Science of the Total Environment 844 (2022) 157189 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Eurasian griffon vultures carry widespread antimicrobial resistant Salmonella and Campylobacter of public health concern ⁎ Johan Espunyes a, , Lucía Illera a, Andrea Dias-Alves a, Lourdes Lobato a, Maria Puig Ribas a, Alicia Manzanares b, Teresa Ayats b, Ignasi Marco a,1, Marta Cerdà-Cuéllar b,c,1 a Wildlife Conservation Medicine Research Group (WildCoM), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain b Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain c IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Vultures are carriers of widespread AMR zoonotic Salmonella and Campylobacter. • Strains of monophasic S. Typhimurium were shared by gulls, livestock and humans. • The majority of isolates showed resistance to at least one antimicrobial. • AMR bacteria in vultures might originate in livestock farming. • The high frequency of AMR to critical an- timicrobials for human medicine is of con- cern. A R T I C L E I N F O A B S T R A C T Editor: Damià Barceló The global emergence of antimicrobial-resistant (AMR) strains of Salmonella and Campylobacter is a serious public health concern. Both bacteria are leading causes of human gastrointestinal foodborne infections and the two most re- Keywords: ported zoonoses in the European Union. By feeding on livestock carcasses, especially from intensive farming, as well as AMR on landfill sites, obligate avian scavengers can become infected with zoonotic pathogens and AMR strains, and can be Bacterial drug resistance considered large-scale sentinels of the environmental burden. In this study, we assessed the occurrence and AMR of Birds Monophasic Salmonella Typhimurium Salmonella spp. and Campylobacter spp. in 218 Eurasian griffon vultures (Gyps fulvus) captured in north-eastern Wildlife Spain. We isolated Salmonella from 8.1 % of individuals and Campylobacter lari from 4.7 %. Among the 10 different Zoonoses Salmonella serovars found, monophasic S. Typhimurium was the most frequent. Genotyping analysis revealed same strains of monophasic S. Typhimurium shared by gulls, livestock and humans. Isolates from both bacterial species pre- sented AMR to important antimicrobials (tetracyclines, fluoroquinolones and β-lactams). In conclusion, this study shows that Eurasian griffon vultures in north-eastern Spain are carriers of widespread AMR zoonotic Salmonella and Campylobacter. More comprehensive analyses are still needed to understand the potential risk of spill-over from those wild birds to humans. 1. Introduction The emergence and spread of antimicrobial-resistant (AMR) bacteria is one of the greatest threats to human medicine, veterinary medicine, and ⁎ Corresponding author. public health (Laxminarayan et al., 2013). This situation impairs our E-mail address: Johan.espunyes@uab.cat (J. Espunyes). efficacy to treat a growing number of infections, increases medical costs, 1 Both authors contributed equally to this work. and could account for as many as 10 million annual deaths worldwide by http://dx.doi.org/10.1016/j.scitotenv.2022.157189 Received 4 February 2022; Received in revised form 30 June 2022; Accepted 2 July 2022 Available online 6 July 2022 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Espunyes et al. Science of the Total Environment 844 (2022) 157189 2050 (O'Neill, 2016). AMR has multifactorial causes which is mainly due to a AMR Salmonella and Campylobacter that are present in other hosts such as combination of inappropriate drug prescriptions and use, lack of appropriate livestock, humans, and other wildlife species in areas with anthropogenic drug regulations and surveillance systems in developing countries and exten- pressure. By comparing the genotypes of the recovered strains with strains sive use of antimicrobials in animal production (Ayukekbong et al., 2017). In from other regions and hosts, we further aimed to gain insight into the order to contain the situation, the World Health Organization (WHO) has potential anthropogenic origin of those strains, the span of those AMR promoted a ban on the use of critically important antimicrobials for zoonotic pathogens, and the role of avian scavengers in their epidemiology. human medicine in livestock disease control (WHO, 2017). Currently, Salmonella and Campylobacter are the leading causes of human gastroin- 2. Material and methods testinal foodborne infections and the two most reported zoonoses in the European Union (EFSA and ECDC, 2021). The recent emergence of the 2.1. Study area and sampling monophasic Salmonella Typhimurium serovar, related with multidrug- resistance (MDR), is now a global public health emergency (Sun et al., Between June 2019 and February 2020, we collected cloacal swabs from 2020). In fact, monophasic Salmonella Typhimurium has become, after 218 Eurasian griffon vultures in Osona (province of Barcelona, Catalonia, Salmonella Enteritidis, one of the most important cause of human salmonello- NE-Spain; 42°4′N, 2°12′E). This capture site is situated in the vicinities of a sis in the EU (EFSA and ECDC, 2021). Overall, the global emergence of AMR landfill site where vultures feed regularly on organic waste. This population strains of both Salmonella and Campylobacter is leading to an increase in of vultures also feed in supplementary feeding stations around the area. mortality rates due to therapeutic failure, and is considered a serious public Twelve animals were captured and sampled twice and two were sampled health concern (Eng et al., 2015; Moore et al., 2006). thrice, obtaining a total of 234 samples. Vultures were captured during While the occurrence of AMR in livestock has been broadly assessed, the monthly ringing activities, using a portable trap with fixed side panels role of wild animal species in the maintenance and transmission of these (5.2 × 5.2 m) and self-locking swinging doors. Two cloacal swabs were resistances is still poorly understood (Vittecoq et al., 2016). In general, obtained from each individual, which were placed in Amies transport wildlife does not naturally come into contact with antimicrobials, but can medium containing charcoal (Deltalab, Rubí, Spain) and were maintained acquire AMR bacteria from human and livestock sources such as agricul- at 4 °C and processed within 36 h. The age of individuals was determined tural facilities, contaminated sewage and refuse dumps. Also, antimicro- according to physical characteristics, and classified as juveniles (<2 years; bials present in human residues and livestock carcasses released to the n = 6), sub-adults (from 2 to 4 years; n = 54), and adults (>4 years; n = environment can promote the development of AMR bacteria in the intesti- 143). The age of 15 individuals couldn't be determined between sub-adult nal microbiota of wildlife (Gómez-Ramírez et al., 2020; Ramey and and adult due to plumage defaults. Ahlstrom, 2020). In that sense, scavengers, due to their feeding habits and their position in the food chain, are expected to be more prone to 2.2. Salmonella and Campylobacter isolation and identification carry Campylobacter and Salmonella and spread AMR bacteria, facilitating their dissemination (Antilles et al., 2021; Marin et al., 2014; Smith et al., We performed the isolation and identification of Salmonella spp. and 2020). Furthermore, wild bird species that can fly great distances may act Campylobacter spp. as previously described by Antilles et al. (2021). Briefly, as a source of bacterial dissemination across broad geographical scales one swab was used for Salmonella isolation using a non-selective pre- (Blanco et al., 2020; Molina-Lopez et al., 2011). enrichment in buffered peptone water (Oxoid, Basingstoke, UK), followed Spain is home for 90 % of the European population of Eurasian griffon by a selective enrichment in Rappaport-Vassiliadis (Oxoid, Basingstoke, vultures (Gyps fulvus) (Del Moral and Molina, 2018). Eurasian griffon UK) and subculturing onto xylose lysine Tergitol 4 agar (Merck, Darmstadt, vultures are obligate avian scavengers that feed from carcasses of wild Germany). Presumptive colonies were subcultured onto MacConkey agar and domestic animals found by flying over extensive areas. The indiscrim- and confirmed as Salmonella with the indole test. Salmonella isolates were inate use of poison up to the 1980s contributed to a critic decline of scaven- serotyped at the Laboratori Agroalimentari (Cabrils, Spain. www.bit.ly/ ger species. To redress the situation, supplementary feeding stations using 3OTqwbi) from the Catalan Governmnet (Departament d'Acció Climàtica, livestock carcasses have been established throughout the Iberian Peninsula Alimentació i Agenda Rural) according to White-Kauffmann-Le Minor to increase the availability of food resources and boost the recovery of the scheme (Grimont and Weill, 2007). We used the second swab for Campylo- European populations of avian scavengers (Margalida et al., 2010). These bacter isolation by streaking it onto modified charcoal cefoperazone “vulture restaurants” are also an inexpensive and efficient alternative to desoxycholate agar (CM739 with selective supplement, SR0155E; Oxoid, the industrial destruction of carcasses. As a consequence, vultures can be- Basingstoke, UK) and subculturing presumptive colonies onto blood agar come infected with zoonotic pathogens while feeding on livestock carcasses plates (BioMérieux, Marcy l'Etoile, France). Identification of Campylobacter at these feeding stations, especially from an intensive farming origin species (C. jejuni, C. coli and C. lari) was performed by a multiplex PCR using (Blanco et al., 2020; Sevilla et al., 2020). At the same time, the disposal of primers targeting the lipid A gene lpxA (Klena et al., 2004). We calculated sick and medicated livestock carcasses may increase the ingestion of veter- the occurrence of each pathogen from the proportion of positives to the inary drugs and AMR pathogens (Blanco et al., 2020). Moreover, vultures total number of individuals examined, with Wilson score confidence inter- forage on organic waste in landfill sites. Such sites, where human waste is vals of 95 %. disposed, are considered a source of zoonotic pathogens and are associated with the presence of pharmaceutical products such as antimicrobials 2.3. Molecular typing of Salmonella and Campylobacter isolates (Matejczyk et al., 2011; Musson and Townsend, 2009). As so, feeding on such sites may favour the transmission of pathogens and antimicrobial We determined the genotypic diversity among bacterial isolates by drugs to scavengers (Plaza et al., 2019). Therefore, obligate avian scaven- pulsed-field gel electrophoresis (PFGE), according to the standard operating gers, such as vultures, can be potential large-scale sentinels of the environ- procedure of PulseNet (www.pulsenetinternational.org). We performed the mental burden of zoonotic pathogens and AMR strains (Blanco and restriction enzyme digests with enzymes XbaI for Salmonella, and with en- Bautista, 2020). zymes SmaI for Campylobacter (Roche Applied Science, Indianapolis, IN, In this study, we aimed to assess the prevalence and genetic diversity of USA). We analysed the resulting patterns using Fingerprinting II v3.0 Salmonella spp. and Campylobacter spp., and their AMR strains in a popula- software (Bio-Rad, Hercules, CA, USA), and compared them with previously tion of Eurasian griffon vultures from north-eastern Spain. As such popula- obtained genotypes from our own database, including isolates from other tion feed on supplementary feeding stations and in a landfill, they might be regions and hosts. Cluster analysis was performed with the unweighted pair more prone to be carriers of zoonotic agents. Our working hypothesis is that group method with arithmetic mean (UPGMA), using the Dice correlation vultures are sentinels of the environmental burden of AMR zoonotic patho- coefficient with a band position tolerance of 1,5 %. We considered PFGE gens. This hypothesis predicts that vultures will be carriers of widespread band patterns with a similarity ≥90 % to be the same pulsotype (PFGE type). 2 J. Espunyes et al. Science of the Total Environment 844 (2022) 157189 2.4. Antimicrobial susceptibility testing of Salmonella and Campylobacter isolates (4.7 %; CI95%: 2.6–8.2) (see supplementary Table S1 for the monthly occur- rence of both pathogens). One individual carried both pathogens. Among Antimicrobial susceptibility testing was performed with a minimum in- the animals sampled more than once, one individual that was negative for hibitory concentration (MIC)-based broth microdilution using EUVSEC both pathogens in December became positive for Salmonella in January, plates for Salmonella spp. and EUCAMP2 plates for Campylobacter spp. one animal that was negative in November and January resulted positive (Sensititre® Susceptibility plates, ThermoFisher Scientific, Spain). For Sal- for Campylobacter in February and one negative in December became posi- monella, the tested antimicrobials were ampicillin (1–64 mg/L), cefotaxime tive to Campylobacter in February. The results of age-related occurrences (0.25–1 mg/L), ceftazidime (0.5–8 mg/L), meropenem (0.03–16 mg/L), can be found in the supplementary Table S2. nalidixic acid (4–128 mg/L), ciprofloxacin (0.015–8 mg/L), gentamycin We identified 10 different serotypes of Salmonella: monophasic Salmonella (0.5–32 mg/L), tetracycline (2–64 mg/L), tigecycline (0,25–8 mg/L), Typhimurium 4,[5],12:i:- (7/19), Muenchen 6,8:d:1,2 (3/19), Lexington 3,10: azithromycin (2–64 mg/L), chloramphenicol (8–128 mg/L), colistin z10:1,5 (2/19), Derby 4,12:f,g:- (1/19), Goelzau 15 + 3,15:a:1,5 (1/19), (1–16 mg/L) and trimethoprim (0,25–32 mg/L). We used E. coli ATCC Infantis 6,7:r:1,5 (1/19), Isangi 6,7:d:1,5 (1/19), Meleagridis 3,10:e,h:l,w 25922 as control strain. For Campylobacter, we tested the antimicrobial sus- (1/19), Muenster 3,10:e,h:1,5 (1/19) and Uganda 3,10:l,z13:1,5 (1/19). All ceptibility to nalidixic acid (1–64 mg/L), ciprofloxacin (0.12–16 mg/L), Campylobacter isolates were identified as Campylobacter lari. gentamycin (0.12–16 mg/L), streptomycin (0.25–16 mg/L), tetracycline All seven monophasic Salmonella Typhimurium isolates grouped in five (0.5–64 mg/L) and erythromycin (1–128 mg/L), and using C. jejuni different clusters at >90 % similarity (Fig. 1). Those isolates clustered to- ATCC 33560 as control strain. We followed the European Committee on gether with isolates from poultry, swine carcasses and griffon vultures sam- Antimicrobial Susceptibility Testing (EUCAST) guidelines to establish the pled in the same and neighbouring regions, as well as with a human clinical epidemiological cut-off values (ECOFF) to consider an isolate as “wild- isolate from Catalonia. The three Salmonella Muenchen isolates showed the type” (hereafter “susceptible”) and “non-wild-type” (hereafter “resistant”). same pulsotype and grouped with an isolate obtained from a kelp gull We used the ECOFF of Salmonella enterica for the Salmonella isolates (except (Larus dominicanus) in Western Cape (South Africa). The two Salmonella in the case of meropenem, tigecycline and colistin where the ECOFF were Lexington isolates showed the same pulsotype. The 11C. lari isolates were not available for Salmonella spp. and we used those for E. coli) and the grouped in six pulsotypes (Fig. 2) and one of them had the same pulsotype ECOFF of C. coli for Campylobacter isolates. We considered an isolate as as an isolate from a yellow-legged gull (Larus michahellis) from the Ebro MDR when showing resistance to three or more nonrelated antimicrobials. Delta, 200 km away from our sampling area. Among Salmonella isolates, the most frequent resistance was to tetracy- 3. Results cline (52 %) and ampicillin (48 %), followed by trimethoprim (10.5 %), whilst resistance to ciprofloxacin, nalidixic acid, chloramphenicol and Out of the 234 samples analysed, we isolated Salmonella from 19 indi- gentamicin was found in a single isolate (5.2 %). MDR was present in one viduals (8.1 %; CI95%: 5.2–12.3) and Campylobacter from 11 individuals monophasic Salmonella Typhimurium isolate (Table 1). A 54.5 % (6/11) of Fig. 1. PFGE dendrogram of XbaI patterns of Salmonella isolates from Eurasian griffon vultures in north-eastern Spain. Isolates from poultry from Catalonia and Aragón regions (north-eastern Spain, SP), swine carcasses and other vultures from the Valencia region (eastern Spain, SP), a human case from Catalonia, and a kelp gull from Western Cape (South Africa, SA) were also included for comparison purposes; only those isolates sharing pulsotype with vulture isolates are included in the figure. m. Typhimurium: monophasic Salmonella Typhimurium. 3 J. Espunyes et al. Science of the Total Environment 844 (2022) 157189 Fig. 2. PFGE dendrogram of SmaI patterns of Campylobacter lari isolates from griffon vultures in north-eastern Spain. A yellow-legged gull isolate from the Ebro Delta (north- eastern Spain) was also included for comparison purposes. C. lari isolates showed resistance to ciprofloxacin. The MIC values of the also the serovar most frequently isolated in our study. This is not surprising, control strains C. jejuni ATCC 33560 and E. coli ATCC 25922 were within as more than two thirds of monophasic Salmonella Typhimurium isolated the limits indicated by the EUCAST. from food and animal sources in Europe are associated with pigs (EFSA and ECDC, 2021), which are one of the main foods supplemented to vul- 4. Discussion tures in feeding stations (Donázar et al., 2010). Marin et al. (2018) reported a high similarity between isolates from vultures and pig carcasses, This study assessed the occurrence of Salmonella spp. and Campylobacter supporting the hypothesis that supplementary feeding stations are an im- spp. in Eurasian Griffon vultures from north-eastern Spain. Overall, we de- portant source of Salmonella for vultures. In fact, four vulture isolates tected a relatively low occurrence of both pathogens in faecal swabs. How- from our study showed the same pulsotype as two isolates of pig carcasses ever, the fact that over a half of these isolates showed resistance to at least from this previous study. We also detected the same profile between vul- one antibiotic class is worrying. tures and poultry isolates, consistent with the fact that, although to a lesser We detected a lower occurrence of Salmonella isolates than previously extent, poultry is also part of the supplemented diet of griffon vultures reported in Eurasian griffon vultures in Spain, using similar laboratorial (Donázar et al., 2010). In contrast, this serovar is scarcely recovered from methodologies (Blanco and Díaz de Tuesta, 2021; Marin et al., 2014, other wild bird groups or even other raptor species (Botti et al., 2013; 2018). Blanco and Díaz de Tuesta (2021) described a seasonal variation Moré et al., 2017; Reche et al., 2003; Troxler et al., 2017). Among the in Salmonella occurrence, but our sampling encompassed their period of Salmonella isolates recovered in our study, 63 % (12/19) are serovars higher occurrence (the pre-breeding period, from December to January). responsible for human salmonellosis cases, including serovars Muenchen, Salmonella typically presents an intermittent excretion (Daoust and Derby, Infantis and the above-mentioned monophasic S. Typhimurium Prescott, 2007; Marin et al., 2018) that could explain these differences in (EFSA and ECDC, 2021). In fact, PFGE analysis showed the same pulsotype occurrence between studies in relatively close areas. In accordance with in one monophasic S. Typhimurium isolate we recovered from a vulture, one the above-mentioned surveys, monophasic Salmonella Typhimurium was strain from a human salmonellosis case in NE Spain, and poultry isolates, suggesting a spill-over from humans or food animals to wildlife. Such Table 1 events highlight the importance of a One Health approach to gain insight Antimicrobial resistance patterns of Salmonella and Campylobacter isolates originat- into the epidemiology of zoonoses and antimicrobial resistance (McEwen ing from cloacal swabs of Eurasian Griffon vultures in north-eastern Spain. and Collignon, 2018). AMR profilea Antimicrobial Isolates The majority of serovars isolated in our study have been also isolated in classb other wild species from NE Spain, such as wild boar, red fox, seagulls and raptors (Antilles et al., 2021; Mentaberre et al., 2013; Molina-López et al., Salmonella serovar monophasic AMP, NAL, CIP, GEN, TET, CHL, 6 1 2015; Ramos et al., 2010) but also in pig, poultry and cattle farming Typhimurium TMP (Marin et al., 2018; Ministry of Climate Action, Food and Rural Agenda of AMP, TET 2 4 Catalonia, personal communication; CESAC-Poultry Health Centre of AMP 1 1 Catalonia and Aragon, personal communication). The wide variety of TET 1 1 Muenchen AMP, TET 2 3 hosts with the same Salmonella serovars than the ones recovered in our Meleagridis TET, TMP 2 1 study suggests a low host-specificity and the presence of different sources Campylobacter lari CIP 1 6 of Salmonella contamination for vultures. As obligate scavengers, vultures a β-lactams: AMP (Ampicillin); Quinolones: NAL (nalidixic acid), CIP (ciproflox- feed on a wide variety of species and therefore are more prone to harbour acin); Aminoglycoside: GEN (gentamycin); Tetracycline: TET (tetracycline); a diversity of serovars. Furthermore, landfill sites —such as the one Phenicol: CHL (Chloramphenicol); Sulfonamide: TMP (Trimethoprim). where the vultures of our study feed regularly— are associated with the b Number of different antimicrobial classes per resistance profile. presence of Salmonella, due to the large quantities of decomposing organic 4 J. Espunyes et al. Science of the Total Environment 844 (2022) 157189 waste (Plaza et al., 2019). Vultures may ingest the pathogen while feeding that are admitted to recovery centres and require the use of antibiotics there, and these sites may therefore also be an important source of contam- (Ramey and Ahlstrom, 2020). Also, scavengers are mostly subclinical car- ination for scavengers. On the other hand, finding the same pulsotypes in riers of Salmonella and Campylobacter, and are mainly considered mainte- vulture isolates from our study and a kelp gull isolate from South Africa nance hosts or reservoirs. However, the direct exposure to antibiotics was surprising, as the distance between both sampling sites and the move- through the consumption of medicated livestock carcasses may have detri- ment patterns of both species prevent the existence of a common site of mental effects on vulture health by unbalancing the gut microbiome and infection, such as a landfill. However, our results may be explained by a cos- enhancing the presence of opportunistic pathogens (Pitarch et al., 2017). mopolitan distribution of such serovar (Muenchen), but further research is In fact, the chronic and indiscriminate consumption of antibiotics could needed on the epidemiology of such pathogens at global level to figure this lead to the development of AMR bacteria in the intestinal microbiota of out. High throughput techniques, such as whole genome sequencing, could vultures, but this potential problem has not been explored and deserves provide complementary and more accurate information on the similarity further investigation. and phylogeny of strains, and would be helpful to confirm our findings. In conclusion, this study shows that wild Eurasian griffon vultures in The occurrence of Campylobacter in wild Eurasian griffon vulture has north-eastern Spain are carriers of widespread AMR zoonotic Salmonella been scarcely studied and only low prevalences of C. jejuni have been and Campylobacter, which in some instances may have an anthropogenic reported (Marin et al., 2014; Molina-Lopez et al., 2011). To the best of origin, due to their scavenging feeding habits. Many isolates found, such our knowledge, this is the first report of C. lari in an avian scavenger in as monophasic Salmonella Typhimurium, are of public health concern and the northern hemisphere. Despite C. lari having been sporadically reported the amount of AMR to critically important antimicrobials for human medi- in raptors (Jurado-Tarifa et al., 2016), the great majority of isolates are cine is worrying. However, more in-depth studies, for example combining cultured from Charadriiform species such as sheathbills and auks (Minias, high throughput genome sequencing with transmission pathways analysis, 2020), or brown skuas in the Southern Ocean (Cerdà-Cuéllar et al., are still needed to understand the potential risk of spill-over from those wild 2019). Campylobacter lari has been associated with enteritis, urinary tract birds to humans. infections and bacteraemia in humans, but it is currently considered a minor zoonotic pathogen (Igwaran and Okoh, 2019). Finding the same CRediT authorship contribution statement pulsotype in a vulture and a yellow-legged gull suggests a widespread strain or a common source of infection, such as landfills, and reinforce the impor- Johan Espunyes: Investigation, Formal analysis, Writing – original tance of these sites as a source of contamination for wildlife. Yellow-legged draft. Lucía Illera: Investigation, Formal analysis, Writing – original gulls, as vultures, are prone to use landfills as feeding sources (Antilles draft. Andrea Dias-Alves: Investigation, Writing – review & editing. et al., 2021). Lourdes Lobato: Investigation, Writing – review & editing. Maria Puig We report that the Eurasian griffon vultures of our study are carriers Ribas: Investigation, Writing – review & editing. Alicia Manzanares: of antibiotic-resistant zoonotic Salmonella serovars and C. lari. The pres- Investigation. Teresa Ayats: Investigation. Ignasi Marco: Writing – review ence of resistances to at least one antimicrobial in all the monophasic & editing, Conceptualization, Methodology, Validation, Supervision. S. Typhimurium isolates is worrying, but also expected. Resistant isolates Marta Cerdà-Cuéllar: Writing – review & editing, Conceptualization, in griffon vultures have been previously reported by Blanco (2018) and Methodology, Validation, Supervision. susceptible isolates of monophasic S. Typhimurium are rarely reported in food-producing animals in Europe (EFSA and ECDC, 2019). Further- Declaration of competing interest more, AMR to ampicillin and tetracycline as well as MDR are usually observed at extremely high levels in fattening pigs (EFSA and ECDC, The authors declare that they have no known competing financial inter- 2019). Concurrently, half of the C. lari isolates in this study were resis- ests or personal relationships that could have appeared to influence the tant to the fluoroquinolone ciprofloxacin. Ciprofloxacin is a critically work reported in this paper. important antibiotic in human medicine and one of the limited available therapies to treat some serious infections in humans (WHO, 2016). Our Acknowledgments results could be due to the partial metabolization of enrofloxacin —an antimicrobial frequently used in veterinary medicine— to ciprofloxacin We are grateful to the Grup d'Anellament de Calldetenes-Osona (GACO) in livestock, allowing the development of resistant bacteria that are then for their invaluable collaboration in the capture and sampling of vultures. transmitted to vultures when feeding on carcasses (García Ovando et al., We thank Phelipe Peres, Ignacio Abadía, Laura Carrera, Nuria Cases and 1999; Schulz et al., 2019). In fact, the prevalence of ciprofloxacin- Pol Sarquella for their technical support in the lab and in the field. Strains resistant enteropathogens is very high in food-producing animals, from poultry (Catalonia region) were kindly provided by the CESAC- despite this antibiotic not being approved for veterinary medicine in Poultry Health Centre of Catalonia and Aragon; strains from swine car- Europe (EFSA and ECDC, 2019). casses and vultures (Valencia region) were kindly provided by Clara Marín Vultures have been long time considered as disease mitigators. By from Instituto de Ciencias Biomédicas (Universidad Cardenal Herrera-CEU, removing decomposing organic matter from the environment, they could Spain). CERCA Programme from the Generalitat de Catalunya is also potentially reduce the spread of pathogens (Ogada et al., 2012). However, acknowledged. even if vultures clearly reduce the availability of rotten organic material, there is still a lack of research to unravel if vultures are effective pathogen Appendix A. Supplementary data regulators (Plaza et al., 2020). At the same time, due to scavenging feeding behaviour, vultures can be colonized by pathogens from a wide variety of Supplementary data to this article can be found online at https://doi. species and are usually considered as sentinels of the environmental patho- org/10.1016/j.scitotenv.2022.157189. genic burden. Our study shows that vultures share some Salmonella and Campylobacter strains with other wildlife and farm animals. The role of vul- References tures, and birds in general, as disseminators of pathogens is also still under study (Smith et al., 2020). Even if our study reports that Eurasian griffon Antilles, N., García-Bocanegra, I., Alba-Casals, A., López-Soria, S., Pérez-Méndez, N., Saco, M., vultures are carriers of AMR zoonotic Salmonella and Campylobacter, there González-Solís, J., Cerdà-Cuéllar, M., 2021. 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