Wildlife living near human settlements may be disproportionately exposed to environmental AMR. Pteropus medius, a large colonial fruit-eating bat species in South Asia, commonly roosts and forages in human-dominated landscapes, exposing it to anthropogenically shaped resistomes across a wide geographic area. Moreover, seasonal variations in environmental conditions and food resources may influence bat movements and their exposure to AMR. We investigated the prevalence of AMR in P. medius and explored AMR risk at seven different roosting sites in Pakistan, while considering variations in seasons and Land Use and Land Cover (LULC) types as predictors for AMR exposure. We tested fecal samples for resistance to twelve antibiotics and sought blaTEM, blaSHV and blaCTX-M resistance genes using PCR. Moderate to high resistance prevalence was identified in five out of twelve tested antibiotics, with resistance to aztreonam, cefradine and gentamicin exhibiting a significant seasonal effect. Similarly, tested antibiotics and genes were positively influenced by land use at 14.5 km buffer around the roosting sites. Approx. 37% of E. coli isolates were ESBL producers in both seasons and carried blaTEM genes (> 90%). Strong correlations were found in AMR patterns between P. medius and livestock (cattle and poultry). However, when resistance prevalence data were organized by season and province, the strength of these correlations varied considerably, highlighting the need for further research. The high level of AMR in P. medius, together with the positive influence of land use, highlights the need for an integrated One Health approach to combat AMR.

Antimicrobial resistance (AMR) is a critical and unprecedented 21st-century health challenge, globally contributing to 5 million human deaths annually either directly or indirectly, with projections suggesting this could reach over 10 million per year by 2050. Growing evidence suggests that the environment plays an underappreciated but critical role in the development, spread, and transmission of AMR, facilitating the transfer of resistant pathogens between humans, animals, and the environment. The primary pathways for environmental AMR include pharmaceutical and hospital wastewater, which, when combined with agricultural and livestock waste, contribute to the contamination of freshwater sources. Untreated wastewater loaded with antimicrobial residues and resistant bacteria from these sources creates a selection pressure for drug-resistant organisms to survive in the environment, shaping what is referred to as the environmental resistome. This selection pressure not only favors the survival of clinically relevant resistant pathogens but also enhances the exchange of resistance genes among commensal environmental bacteria such as E. coli, which can act as silent reservoirs and facilitators of resistance dissemination across diverse environmental microbial communities.

The WHO report on AMR surveillance highlights global geographical gaps in AMR surveillance and acknowledges limited research on AMR in wildlife. The AMR Quadripartite Joint Secretariat that includes four global organizations (FAO, UNEP, WHO and WOAH) is tasked with global AMR control response, including environmental and wildlife AMR, under a One Health framework. Wildlife act as a sentinel of environmental AMR and resistome by providing insights into how both are distributed and transmitted across the landscape, as indicated by very low AMR prevalence in wildlife in areas that are distant from human activity. In contrast, wildlife species, including some bats (order Chiroptera) that are adapted to human-modified habitats, showed elevated levels of AMR. Research has also shown that bats carry extended-spectrum β-lactamase (ESBL) producer bacteria and resistance genes, suggesting that bats could not only be a key indicator but also a propagator of AMR across ecosystems.

Despite the integral role that some wildlife species play in human-modified landscapes, there is limited understanding of how they shape or influence the environmental resistome. Specifically, few studies have incorporated spatial land use variables into wildlife AMR research, yet these are likely to be key predictors of AMR dynamics. The spatial distribution of land uses, such as agriculture and urbanization, can create environments that promote the development and spread of resistant microbes. Wildlife, acting as reservoirs and vectors, may interact with these modified landscapes in ways that influence the persistence and transmission of AMR. Furthermore, the movement of wildlife across diverse land use types can facilitate resistance gene flow between ecosystems, potentially exacerbating the spread of resistant strains.

Pteropus medius (Indian flying fox) is a large frugivorous bat with a range that spans from Pakistan in the west to Myanmar in the east and Bhutan in the north to the Maldives in the south. Large populations reside in India, Sri Lanka, Nepal, and Bangladesh and, in many cases, migrate seasonally within and among countries. These flying foxes form colonies of hundreds to thousands of individuals, roosting in large, exposed trees that are frequently near human settlements. As a result, these bats commonly engage in synanthropic behaviors such as consuming leftover fruits and thermoregulatory behaviors that rely on soaking their belly and drinking from water resources that are designated for domestic animals. Together these behaviors increase their vulnerability to environmental AMR. At the same time, colonies generate a rain of ejecta, feces and urine that accumulates under the roost - accessible to any roaming livestock and wildlife, and possibly to people, creating a propagation pathway for AMR in the environment. Furthermore, spatiotemporal variation in the population density of P. medius can potentially provide an influx of antimicrobial resistance, contributing toward environmental AMR propagation.

P. medius forage over mixed landscapes, promoting plant gene flow and forest regeneration in fragmented habitats. However, these daily movements also expose bats to diverse sources of environmental AMR associated with human-modified landscapes, such as agricultural run-off and contaminated water sources. Long distance seasonal migration further exposes bats to environmental AMR sources but critically facilitates the dissemination of AMR and resistant genes over a large geographic scale, with serious One Health implications. Therefore, we consider P. medius as an effective sentinel mammalian species to assess environmental AMR due to its extensive synanthropic behavior, large colony size and regional significance in Pakistan.

The emergence and dissemination of drug-resistant microbes are steadily increasing and are aggravated by climate change. Studies from around the globe indicate that an increase in average minimum temperatures is linked to higher rates of antibiotic resistance. Extreme temperatures could contribute to antibiotic resistance indirectly by changing the behaviors of people, livestock, and wildlife and the interactions among them and the environmentincreasing exposure and vulnerability to the environmental resistome. For instance, P. medius roosts colonially in exposed large trees, and mitigates heat stress through a series of cooling thermoregulatory behaviors that intensify as temperatures increase. Behaviors used at the highest temperatures rely on evaporative cooling (wrist licking, panting, opportunistic belly soaking in nearby water bodies). Drinking to restore water balance or belly soaking increases bats' exposure to water resources that are often contaminated with antimicrobial residues and resistant bacteria (Supplementary Fig. 1).

This cross-sectional study aimed to determine the seasonal prevalence of AMR in E. coli, ESBL producing E. coli and its commonly associated blaCTX-M, blaSHV and blaTEM resistance genes in P. medius. We tested for resistance against twelve antibiotic classes that are widely used to treat human and livestock infections and are expected to have a higher prevalence in the environment. To ascertain the severity of AMR in P. medius, we used the WHO Medically Important Antibiotics (MIA) 2024 classification to classify the twelve antibiotics studied into six different classes. The MIA classification is an AMR risk management tool by minimizing the impact of antimicrobial use coming from non-human sources to humans.

We expected to see higher seasonal AMR prevalence in summer than in winter due to the higher flow of water in the rivers and streams, which can spread AMR to a wider geographic area. The water flow in summer is governed by seasonal variation in rainfall and the melting of glaciers and ice caps in northern regions of Pakistan. More importantly, demands for hydration to offset evaporative cooling losses associated with thermoregulation, as well as belly soaking, increase in summer, which may increase exposure of P. medius to the environmental resistome. Owing to general synanthropic behavior, we predict a higher prevalence of AMR in P. medius in human-modified landscapes, particularly those with a higher proportion of human settlement area. In contrast, bats in roosts surrounded by greater natural vegetation and forests are expected to have lower AMR prevalence. Relying on publicly available data on AMR in poultry and cattle that is spatiotemporally consistent with our P. medius AMR data, we performed spatiotemporal correlation of resistance profiles of P. medius with those of cattle and poultry, to ascertain the major source of environmental AMR in P. medius. These comparisons of AMR across seven roosts, two seasons, and key sources of environmental AMR provide valuable insights into the environmental dynamics of AMR pollution affecting P. medius.