated and environmental microbiota. In a previous study on these same ring-tailed lemur populations, we showed that exposure to and acquisition of soil microbes likely contributed to interpopulation variation in gut microbiota (Bornbusch et al., 2021). Here, we expand on this finding, showing that the same pattern holds for ARGs: significant covariation between lemur gut and soil resistomes was found in two disturbed sites, ISA and BEZ. Although based on a directional, source-sink analysis, this covariation is likely to be bidirectional, such that lemurs can acquire ARGs from their environment and also shed them into the environment. Although ISA lemurs had relatively low-abundance resistomes and neither human contact nor antibiotic exposure, the significant covariation between lemur and soil ARGs indicates that the presence of non-wildlife animals and humans might contribute to ARG reservoirs both in lemurs and their environments.

Although antibiotic stewardship has rightly become a focus of western medicine, our study highlights that the resistance crisis extends outside of traditional clinical settings; antibiotic treatment should be considered as only one of many possible mechanisms that facilitate ARG enrichment. Indeed, unlike the curbing of antibiotic use, the exploitation and anthropogenic disturbance of natural ecosystems is accelerating. We show that an endangered primate harbors diverse and, in some cases, anthropogenically enriched resistomes. Our results reinforce the recent call for greater preservation of natural microbial communities (Trevelline et al., 2019; Banerjee et al., 2020) and highlight the need to incorporate antibiotic resistance into in vivo and ex vivo conservation strategies, particularly those that could facilitate the propagation of ARGs (e.g., animal reintroductions, translocations, and transfers). As a premier One Health issue, with relevance to the health of humans, other animals, and the environment, antibiotic resistance demands greater study in a wider range of systems, specifically in the context of wildlife and environmental reservoirs of ARGs.

The data presented in the study are deposited in the Open Science Framework repository, link: https://osf.io/vkr2f/, doi: 10.17605/OSF.IO/VKR2F. The full metagenomic library is available upon reasonable request.

The animal study was reviewed and approved by Duke University Institutional Animal Care and Use Committee. Written informed consent for participation was not obtained from the pet owners because verbal consent was provided for collection of feces from pet lemurs.

SB and CD conceived of this study. SB collected and analyzed samples, and drafted the original manuscript. Both authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.