Espunyes et al. BMC Veterinary Research (2021) 17:79 https://doi.org/10.1186/s12917-021-02784-2 RESEARCH ARTICLE Open Access Assessing the role of livestock and sympatric wild ruminants in spreading antimicrobial resistant Campylobacter and Salmonella in alpine ecosystems Johan Espunyes1,2* , Oscar Cabezón1,3, Andrea Dias-Alves1, Pol Miralles3, Teresa Ayats4 and Marta Cerdà-Cuéllar4 Abstract Background: Livestock play an important role as reservoir of enteric pathogens and antimicrobial resistance (AMR), a health and economic concern worldwide. However, little is known regarding the transmission and maintenance of these pathogens at the wildlife-livestock interface. In this study, we assessed the occurrence, genetic diversity and AMR of Campylobacter spp. and Salmonella spp. shed by sympatric free-ranging livestock and a wild herbivore in an alpine ecosystem. Results: Campylobacter spp. was isolated from 23.3 % of cattle and 7.7 % of sheep but was not isolated from horses nor Pyrenean chamois (Rupicapra pyrenaica). Campylobacter jejuni was the most frequent species. A high genetic diversity and certain host specificity of C. jejuni isolates was observed. The main AMR detected in Campylobacter isolates was to nalidixic acid (88.2 %), ciprofloxacin (82.4 %) and tetracycline (82.4 %); only 11.7 % of the isolates were pan-susceptible and 17.6 % were multi-resistant. Salmonella ser. Newport was isolated only from one Pyrenean chamois and was pan-susceptible. Conclusions: Results show that free-ranging cattle and sheep are spreaders of Campylobacter as well as their AMR strains in the alpine environment. Therefore, contaminated alpine pastures or streams may constitute a source for the dissemination of AMR enteropathogens. However, apparently, alpine wild ungulates such as Pyrenean chamois play a negligible role in the epidemiology of zoonotic enteropathogens and AMR, and are not potential bioindicators of the burden of alpine environments. Keywords: Campylobacter, Salmonella, Antimicrobial resistance, Free-ranging livestock, Pyrenean chamois * Correspondence: johan.espunyes@uab.cat 1 Wildlife Conservation Medicine Research Group (WildCoM), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain 2 Research and Conservation Department, Zoo de Barcelona, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 2 of 8 Background However, they may acquire resistant bacteria from con- Gastrointestinal foodborne infections are challenging is- taminated environments, becoming new host reservoirs sues for humanity. They cause an estimated number of [21]. Despite the knowledge regarding the potential 600 million illnesses and 420,000 deaths worldwide every spill-over from free-ranging livestock to wildlife is still year [1]. Campylobacter spp. and Salmonella spp. are limited [21], some authors have detected a significant the leading causes of these diarrhoeal diseases [1] and gradient of antibiotic resistance from wildlife less im- are also the two most reported zoonosis in the European pacted by livestock production to wildlife in close con- Union (94 % of all the reported cases of zoonotic diseases tact with farm animals [22]. In fact, wildlife has been in 2018; [2]). Humans get infected by these enteric bac- suggested as bioindicators or sentinels of the AMR bur- teria mostly through handling and consumption of con- den present in the environment, even if the specific taminated foods but also through direct contact with transmission routes still have to be elucidated [23, 24]. carrier animals and contaminated environments [3, 4]. When considering less impacted habitats and remote re- Livestock act as carriers of these enteric pathogens gions of the world, the origin of the burden of resistance and they play an important role in their dissemination, within the local environment can be narrowed down to particularly in farm environments [5, 6]. However, there limited inputs. In high-altitude alpine environments, is a lack of knowledge regarding the contribution of where human waste disposal, wastewater runoff and free-ranging livestock in the epidemiology of zoonotic sewage contamination can be considered absent, the bacterial enteropathogens in natural environments. Live- only apparent important source of AMR is the presence of stock is usually considered to be the primary source of free-ranging livestock during the summer months. In freshwater bacterial contamination but their effect on mountainous areas of Europe, traditional farming prac- the bacterial quality of soils is rarely assessed [7]. The tices are based on livestock freely grazing on alpine grass- role of wildlife in the ecology of Campylobacter and Sal- lands from after the snowmelt until the decay of grass monella is also poorly understood, mostly due to the un- quality in autumn, usually from May to October. This known carrier status of many wild species. Several practice is frequently applied to reduce the economic costs studies have identified wild animals as potential environ- involved in livestock maintenance. As such, it is easier to mental reservoirs of food borne pathogens [8, 9]. For ex- assess the contribution of livestock to the prevalence of ample, wild birds such as seagulls and waterfowl have AMR pathogens in wildlife, as numerous other anthropo- been shown to have a role in the dispersion of these genic inputs are absent. diseases [10, 11]. However, the wildlife compartment In this study, we assessed the shedding of Campylo- is also considered a “net sink” of enteric pathogens bacter spp. and Salmonella spp. and their AMR strains due to a contamination from domestic species or hu- by sympatric free-ranging livestock (cattle, sheep and man waste [12, 13]. Nevertheless, little is known re- horses) and a wild herbivore (Pyrenean chamois; Rupica- garding the transmission of those pathogens at the pra pyrenaica) in an alpine ecosystem of the Eastern wildlife-livestock interface. Transmission may be bi- Pyrenees. We further explored the transfer of these bac- directional [14], but at the same time some wild spe- teria between species by investigating the genetic rela- cies are independent from this transmission cycle due tionship among isolates. to ecological and behavioural differentiations [15]. A more recent public health issue is related with the Results emergence and spread of antimicrobial-resistant patho- We isolated Campylobacter spp. from 17 cattle (23.3 %; gens in natural environments. This phenomenon has ser- CI95 %: 15.1–34.1) and 3 sheep (7.7 %; CI95 %: 2.6–20.3), ious implications for human as well as animal health and whilst Salmonella was isolated from only one Pyrenean it is considered one of the biggest threats to modern medi- chamois (1.2 %; CI95 %: 0.2–6.3) (Table 1). None of the cine [16]. Antimicrobial resistance (AMR) can be dis- two pathogens were isolated from horses. At the herd persed in the environment through several mechanisms, level, 11 cattle herds (57.8 %) and 2 sheep flocks (66.6 %) including water contaminated by sewage containing anti- presented at least one positive individual. microbials and resistant bacteria, run-off from fertilized land, or through the faeces of treated livestock [17]. In In cattle, Campylobacter jejuni was the most fre- fact, the occurrence of AMR in livestock is considered ex- quently isolated species (16/17), followed by Campylo- tremely high, especially for tetracyclines, quinolones and bacter coli (2/17; Table 1). One animal was coinfected sulphonamides [18]. The agricultural use of antibiotics with both species. In sheep, C. jejuni was the only spe- can have a dramatic impact on human health and is one cies isolated (3/3). We isolated Salmonella from only of the major driver of AMR worldwide [19, 20]. one Pyrenean chamois and all Salmonella isolates from On the contrary, wildlife is not usually exposed to an- this positive individual showed the same ERIC-PCR pro- timicrobials used in veterinary or human medicine. file and were identified as Salmonella ser. Newport. Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 3 of 8 Table 1 Frequency of Campylobacter species and Salmonella same host species clustered together at different simi- serovars in faecal samples from livestock and Pyrenean chamois larity levels, except a C. jejuni isolate from cattle in the Freser-Setcases National Game Reserve, Eastern Pyrenees, showing > 90 % similarity with a sheep isolate. Besides, Spain in most cases, closely related isolates originated from Cattle Sheep Horse Chamois individuals from the same herd. Campylobacter spp. C. jejuni 16/74 3/39 0/30 0/86 We tested eighteen isolates from the 13 different flaA- C. coli 2/74 0/39 0/30 0/86 RFLP profiles (Fig. 1) for antimicrobial susceptibility to a panel of six antimicrobials. One of them showed no Total 17/74 3/39 0/30 0/86 growth in any of the wells (including the control wells) Salmonella spp. and thus no data could be obtained. MIC test was re- S. ser. Newport 0/74 0/39 0/30 1/86 peated three times with this strain, obtaining the same Total 0/74 0/39 0/30 1/86 results. In overall, the main AMR detected was to nali- dixic acid (88.2 %, 15/17), ciprofloxacin (82.4 %, 14/17) To assess the genetic relationship among Campylobac- and tetracycline (82.4 %, 14/17), consequently the most ter isolates, 32 C. jejuni isolates (from twelve cattle and frequent AMR profile was NalCipTet (Table 2). Two iso- three sheep) and one C. coli isolate (from one cattle) lates, both from cattle, were pan-susceptible and three were genotyped by flaA-RFLP (restriction fragment isolates, all from cattle, were multidrug resistant (MDR). length polymorphism of the flaA gene). The remaining The Salmonella ser. Newport tested was pansusceptible. isolates could not be analysed since they were non- typeable and no band patterns were obtained. A high genetic diversity was observed, since almost each Campylobacter-positive individual carried a single Discussion and unique genotype. Overall, the flaA-RFLP analysis re- The role of livestock and a sympatric wild ruminant in vealed 13 different profiles (10 from cattle, 2 from sheep, the epidemiology of two relevant zoonotic agents—Cam- and one shared between both host species; Fig. 1). A cer- pylobacter and Salmonella—in an alpine ecosystem has tain host specificity was observed, as isolates from the been assessed. It has been found a differential prevalence Fig. 1 Dendrogram of flaA-RFLP banding patterns of Campylobacter spp. isolates originating from faecal samples from cattle and sheep in the Freser-Setcases National Game Reserve, Eastern Pyrenees, Spain. One different profile per animal is shown Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 4 of 8 Table 2 Antimicrobial resistance patterns of C. jejuni and C. coli this spatial avoidance has not been documented between isolates originating from faecal samples from cattle and sheep livestock species and the lack of transmission may be in the Freser-Setcases National Game Reserve, Eastern Pyrenees, due to other variables. In fact, studies have shown that Spain in faeces shed on pastures, Campylobacter rapidly inacti- AMR profilea Antimicrobial classb Isolates vates in spring and summer due to desiccation, which NAL, TET, ERY, GEN 4 1 reduces the risk of transmission [34]. NAL, CIP, TET, ERY 3 2 The high genetic diversity we have found among C. NAL, CIP, TET 2 11c jejuni isolates is in accordance with other reports in live- stock [25], and is common in this bacterial species due NAL, CIP 1 1 to its genetic instability [35]. In situation of environmen- Pan-susceptible - 2 tal stress, this genomic variability seems to be relevant, a Quinolones: NAL (nalidixic acid), CIP (ciprofloxacin); Tetracyclines: TET (tetracycline); Macrolides: ERY (erytromycin); Aminoglycosides: as it generates a population heterogeneity to improve fit- GEN (gentamycin) ness and survival in hostile environments, increasing the b Number of different antimicrobial classes per resistance profile colonization potential [36]. On the other hand, the host- c It includes the C. coli isolate species specificity of C. jejuni we have found in ungu- lates has also been reported in other host species, par- of these pathogens, and a distinct relevance of the host ticularly birds [37]. species studied. Salmonella spp. was not found in the livestock species Of the four host species included in our study, cattle from our study area. Even if Salmonella is principally were the main carrier of Campylobacter in alpine envi- isolated from poultry or pigs [38, 39], low to moderate ronments. The prevalence found in our study area was prevalence are usually expected in cattle and sheep in higher than that previously reported in cattle under free- Spain [6, 40]. In wild ruminants, the occurrence of Sal- ranging and extensive farming management in Spain [15, monella is frequently low or null [40, 41]. Also, this zoo- 25]. However, similar frequencies have been described in notic agent has been seldom isolated in chamois species Spain as well as in other European countries for dairy cat- [42, 43], although mortality due to septicaemia has been tle, either under indoor or intensive farming management described [44]. It has also been reported a reduction in [26–28]. Nevertheless, seasonal, environmental and the reproductive success of alpine chamois (Rupicapra specific management variables must be taken into ac- rupicapra) in relation with the prevalence of antibodies count as they all can affect the carriage rate of Cam- against this pathogen [45]. The low prevalence in our pylobacter [28]. In our study, the high burden study area seems to indicate that this pathogen is not a detected can be due to the winter farming conditions. problem for chamois demography. In fact, the overall During this season, animals are gathered in the lower lack of detection of Salmonella in livestock suggest a mountain pastures and may be kept indoor for long very low burden in alpine environments. periods of time during colder days, increasing the In our study, cattle and sheep showed to be reservoirs chance of infection from faecal material [25]. of AMR bacteria in alpine environments, and therefore The low prevalence detected in sheep indicates that can contribute to their spread and maintenance in these this species has a moderate role in the epidemiology of habitats. We found resistance to antimicrobial agents in Campylobacter in alpine environments. Sheep, as cattle, all but two Campylobacter isolates tested, with high fre- is usually pointed as an important source of enterobacte- quencies of resistance to nalidixic acid, ciprofloxacin and rial contamination in the environment and the recovery tetracycline. These findings are similar to what has been rates are usually higher than in our study ([5, 29] but see reported for Campylobacter in the EU survey on AMR [25]). Findings of enteropathogens in horses are rarely in zoonotic bacteria, where resistance to these agents reported, particularly in free-ranging horses for meat were the most frequently detected in livestock in Europe production, but studies have shown that horses are po- in 2017 [18]. Results regarding tetracycline resistance are tential carriers of Campylobacter [30] and have the po- not surprising as they are the most used antimicrobials tential to cause disease outbreaks in humans by in livestock in Europe [46]. However, the frequencies of contaminating the water supply [31]. However, in our resistance to quinolones (i.e. nalidixic acid) and fluoro- study we did not detect any positive horse, highlighting quinolones (i.e. ciprofloxacin) are alarming as they are the null or negligible role of free-ranging horses in the higher than those previously described in Europe for epidemiology of Campylobacter in alpine environments. livestock [18, 47, 48]. On the other hand, a very low oc- The lack of transmission from livestock to chamois currence of resistance to macrolides and aminoglyco- may be due to a spatial behaviour adjustment of chamois sides was found, probably due to the relatively to avoid pastures when livestock is present [32], or even infrequent use of these antibiotics in food-producing an avoidance of dung-contaminated areas [33]. However, species [46]. Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 5 of 8 Wildlife is frequently considered sentinel of the bur- every year.. In the FSNGR, around 4,660 cattle, 1,450 den and distribution of pathogens and AMR in the en- sheep and 310 horses benefit from these conditions [56]. vironment [49, 50]. Due to their life history, synurbic We collected 86 faecal samples from harvested Pyrenean wildlife [23], small mammals near aquatic environments chamois during the hunting season. We obtained samples [24] and even seabirds around human settlements in from the rectum of the animal using disposable gloves, Antarctica [12] show a strong link between environmen- minutes after being hunted. Additionally, we collected tal contamination and the wildlife carriage of fresh faeces from livestock (up to 100 g) from the ground antimicrobial-resistant pathogens. However, data ob- just after deposition. We obtained samples from 74 cattle tained in our study in Pyrenean chamois does not reflect from 19 herds, 39 sheep from 3 flocks and 30 horses from the high levels of AMR enteropathogens spreading by 10 herds. Special care was taken to avoid sample con- sympatric livestock. In fact, the scarce information in al- tamination and we used individual disposable plastic pine environments suggests that wild mammals are un- bags for every sample. All samples were refrigerated commonly infected by Campylobacter and Salmonella and processed within 24 h. [51, 52], supporting that the pathogenic burden of alpine environments is low, despite the high levels of excretion Isolation and identification of Campylobacter spp. and by livestock. Even fewer studies have investigated the Salmonella spp presence of AMR pathogens in those species and have For each sample, we introduced ten grams of faeces into reached similar conclusions [53]. sterile test tubes to which we added between three to The recent agricultural shift in European mountains, five mL of sterile water to homogenize and facilitate its where primary production is gradually abandoned [54] handling using a sterile swab. and the replacement of sheep for cattle by the remaining farmers [55], is probably producing constant changes in Campylobacter isolation and identification the epidemiology of zoonotic enteopathogens in these We performed Campylobacter isolation by direct areas. Locally we may predict that the presence of Cam- streaking onto blood-free selective medium (mCCDA, pylobacter will increase in areas where the densities of modified charcoal cefoperazone desoxycholate agar, cattle increase but may decrease in areas of farm aban- CM739 with selective supplement, SR0155E; Oxoid, donment. However, additional studies on Campylobacter Basingstoke, UK) as described by Urdaneta et al. [57]. and Salmonella survival in alpine environments are Plates were incubated at 42ºC for 48 h under microaerobic needed to better understand the cycles of environmental conditions, and up to four presumptive colonies per posi- contamination and AMR spread. tive sample were subcultured onto blood agar plates (Bio- Merieux, Marcy l’Etoile, France) at 37ºC during 48 h in Conclusions microaerobic conditions. We identified Campylobacter In conclusion, we show that free-ranging cattle and species by multiplex PCR using primers targeting the lipid sheep are spreaders of enteric zoonotic bacteria of public A gene lpxA [58]. All isolates were preserved in brain- health concern as well as their AMR strains in the alpine hearth infusion broth with 20 % of glycerol at -75ºC for environment. Therefore, contaminated alpine pastures later analysis. or streams may constitute a source for the dissemination of these organisms. However, alpine wild ungulates such Salmonella isolation and identification as Pyrenean chamois might not be considered as poten- Salmonella isolation procedure was as described by An- tial bioindicators or sentinels of the pathogen (AMR or tilles et al. [10]. Briefly, we pre-enriched the swabs in susceptible) burden of this environment. buffered peptone water (Oxoid, Basingstoke, UK) at 37ºC for 20 ± 2 h, followed by a selective enrichment in Methods Rappaport-Vassiliadis broth (Oxoid, Basingstoke, UK) at Study area and sampling 42ºC for 24–48 h and subculturing onto XLT4 (Xylose The study was performed from 2015 to 2017 in the Lysine Tergitol 4) agar plates (Merck, Darmstadt, Freser-Setcases National Game Reserve (FSNGR), east- Germany) at 37ºC for 24 h. Finally, up to four presump- ern Pyrenees, Catalonia, Spain (42˚ 22’ N, 2˚ 09’ E). The tive colonies were selected and streaked onto MacConkey FSNGR is a mountainous area of 20.200 ha where sub- agar and incubated at 37ºC for 24 h. We confirmed the alpine and alpine ecosystems predominate with an aver- lactose-negative colonies as Salmonella spp. with the age altitude of 2,000 meters above sea level (m.a.s.l.), Mucap (Biolife, Milano, Italy) and indole tests. Salmonella ranging from 1,200 to 2,910 m.a.s.l. The area is inhabited serotyping was carried out according to the White- by a population of around 3,000 Pyrenean chamois Kauffmann-Le Minor scheme [59] at the Laboratori (Rupicapra p. pyrenaica), the most abundant wild ungu- Agroalimentari (Cabrils, Spain) of the Departament late present. Around 300 specimens are legally hunted d’Agricultura, Ramaderia, Pesca i Alimentació. Isolates Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 6 of 8 were preserved in brain-hearth infusion broth with 20 % 32 mg/L), sulfamethoxazole (8–1,024 mg/L), and colistin of glycerol at -75ºC for later analysis. (1–16 mg/L). We used E. coli ATCC 25,922 as control strain. Plates were incubated at 37ºC for 24 h. Molecular typing of Campylobacter and Salmonella An isolate was considered MDR when showing re- isolates sistance to three or more nonrelated antimicrobials. We determined the genotypic diversity among Campylo- We used epidemiological cut-off values according to bacter isolates by flaA-RFLP, following the CAMPYNET EUCAST guidelines (www.eucast.org) to consider an protocol as previously described by Harrington et al., isolate susceptible or resistant. When reporting data [60]. The flaA gene was amplified using the forward A1 using EUCAST epidemiological cut-off values, bacteria (5’-GGA TTT CGT ATT AAC ACA AAT GGT GC -3’) should be reported as ‘‘wild-type’’ (WT) or ‘‘non-wild- and reverse A2 (5’- CTG TAG TAA TCT TAA AAC ATT type’’ (non-WT) [63]. For simplicity, the terms “sus- TTG-3’) primers [61]. The amplified product of 17.7 kb ceptible” and “resistant” have been used here. was digested using the restriction enzyme DdeI (HypF3I; FastDigest, Thermo Fisher Scientific, Waltham, MA, USA). Data analyses The digest products were separated by electrophoresis on The prevalence of each pathogen was calculated from 2.5 % agarose gel in 1x TAE buffer at 90V for 3 h. the proportion of positives to the total number of ani- We analysed the RFLP band patterns using Finger- mals examined, with Wilson score confidence intervals printing II v3.0 software (Bio-Rad, Hercules, CA, USA). of 95 %. We calculated the similarity matrices using the Dice co- efficient with tolerance and optimization values of 1.0 %. Abbreviations We constructed a dendrogram based on an unweighted- AMR: Antimicrobial resistance; EUCAST: European Committee on pair group method with arithmetic mean (UPGMA) Antimicrobial Susceptibility Testing; flaA-RFLP: Restriction fragment length cluster analysis. We used a cut-off of 90 % for the deter- polymorphism of the flaA gene; MDR: Multidrug resistant; WT: Wild-type mination of the different profiles. Acknowledgements We genotyped the Salmonella isolates by ERIC-PCR We are grateful to the gamekeepers and hunters from the Freser-Setcases as previously described [10], except that we used a 50ºC National Game Reserve as well as all our fieldwork team (particularly Xavier Fernandez-Aguilar, Andreu Colom-Cadena, Maria Puig-Riba and Gregorio annealing temperature that is more adequate for Entero- Mentaberre), for their valuable help with the sampling of Pyrenean chamois. bacteriaceae. We used the primer pairs ERIC-F (5’-AAG CERCA Programme from the Generalitat de Catalunya is also acknowledged. TAA GTG ACT GGG GTG AGC G-3’) and ERIC-R (5’- ATG TAA GCT CCT GGG GAT TCA C-3’) [62]. Authors’ contributions JE, OC and MC contributed to the study design, data analysis and manuscript writing. JE and AD-A contributed in the sample collection. JE, PM Antimicrobial susceptibility testing and TA contributed to the sample analysis. All authors read and approved We considered all Campylobacter isolates from the same the final manuscript. individual showing identical flaA-RFLP profiles as the Funding same strain and we only selected one of them for the The authors received no specific funding for this work. antimicrobial susceptibility testing. We tested the iso- Availability of data and materials lates using a minimum inhibitory concentration (MIC)- The datasets used and/or analysed during the current study are available based broth microdilution (EUCAMP2 Sensititre® Sus- from the corresponding author on reasonable request. ceptibility plates, ThermoFisher Scientific, Spain). The Ethics approval and consent to participate antimicrobials tested were: nalidixic acid (1–64 mg/L), The authors confirm that the ethical policies of the journal have been ciprofloxacin (0.12–16 mg/L), tetracycline (0.5–64 mg/ adhered to. No ethical approval was required as the samples from livestock L), gentamycin (0.12–16 mg/L), streptomycin (0.25– were collected from the ground and Pyrenean chamois were not purposefully hunted for this study. Samples were collected from individuals 16 mg/L) and erythromycin (1–128 mg/ L). We used C. legally hunted by authorized hunters with the correct permit of the Catalan jejuni ATCC 33,560 as control strain. Plates were incu- government. bated at 37ºC for 48 h. Salmonella susceptibility testing was performed using Consent for publication Not applicable. EUVSEC plates (Sensititre® Susceptibility plates, Ther- moFisher Scientific, Spain). Antimicrobials tested were: Competing interests ampicillin (1–64 mg/L), cefotaxime (0.25-1 mg/L), cef- The authors declare that they have no competing interests. tazidime (0.5–8 mg/L), meropenem (0.03–16 mg/L), Author details nalidixic acid (4–128 mg/L), ciprofloxacin (0.015–8 mg/ 1 Wildlife Conservation Medicine Research Group (WildCoM), Departament de L), tetracycline (2–64 mg/L), tigecycline (0,25–8 mg/L), Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain. 2Research and Conservation Department, Zoo de Barcelona, Barcelona, gentamycin (0.5–32 mg/L), azithromycin (2–64 mg/L), Spain. 3UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), chloramphenicol (8–128 mg/L), trimethoprim (0,25– Campus de la Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 7 of 8 Spain. 4IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), 21. Ramey AM, Ahlstrom CA. Antibiotic resistant bacteria in wildlife: Campus de la Universitat Autònoma de Barcelona, Bellaterra, Barcelona, perspectives on trends, acquisition and dissemination, data gaps, and future Spain. directions. J Wildl Dis. 2020;56(1):1. 22. Mercat M, Clermont O, Massot M, Ruppe E, De Garine-Wichatitsky M, Miguel Received: 18 September 2020 Accepted: 2 February 2021 E, et al. Escherichia coli population structure and antibiotic resistance at a buffalo/cattle interface in southern Africa. Appl Environ Microbiol. 2016; 82(5):1459–67. 23. Jobbins SE, Alexander KA. From whence they came—Antibiotic-resistant References Escherichia coli in African wildlife. J Wildl Dis. 2015;51(4):811–20. 1. Havelaar AH, Kirk MD, Torgerson PR, Gibb HJ, Hald T, Lake RJ, et al. World 24. Furness LE, Campbell A, Zhang L, Gaze WH, McDonald RA. Wild small Health Organization global estimates and regional comparisons of the mammals as sentinels for the environmental transmission of antimicrobial burden of foodborne disease in 2010. PLOS Med. 2015;12(12):e1001923. von resistance. Environ Res. 2017;154(May 2016):28–34. Seidlein L, editor 25. Oporto B, Esteban JI, Aduriz G, Juste RA, Hurtado A. Prevalence and strain 2. EFSA and ECDC (European Food Safety Authority and European Centre for diversity of thermophilic campylobacters in cattle, sheep and swine farms. J Disease Prevention and Control). The European Union summary report on Appl Microbiol. 2007;103(4):977–84. trends and sources of zoonoses, zoonotic agents and food-borne outbreaks 26. Vilar M, Peña F, Pérez I, Diéguez F, Sanjuán M, Rodríguez-Otero J, et al. in 2017. EFSA J 2018. 2018;16(12). Presence of Listeria, Arcobacter, and Campylobacter spp. in dairy farms in 3. Pires SM, Vigre H, Makela P, Hald T. Using outbreak data for source Spain. Berl Munch Tierarztl Wochenschr. 2010;123(1–2):58–62. attribution of human salmonellosis and campylobacteriosis in Europe. 27. Nielsen EM. Occurrence and strain diversity of thermophilic campylobacters Foodborne Pathog Dis. 2010 Nov;7(11):1351–61. in cattle of different age groups in dairy herds. Lett Appl Microbiol. 2002; 4. Taylor EV, Herman KM, Ailes EC, Fitzgerald C, Yoder JS, Mahon BE, et al. 35(1):85–9. Common source outbreaks of Campylobacter infection in the USA, 1997– 28. Ellis-Iversen J, Cook AJC, Smith RP, Pritchard GC, Nielen M. Temporal 2008. Epidemiol Infect. 2013;141(5):987–96. Patterns and Risk Factors for Escherichia coli O157 and Campylobacter 5. Stanley K, Jones K. Cattle and sheep farms as reservoirs of Campylobacter. J spp Young Cattle. J Food Prot. 2009;72(3):490–6. Appl Microbiol. 2003;94(s1):104–13. 6. Hurtado A, Ocejo M, Oporto B. Salmonella spp. and Listeria monocytogenes 29. Uaboi-Egbenni PO, Bessong PO, Samie A, Obi CL. Campylobacteriosis in shedding in domestic ruminants and characterization of potentially sheep in farm settlements in the Vhembe District of South Africa. African J pathogenic strains. Vet Microbiol. 2017 Oct;210(September):71–6. Microbiol Res. 2010;4(20):2109–17. 7. Dorner SM, Huck PM, Slawson RM. Estimating potential environmental 30. Moriarty E, Downing M, Bellamy J, Gilpin B. Concentrations of faecal loadings of Cryptosporidium spp. and Campylobacter spp. from livestock in coliforms, Escherichia coli, enterococci and Campylobacter spp. in equine the Grand River Watershed, Ontario, Canada. Environ Sci Technol. 2004; faeces. N Z Vet J. 2015;4(2):104–9. 38(12):3370–80. 31. Paruch L, Paruch AM, Sørheim R. DNA-based faecal source tracking of 8. Carbonero A, Paniagua J, Torralbo A, Arenas-Montes A, Borge C, García- contaminated drinking water causing a large Campylobacter outbreak in Bocanegra I. Campylobacter infection in wild artiodactyl species from Norway 2019. Int J Hyg Environ Health. 2020;224(November):113420. southern Spain: Occurrence, risk factors and antimicrobial susceptibility. 32. Chirichella R, Ciuti S, Apollonio M. Effects of livestock and non-native Comp Immunol Microbiol Infect Dis. 2014;37(2):115–21. mouflon on use of high-elevation pastures by Alpine chamois. Mamm Biol - 9. Hilbert F, Smulders FJM, Chopra-Dewasthaly R, Paulsen P. Salmonella in the Zeitschrift für Säugetierkd. 2013;78(5):344–50. wildlife-human interface. Food Res Int. 2012;45(2):603–8. 33. Fankhauser R, Galeffi C, Suter W. Dung avoidance as a possible mechanism 10. Antilles N, Sanglas A, Cerdà-Cuéllar M. Free-living waterfowl as a source of in competition between wild and domestic ungulates: Two experiments zoonotic bacteria in a dense wild bird population area in northeastern with chamois Rupicapra rupicapra. Eur J Wildl Res. 2008;54(1):88–94. Spain. Transbound Emerg Dis. 2015;62(5):516–21. 34. Moriarty EM, Mackenzie ML, Karki N, Sinton LW. Survival of Escherichia coli, 11. Moré E, Ayats T, Ryan PG, Naicker PR, Keddy KH, Gaglio D, et al. Seabirds Enterococci, and Campylobacter spp. in Sheep Feces on Pastures. Appl (Laridae) as a source of Campylobacter spp., Salmonella spp. and antimicrobial Environ Microbiol. 2011;77(5):1797–803. resistance in South Africa. Environ Microbiol. 2017;19(10):4164–76. 35. de Boer P, Wagenaar JA, Achterberg RP, van Putten JPM, Schouls LM, Duim 12. Cerdà-Cuéllar M, Moré E, Ayats T, Aguilera M, Muñoz-González S, Antilles N, B. Generation of Campylobacter jejuni genetic diversity in vivo. Mol et al. Do humans spread zoonotic enteric bacteria in Antarctica? Sci Total Microbiol. 2002;44(2):351–9. Environ. 2019;654:190–6. 36. Ridley AM, Toszeghy MJ, Cawthraw SA, Wassenaar TM, Newell DG. Genetic 13. Hassell JM, Ward MJ, Muloi D, Bettridge JM, Robinson TP, Kariuki S, et al. instability is associated with changes in the colonization potential of Clinically relevant antimicrobial resistance at the wildlife–livestock–human Campylobacter jejuni in the avian intestine. J Appl Microbiol. 2008;105(1): interface in Nairobi: an epidemiological study. Lancet Planet Heal. 2019;3(6): 95–104. e259-69. 37. Griekspoor P, Colles FM, McCarthy ND, Hansbro PM, Ashhurst-Smith C, 14. Sippy R, Sandoval-Green CMJ, Sahin O, Plummer P, Fairbanks WS, Zhang Q, Olsen B, et al. Marked host specificity and lack of phylogeographic et al. Occurrence and molecular analysis of Campylobacter in wildlife on population structure of Campylobacter jejuni in wild birds. Mol Ecol. 2013; livestock farms. Vet Microbiol. 2012;157(3–4):369–75. 22(5):1463–72. 15. Navarro-Gonzalez N, Ugarte-Ruiz M, Porrero MC, Zamora L, Mentaberre G, 38. Davies RH, Dalziel R, Gibbens JC, Wilesmith JW, Ryan JMB, Evans SJ, et al. Serrano E, et al. Campylobacter Shared Between Free-Ranging Cattle and National survey for Salmonella in pigs, cattle and sheep at slaughter in Sympatric Wild Ungulates in a Natural Environment (NE Spain). Ecohealth. Great Britain (1999–2000). J Appl Microbiol. 2004;96(4):750–60. 2014;5(3):333–42. 39. Solveig J, Trude ML, Merete H, Bjarne B, Torkjel B, Michaela F, et al. The 16. Jasovský D, Littmann J, Zorzet A, Cars O. Antimicrobial resistance—a threat surveillance and control programme for Salmonella in live animals, eggs to the world’s sustainable development. Ups J Med Sci. 2016;121(3):159–64. and meat in Norway. Annual Report. Oslo; 2008. 17. Bengtsson-Palme J, Kristiansson E, Larsson DGJ. Environmental factors 40. Navarro-Gonzalez N, Velarde R, Porrero MC, Mentaberre G, Serrano E, Mateos A, influencing the development and spread of antibiotic resistance. FEMS et al. Lack of Evidence of Spill-Over of Salmonella enterica Between Cattle and Microbiol Rev. 2018;42(1):68–80. Sympatric Iberian ibex (Capra pyrenaica) from a Protected Area in Catalonia, NE 18. EFSA (European Food Safety Authority) and ECDC (European Centre for Spain. Transbound Emerg Dis. 2014;61(4):378–84. Disease Prevention and Control). The European Union summary report on 41. Díaz-Sánchez S, Sánchez S, Herrera-León S, Porrero C, Blanco J, Dahbi G, antimicrobial resistance in zoonotic and indicator bacteria from humans, et al. Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. animals and food in 2017. EFSA J 2019. 2019;17(2). and Campylobacter spp. in large game animals intended for consumption: 19. Smith DL, Dushoff J, Morris JG. Agricultural antibiotics and human health. Relationship with management practices and livestock influence. Vet PLoS Med. 2005;5(8):e232. Microbiol. 2013;163(3–4):274–81. 20. Silbergeld EK, Graham J, Price LB. Industrial food animal production, 42. Obwegeser T, Stephan R, Hofer E, Zweifel C. Shedding of foodborne antimicrobial resistance, and human health. Annu Rev Public Health. 2008; pathogens and microbial carcass contamination of hunted wild ruminants. 29(1):151–69. Vet Microbiol. 2012;159(1–2):149–54. Espunyes et al. BMC Veterinary Research (2021) 17:79 Page 8 of 8 43. Dumitrescu V, Borlea F, Nichita I, Bucur IM, Tîrziu E. Comparative research on antimicrobial resistance in bacteria isolated from domestic and wild animals (chamois - Rupicapra rupicapra). In: Young People and Veterinary Medicine Research. Timișoara, Romania; 2018. p. 53. 44. Glawischnig W, Khaschabi D, Schöpf K, Schönbauer M. An outbreak of Salmonella Dublin in chamois (Rupicapra rupicapra). Wien Tierarztl Monatsschr. 2000;87(1):21–5. 45. Pioz M, Loison A, Gibert P, Jullien J-M, Artois M, Gilot-Fromont E. Antibodies against Salmonella is associated with reduced reproductive success in female alpine chamois (Rupicapra rupicapra). Can J Zool. 2008;86(10):1111–20. 46. EMA (European Medicines Agency). Sales of veterinary antimicrobial agents in 31 European countries in 2017. Trends from 2010 to 2017 (EMA/294674/ 2019). Amsterdam; 2019. 47. Ocejo M, Oporto B, Hurtado A. Occurrence of Campylobacter jejuni and Campylobacter coli in cattle and sheep in northern Spain and changes in antimicrobial resistance in two studies 10-years apart. Pathogens. 2019;8(3):98. 48. Sproston EL, Wimalarathna HML, Sheppard SK. Trends in fluoroquinolone resistance in Campylobacter. Microb Genomics. 2018;4(8). 49. White A, Hughes JM. Critical Importance of a One Health Approach to Antimicrobial Resistance. Ecohealth. 2019;16(3):404–9. 50. Conrad PA, Meek LA, Dumit J. Operationalizing a One Health approach to global health challenges. Comp Immunol Microbiol Infect Dis. 2013;36(3): 211–6. 51. Marreros N, Hüssy DH, Albini S, Frey CF, Abril C, Vogt HR, et al. Epizootiologic investigations of selected abortive agents in free-ranging alpine ibex (Capra ibex ibex) in Switzerland. J Wildl Dis. 2011;47(3):530–43. 52. Pagano A, Nardi G, Bonaccorso C, Falbo V, Passi C, Sanguinetti V, et al. Faecal bacteria of wild ruminants and the alpine marmot. Vet Res Commun. 1985;9(1):227–32. 53. Caprioli A, Donelli G, Falbo V, Passi C, Pagano A, Mantovani A. Antimicrobial resistance and production of toxins in Escherichia coli strains from wild ruminants and the alpine marmot. J Wildl Dis. 1991;27(2):324–7. 54. MacDonald D, Crabtree J, Wiesinger G, Dax T, Stamou N, Fleury P, et al. Agricultural abandonment in mountain areas of Europe: Environmental consequences and policy response. J Environ Manage. 2000;59(1):47–69. 55. Garcia-Ruiz JM, Lasanta-Martinez T. Land-Use Changes in the Spanish Pyrenees. Mt Res Dev. 1990;10(3):267. 56. Idescat. (Institut d’Estadística de Catalunya). Ramaderia: Anuari estadístic de Catalunya; 2009. 57. Urdaneta S, Dolz R, Cerdà-Cuéllar M. Assessment of two different types of sample for the early detection and isolation of thermophilic Campylobacter in broiler farms. Avian Pathol. 2015;44(4):103–5. 58. Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PML, Horn ST, et al. Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol. 2004;42(1): 5549–57. 59. Grimont P, Weill F-X. Antigenic formulae of the Salmonella servovars. 9th Edition. WHO Collaborating Centre for reference and research on Salmonella (WHOCC-SALM). Paris:; 2007. 60. Harrington CS, Moran L, Ridley AM, Newell DG, Madden RH. Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience. J Appl Microbiol. 2003;95(6): 1321–33. 61. Nachamkin I, Bohachick K, Patton CM. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J Clin Microbiol. 1993;31(6):1531–6. 62. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991;19(24):6823–31. 63. Schwarz S, Silley P, Simjee S, Woodford N, van Duijkeren E, Johnson AP, et al. Editorial: Assessing the antimicrobial susceptibility of bacteria obtained from animals. J Antimicrob Chemother. 2010;65(4):601–4. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.