BRIEF RESEARCH REPORT published: 22 March 2021 doi: 10.3389/fvets.2021.651781 Occurrence and Quantification of Antimicrobial Resistance Genes in the Gastrointestinal Microbiome of Two Wild Seabird Species With Contrasting Behaviors Edited by: Alain Hartmann, Ana Carolina Ewbank 1*†, Fernando Esperón 2†, Carlos Sacristán 1, Irene Sacristán 3, Institut National de Recherche pour 2 4 4 4 l’agriculture, l’alimentation et Elena Neves , Samira Costa-Silva , Marzia Antonelli , Janaina Rocha Lorenço , 4 1 l’environnement (INRAE), France Cristiane K. M. Kolesnikovas and José Luiz Catão-Dias Reviewed by: 1 Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Hazem Ramadan, Sciences, University of São Paulo, São Paulo, Brazil, 2 Group of Epidemiology and Environmental Health, Animal Health Mansoura University, Egypt Research Centre (INIA-CISA), Madrid, Spain, 3 Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago, Chile, Getahun E. Agga, 4 Associação R3 Animal, Florianópolis, Brazil United States Department of Agriculture, United States Antimicrobial resistance genes (ARGs) are environmental pollutants and anthropization *Correspondence: Ana Carolina Ewbank indicators. We evaluated human interference in the marine ecosystem through the [email protected] ocurrence and quantification (real-time PCRs) of 21 plasmid-mediated ARGs in †These authors have contributed enema samples of 25 wild seabirds, upon admission into rehabilitation: kelp gull equally to this work and share first (Larus dominicanus, n = 14) and Magellanic penguin (Spheniscus magellanicus, authorship n = 11). Overall, higher resistance values were observed in kelp gulls (non-migratory Specialty section: coastal synanthropic) in comparison with Magellanic penguins (migratory pelagic This article was submitted to non-synanthropic). There were significant differences between species (respectively, kelp Veterinary Infectious Diseases, a section of the journal gull and Magellanic penguin): ARGs occurrence (blaTEM [p = 0.032]; tetM [p = 0.015]; Frontiers in Veterinary Science tetA[p = 0.003]; and sulII [p = 0.007]), mean number of ARGs per sample (p = 0.031), Received: 11 January 2021 ARGs mean load percentage (aadA [p = 0.045], tetA [p = 0.031], tetM [p = 0.016], Accepted: 24 February 2021 blaTEM [p = 0.032], sulII [p = 0.008]), percentage of genes conferring resistance to Published: 22 March 2021 an antimicrobial class (betalactams [p = 0.036] and sulfonamides [p = 0.033]), mean Citation: Ewbank AC, Esperón F, Sacristán C, number of genes conferring resistance to one or more antimicrobial classes (p = 0.024]), Sacristán I, Neves E, Costa-Silva S, percentage of multiresistant microbiomes (p = 0.032), and clustering (p = 0.006). These Antonelli M, Rocha Lorenço J, Kolesnikovas CKM and Catão-Dias JL differences are likely due to these species’ contrasting biology and ecology - key factors (2021) Occurrence and Quantification in the epidemiology of ARGs in seabirds. Additionally, this is the first report of mecA of Antimicrobial Resistance Genes in in seabirds in the Americas. Further studies are necessary to clarify the occurrence and the Gastrointestinal Microbiome of Two Wild Seabird Species With diversity of ARGs in seabirds, and their role as potential sources of infection and dispersal Contrasting Behaviors. within the One Health chain of ARGs. Front. Vet. Sci. 8:651781. doi: 10.3389/fvets.2021.651781 Keywords: anthropization, marine pollution, antibiotic resistance, wildlife, gull, penguin, One Health Frontiers in Veterinary Science | www.frontiersin.org 1 March 2021 | Volume 8 | Article 651781 Ewbank et al. Antimicrobial Resistance Genes in Seabirds INTRODUCTION METHODS Antimicrobial resistance is an issue of serious public Sample Collection health concern with global economic, social and political Fresh fecal samples were immediately obtained by enema (16) in implications affecting human and animal populations, as well 25 physically restrained birds (14 kelp gulls and 11 Magellanic as the environment (1–3). This worldwide phenomenon is penguins) upon admission at the wildlife rehabilitation center compromising our ability to treat infectious diseases, and (Associação R3 Animal, Florianópolis, Santa Catarina state, ◦ undermining or preventing advances in health and medicine southern Brazil), and stored at −20 C until analyses. All (4). Microbial resistance is the result of natural bacteria genetic birds included in the study came directly from their rescue plasticity and interactions between microbial agents, host sites (beach), and did not receive previous veterinary care organisms and the environment (1, 5), enhanced by the selective prior to their arrival at the center. Total DNA extraction pressure exerted by antimicrobial usage and over-prescription was carried out by a pressure filtration technique (QuickGene in human and veterinary medicine treatments, animal and fish DNA tissue kit S, Fujifilm, Tokyo, Japan), according with the production (i.e., growth promoters and prophylaxis), agriculture manufacturer’s instructions. The 16S rRNA gene was amplified and food technologies (1, 5, 6). The consequent remodeling of by real time PCR (rtPCR) in 10-fold dilutions of each extracted the existing microbiomes (group of all the genomic elements sample [(27, 28), Supplementary Materials] to verify adequate of a specific microbiota), associated with their dissemination concentration of bacterial DNA. A sample was considered capacity, confer antimicrobial resistance genes (ARGs) the role of validated when its 10-fold dilution showed a cycle threshold environmental pollutants (7, 8) and indicators of environmental (Ct) <25 (29). To normalize the study, ct was obtained anthropization (2, 9, 10). based on the fluorescence variation value [(F/C) = 0.02] Seabirds are long-lived, wide-ranging, and upper trophic level (30). Once validated, samples were analyzed by rtPCR for marine predators present in all marine ecosystems and oceans 21 selected ARGs encoding resistance to eight antimicrobial of the world, from coastline to pelagic and open seas (11). By classes: tetracyclines (tet(A), tet(B), tet(Y), tet(K), tet(M), tet(Q), acting as predators, scavengers and cross-ecosystem nutrient tet(S), and tet(W) (28), aminoglycosides [aadA (31) and str ancillaries, seabirds play important roles in the processes, (32)], sulfonamides (sulI, sulII), chloramphenicols [catI and function and resilience of island and marine ecosystems (12). catII (28)], macrolides [erm(B), erm(F) (33)], quinolones [qnrB Essentially, seabirds respond rapidly to environmental changes, (34) and qnrS (35)]; betalactams [blaTEM (31) and mecA and due to their behavior and population dynamics, are (36)], and polymyxins [mcr-1(30)] (Supplementary Materials). excellent sentinels of the marine ecosystem health, reflecting The estimation of the percentage of bacteria harboring ARGs natural and anthropogenic changes to the environment (13), (mean load percentage of each ARG), was based on the + − including pollution by ARGs (14–16). In seabirds, most ARGs formula % gene X = 10[2 0.33(ct16S ctgeneX)], with ct as the studies have focused on synanthropic species, due to their cycle threshold (16S rRNA regarding bacterial determination proximity to anthropized areas and feeding habits, and relied and X for each evaluated gene), and 0.33 as the mean slope on classic microbiological techniques (bacterial culture and for all the evaluated genes. Results were expressed in log10 sensitivity testing) (9, 17, 18). Nevertheless, recent studies have scale of the hypothetical percentage of bacteria presenting shown that biological and ecological factors (e.g., migration each gene, ranging from −8 (sample considered negative) and feeding niche) are also relevant to the issue of ARGs in to +2 (when 100% of the bacteria in the sample presented wild birds (16, 19, 20). Additionally, only a small fraction of the ARG) (30). The same thermal cycle was used for all ′ ◦ ′′ ◦ ′′ ◦ bacteria are cultivable (21, 22). Thus, in order to promote a rtPCR reactions [6 95 C, 40x (10 95 C, 30 60 C)], with ◦ more comprehensive approach, we employed highly sensitive alignment and extension in the same step, at constant 60 C. real time polymerase chain reaction (rtPCR) methods (10, 23) A melting curve step was performed at the end of the rtPCR to directly detect and quantify 21 selected plasmid-mediated reaction (30). As per (37), we applied the term “multiresistant ARGs in the gastrointestinal microbiome of two wild seabirds microbiome” when a fecal sample presented at least three species (kelp gulls [Larus dominicanus] and Magellanic penguins ARGs encoding resistance to different classes of antimicrobials [Spheniscus magellanicus]) upon admission to a rehabilitation (10, 29, 30). All samples used in this study were collected as center. The goals of this study were to (i) assess the presence part of the Santos Basin Beach Monitoring Project (Projeto and load of ARGs in these individuals and (ii) evaluate our de monitoramento de Praias da Bacia de Santos - PMP-BS), findings in light of selected biological and ecological parameters licensed by the Brazilian Institute of the Environment and (i.e., dispersal [migratory and non-migratory], feeding niche Renewable Natural Resources (IBAMA) of the Brazilian Ministry ◦ [coastal and pelagic], and interaction with human-impacted areas of Environment (ABIO N 640/2015), and in full compliance [synanthropic and non-synanthropic]). We hypothesized that with