ORIGINAL RESEARCH published: 28 July 2020 doi: 10.3389/fmicb.2020.01397 Foraging at Solid Urban Waste Disposal Sites as Risk Factor for Cephalosporin and Colistin Resistant Escherichia coli Carriage in White Storks (Ciconia ciconia) Edited by: Ursula Höfle 1* † , Juan Jose Gonzalez-Lopez 2 , Maria Cruz Camacho 1 , Marc Solà-Ginés 3 , Sebastian Guenther, Albert Moreno-Mingorance 2 , Jose Manuel Hernández 4 , Javier De la Puente 5 , University of Greifswald, Germany Javier Pineda-Pampliega 6 , José Ignacio Aguirre 6 , Fernando Torres-Medina 7,8 , Reviewed by: Antoni Ramis 3,9 , Natalia Majó 3,9 , Julio Blas 7 and Lourdes Migura-Garcia 3* † Jian-Hua Liu, 1 South China Agricultural University, SaBio (Health and Biotechnology) Working Group, Instituto de Investigación en Recursos Cinéticos IREC China (CSIC-UCLM-JCCM), Ciudad Real, Spain, 2 Servei de Microbiologia, Hospital Vall d’Hebron, Universitat Autònoma Dorina Timofte, de Barcelona, Barcelona, Spain, 3 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Universitat Autònoma de The University of Liverpool, Barcelona (UAB), Bellaterra, Spain, 4 Independent Researcher, Cañada de Calatrava, Spain, 5 Bird Monitoring Unit, United Kindgom SEO/BirdLife, Madrid, Spain, 6 Department of Biodiversity, Ecology and Evolution, Faculty of Biology, Complutense University of Madrid, Madrid, Spain, 7 Departamento de Biología de la Conservación, Estación Biológica de Doñana, Consejo Superior *Correspondence: de Investigaciones Científicas, Seville, Spain, 8 Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada, Ursula Höfle 9 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain ursula.hofle@uclm.es Lourdes Migura-Garcia lourdes.migura@irta.cat White stork (Ciconia ciconia) may act as a reservoir and vehicle of cephalosporin † These authors have contributed resistant (CR) Escherichia coli. Between 2011 and 2014, we sampled white storks equally to this work from colonies exposed to different degrees of anthropic pressure across the major Specialty section: areas of natural distribution of white storks in Spain. Cloacal swab samples (n = 467) This article was submitted to were obtained from individuals belonging to 12 different colonies from six different Antimicrobials, Resistance and Chemotherapy, regions. Additionally, 70 samples were collected from recently deposited droppings a section of the journal at the base of nesting platforms. We phenotypically characterized E. coli isolates, Frontiers in Microbiology confirmed presence of CR genes and classified plasmids. Risk factors for acquiring Received: 30 December 2019 Accepted: 29 May 2020 these genes were assessed. Overall, 8.8% (41 out of 467) storks carried CR E. coli Published: 28 July 2020 in their cloaca and five (7.1%) were identified from recently deposited droppings; Citation: therefore, 46 isolates were further characterized. Of them, 20 contained blaCTX−M−1 , Höfle U, Jose Gonzalez-Lopez J, nine blaCMY−2 , six blaCTX−M−14 , four blaSHV−12 , three blaCTX−M−15 , two blaCTX−M−32 , Camacho MC, Solà-Ginés M, Moreno-Mingorance A, one blaCTX−M−1 together with blaCMY−2 , and one blaCTX−M−1 together with blaSHV−12 . Manuel Hernández J, De La Puente J, All were multidrug-resistant, and four harbored the plasmid-mediated colistin resistance Pineda-Pampliega J, Aguirre JI, Torres-Medina F, Ramis A, Majó N, mcr-1 gene. CR genes were associated with the presence of IncI1, IncFIB, and IncN Blas J and Migura-Garcia L (2020) replicon families. XbaI-macrorestriction analysis revealed a great diversity among most of Foraging at Solid Urban Waste the XbaI-PFGE types, but indistinguishable types were also seen with isolates obtained Disposal Sites as Risk Factor for Cephalosporin and Colistin from different locations. Clonal complex 10 was the most common among CR E. coli Resistant Escherichia coli Carriage and two blaCTX−M−15 positive isolates were identified as B2-ST131. Carriage of CR in White Storks (Ciconia ciconia). Front. Microbiol. 11:1397. E. coli was significantly higher in colonies located close to solid urban waste disposal doi: 10.3389/fmicb.2020.01397 sites in which foraging on human waste was more likely and in one case to cattle grazing. Frontiers in Microbiology | www.frontiersin.org 1 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli The co-occurrence of blaCMY−2 and mcr-1 on plasmids of E. coli isolated from wild birds as early as 2011 is of note, as the earliest previous report of mcr-1 in wild birds is from 2016. Our study shows that foraging at landfills and in association with cattle grazing are important risk factors for the acquisition of CR E. coli in white storks. Keywords: Escherichia coli, virulence factors, white stork Ciconia, solid urban waste landfills, cephalosporin resistance INTRODUCTION they cohabit with humans, making use of manmade facilities such as roofs of buildings, telephone or electric power line poles, or Over the past years, the presence of antimicrobial resistant other constructions for their nests. If such individuals acquire bacteria in wildlife, particularly cephalosporin resistant (CR) CR E. coli during feeding, they can act as potential reservoirs of Escherichia coli has become a problem of increasing concern resistant bacteria (Liakopoulos et al., 2016a,b). in public health (Guenther et al., 2011). The genes encoding There have been several studies reporting detection of CR antimicrobial resistance are frequently located on plasmids, E. coli in wild birds (Veldman et al., 2013; Zurfluh et al., 2013; which may mediate the horizontal transfer of such genes to Báez et al., 2015) and Alcalá et al. (2016) described for the other bacteria (Carattoli, 2013; Dierikx et al., 2013). The overuse first time the presence of CR E. coli in a white stork in Spain. of antimicrobials in both human and veterinary medicine has However, to our knowledge this is the first study to analyze been considered one of the main factors contributing to the carriage of CR E. coli in white storks, in detail, in a large dissemination of antimicrobial resistant bacteria, increasing their number of colonies subjected to different degrees of anthropic detection in humans, food-producing animals, and food (Jouini pressure with the aim of determining the epidemiology of CR et al., 2007; Smet et al., 2010). Furthermore, antimicrobial E. coli in this species and their role as potential spreaders of residues from urban and livestock sources may persist over CR E. coli. For this purpose, the isolates have been extensively time in soil and aquatic environments (Kemper, 2008). As a characterized by antimicrobial susceptibility testing (AST), the consequence, the emergence of antimicrobial resistant bacteria identification of extended-spectrum β-lactamase (ESBL) and in the environment may be facilitated (Martínez, 2009), plasmid-mediated AmpC β-lactamase (pAmpC) genes, and representing a global threat of major concern for human, animal, the plasmids harboring these genes, phylotyping, genotypic and environmental health. relationships and the detection of virulence genes associated to Some studies have demonstrated the presence of biologically avian pathogenic E. coli (APEC). active antibiotic residues in animal and human waste, such as sewage and manure (Kummerer, 2009; Cycoń et al., 2019; Menz et al., 2019). In the case of wild birds, most of the studies have MATERIALS AND METHODS associated the influence of human activities such as farming, presence of dumpsites, or even tourism with the detection of Ethics Statement antibiotic resistant bacteria (Allen et al., 2010; Ahlstrom et al., Sampling of white storks was in all cases associated to ringing 2019). In addition, several reports have suggested the importance or radio-tagging activities. None of the storks were specifically of wildlife in the dissemination of CR E. coli (Pinto et al., 2010; captured or handled for the purpose of this study. All ringing Veldman et al., 2013; Báez et al., 2015). Since wild birds in their and radio-tagging activities were carried out under the pertinent natural environment are not treated with antibiotics (Santos et al., permits from the local authorities (regional governments of 2013), they are potential sentinels of multidrug resistant bacteria Castilla – La Mancha, Madrid, Extremadura, Castilla y Leon, and discharged into the environment. Andalucía) and by registered specialized personnel. Handling and The role of wild birds as disseminators of antibiotic-resistant sampling of the storks was carried out following all applicable bacteria between distant ecosystems is difficult to estimate (Báez international, national, and/or institutional guidelines for the et al., 2015; Ahlstrom et al., 2019). White storks (Ciconia care and ethical use of animals, specifically directive 2010/63/EU ciconia), like many other free-living migratory birds, can become and Spanish laws 9/2003 and 32/2007, and Royal decrees long-distance vectors of CR E. coli. They feed nearby pastures 178/2004 and 1201/2005. and plowed fields, marshy wetlands, rice fields, and more recently on solid human waste disposal sites, and display long-distance Field Sampling movement patterns within and between continents (Flack et al., The study area covered most of the natural distribution of white 2016; Bécares et al., 2019). Due to their mobility and contact storks in Spain. We collected cloacal swab samples (n = 467) from with fecal contamination in pastures and surface waters, they may storks from 12 different colonies located in five different regions. effectively acquire and spread disease and antimicrobial-resistant Sampling occurred more intensively and in consecutive years in bacteria (Szczepanska et al., 2015) and become a potential source colonies situated in South-central Spain. Hence, six colonies in of CR E. coli for humans and farm animals (Tryjanowski et al., South-central Spain were sampled in 2013 and 2014. From two 2006; Keller et al., 2011). White storks may breed in open colonies, samples were obtained only in 2013 and four additional farmland with access to marshy wetlands but also, very often, colonies were sampled only in 2014 (Figure 1 and Table 1). Frontiers in Microbiology | www.frontiersin.org 2 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli FIGURE 1 | Distribution of studied colonies and the closest solid urban waste landfills in Spain. The size of the circles reflect sample size and the lighter shaded areas reflect CR E. coli prevalence. The shaded solid urban waste landfill silhouette shows a landfill that was sealed in 2005. In addition, recently deposited droppings were recovered at the Samples included, on one hand recently deposited droppings base of the nesting platforms in three of the six colonies in collected below nesting platforms in three colonies in South-central Spain in 2011. South-central Spain (n = 70) in March 2011 (just prior to To take specific aspects of the feeding and spatial ecology egg-laying), thus presumably belonging to the adults breeding of white storks into account, samples were obtained during on each particular platform. On the other hand, sampling the reproductive season (approximately February–July), when comprised cloacal swab samples from adult and nestling white white storks are spatially bound to their colony/nest. During storks collected during the breeding period (May–July) in 2013 this period, foraging occurs mostly in a spatially defined area (n = 226) and 2014 (n = 241). and can be linked fairly clearly to a specific type of habitat/food The largest part of the cloacal swab samples (one per source, allowing for the comparison of the effect of feeding on bird n = 441) was collected from chicks during the ringing natural and/or human origin (waste) resources (Alonso et al., activities performed at their nests (nestlings). This allowed for 1991; Tortosa et al., 2002). In contrast, during the migration and the collection of a considerable number of samples without the wintering periods, the spatial distribution of foraging increases, need of stressful, costly and time-consuming capture. In addition, as well as the intensity of use of human waste disposal sites as a cloacal swab samples were obtained from 26 breeding adults predictable food source, making association of storks to a specific captured for satellite/gps data logger transmitter fitting, in their habitat more difficult (Ciach and Kruszyk, 2010; Bécares et al., nests (the presence of small chicks at their nest was certified), 2019). The continuous predictable availability of food on landfills meaning that the samples were obtained in a period were foraging has a profound effect on the migratory patterns of several avian was also concentrated in a defined type of habitat. Tarsus length species including white storks (Plaza and Lambertucci, 2017). and body weight were recorded in all white storks in order to While juvenile white storks and some adult birds still migrate enable calculation of a scaled mass index (SMI), subsequently to Africa, the majority have shortened their migratory distance used as a proxy for body condition. considerably. As a result, many Spanish white storks winter in Recently deposited droppings were collected using sterile Spain on, or close to, landfills and are joined by birds from central cotton tip swabs and small zip-lock bags. The samples were and northern Europe, that either are on stopover on their route kept at 4◦ C until arrival at the laboratory and divided into a toward Africa, or remain in Spain for the winter (Gilbert et al., subsample for storage at −80◦ C and a sample for immediate 2016; Bécares et al., 2019). processing. Cloacal swab samples were obtained using sterile Frontiers in Microbiology | www.frontiersin.org 3 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli TABLE 1 | Location, distance to closest human waste disposal site, presence of livestock, and type, and number of samples and presence of CR E. coli obtained from the different white stork colonies. Colony designation Province Distance to Live-stock Year Age Number of CR E. coli human waste samples disposal site (km) Abenójar Ciudad Real 60.95 Extensive sheep 2011 Droppings 23 0 (0%) 2013 Nestling 28 0 (0%) Adult 2 0 (0%) 2014 Nestling 29 0 (0%) Almodóvar Ciudad Real 50.58 Extensive sheep 2011 Droppings 10 0 (0%) (formerly 1 km) 2013 Nestling 30 2 (6.7%) Adult 2 0 (0%) 2014 Nestling 30 3 (10%) Alcázar de San Juan Ciudad Real 0.77 Extensive sheep 2011 Droppings 37 5 (13.5%) 2013 Nestling 8 0 (0%) Adult 3 0 (0%) 2014 Nestling 13 1 (7.7%) Cabañeros Ciudad Real 64.91 No 2013 Nestling 15 0 (0%) Adult 2 0 (0%) 2014 Nestling 19 0 (0%) Alcalá de Henares Madrid 2.609 No 2014 Nestling 14 0 (0%) Pinilla del Valle Madrid 30.417 2013 Nestling 31 0 (0%) Extensive cattle Adult 2 0 (0%) 2014 Nestling 29 0 (0%) Prado Herrero Madrid 11.425 Cattle (breeder) 2013 Nestling 30 6 (20%) 2014 Nestling 57 8 (14%) Perales del Rio Madrid 6.343 No 2014 Nestling 20 2 (10%) Malpartida Cáceres 6.547 Extensive sheep 2013 Nestling 43 16 (37.2%) Adult 1 0 (0%) Doñana, Dehesa de Abajo Sevilla 33.203 Extensive cattle 2014 Nestling 18 1 (5.6%) Adult 7 2 (28.6%) Doñana, Matasgordas Huelva 40.370 Extensive cattle 2014 Adult 6 0 (0%) León León 38.279 Extensive cattle 2013 Nestling 28 0 (0%) Adult 1 0 (0%) cotton swabs in AMIES transport medium (Deltalab, Barcelona, characterization in the present study and confirmed as E. coli by Spain). They were kept at 4◦ C until arrival to the laboratory, PCR (Heininger et al., 1999). where they were processed in less than 12 h after sampling. Samples were plated onto MacConkey agar (Oxoid, Basingstoke, Antimicrobial Susceptibility Testing United Kingdom) supplemented with 4 mg/L cefotaxime The combined disc method was used for the confirmatory tests (Sigma-Aldrich Chemical, Madrid, Spain), and incubated for ESBL-producing E. coli (CLSI, 2020). Therefore, the following overnight at 37◦ C. Lactose positive colonies morphologically discs were used: cefotaxime, 30 µg, cefotaxime + clavulanic acid, compatible with E. coli were considered indicative of growth 30 + 10 µg; ceftazidime, 30 µg; ceftazidime + clavulanic acid, above breakpoint concentrations of cefotaxime based on 30 + 10 µg (Oxoid, Basingstoke, United Kingdom). For the the Clinical and Laboratory Standards Institute (CLSI) identification of pAmpC-producing E. coli, the cefoxitin disc document, describing clinical resistance for cefotaxime ≥4 (30 µg) was used. Additionally, the cefepime disc (30 µg) was (CLSI, 2020). From these plates, three lactose-positive colonies also used. Moreover, all isolates were subjected to susceptibility were stored at −80◦ C in 30% glycerol BHI (Brain-Heart testing by broth microdilution (VetMIC GN-mo, National Infusion broth, Scharlau Microbiología, Barcelona, Spain). Veterinary Institute, Uppsala, Sweden) to the following Subsequently, one representative colony was selected for further antimicrobial agents: ampicillin (1–128 mg/L), cefotaxime Frontiers in Microbiology | www.frontiersin.org 4 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli (0.016–2 mg/L), ceftazidime (0.25–16 mg/L), nalidixic acid The obtained sequences were submitted to Enterobase3 to obtain (1–128 mg/L), ciprofloxacin (0.008–1 mg/L), gentamicin new allele/STs designation. (0.12–16 mg/L), streptomycin (2–256 mg/L), kanamycin (8–16 mg/L), chloramphenicol (2–64 mg/L), florfenicol Detection of Virulence-Associated Genes (4–32 mg/L), trimethoprim (1–128 mg/L), sulfamethoxazole All 46 isolates were tested by multiplex PCR for the genes (8–1,024 mg/L), tetracycline (1–128 mg/L), and colistin described previously by Johnson et al. (2008) as the minimal (0.5–4 mg/L). The E. coli ATCC 25922 was used as a control predictors of APEC virulence; iroN, ompT, hlyF, iutA, and strain. Isolates were considered to be wild-type or non-wild-type iss (Figure 2). based on epidemiological cut-off (ECOFF) values defined by EUCAST1 . ECOFF separates wild-type populations from isolates Mating Experiments and Plasmid that have developed reduced susceptibility for the antimicrobial and may differ from clinical breakpoints used for clinical settings Characterization (EFSA, 2017). Filter mating experiments were carried out to assess the transferability of ESBL-encoding plasmids by conjugation. For Antimicrobial Resistance Genes the analysis, one representative isolate from each PFGE-type All cefotaxime resistant (CR) isolates were tested by PCR was selected. Mating assays were performed as previously methods for the presence of the blaCTX−M , blaSHV , blaCMY−1 , described (Bielak et al., 2011), using the isolates as donors and and blaCMY−2 genes as previously described by Hasman et al. rifampicin-resistant E. coli HB101 as a recipient. Transconjugants (2005). The isolates showing resistance to colistin were also tested were selected on LB agar plates supplemented with rifampicin for the presence of mcr genes (Rebelo et al., 2018). Sequencing of (150 mg/L) and ceftriaxone (1 mg/L). The identity of the both strands of amplicons was performed. transconjugants was confirmed by PFGE and the presence of the corresponding CR gene was confirmed by PCR. Plasmid DNA was purified from the subset of 42 isolates Phylogeny, Macrorestriction Analysis (one isolate as representative of PFGE-type) using a Qiagen and Multi Locus Sequence Typing Plasmid Midi kit (Qiagen, Hilden, Germany) according to The isolates were classified in phylogenetic groups (A, B1, B2, C, the manufacturer’s recommendations. Plasmids were introduced D, E, or F) by PCR, according to a method previously described to electrocompetent plasmid free E. coli BL21 cells by by Clermont et al. (2000, 2013). electroporation. Transformants were selected in LB agar plates Genomic DNA macrorestriction analysis by pulsed-field containing ceftriaxone (1 mg/L), and PCR assays were performed gel electrophoresis (PFGE) was performed to determine the to confirm the presence of ESBL and/or pAmpC genes. genomic relatedness among the isolates. The experiments The presence of a unique plasmid in the transformants and were carried out as described in the PulseNet protocol their corresponding sizes were analyzed using S1 nuclease (Ribot et al., 2006). Salmonella enterica serovar Branderup digestion followed by PFGE (S1-PFGE) (Barton et al., 1995). H9812 was used as a size marker. The results were analyzed Additionally, plasmids were classified by PCR-based replicon by Fingerprinting II Informatix software (Applied Maths, typing (Carattoli et al., 2005). Sint-Martens-Latem, Belgium). Isolates were considered to have a different PFGE-type when changes in at least one band were detected after digestion with XbaI. The analysis Statistical Analysis As a proxy to the degree of exposure of storks to human of the PFGE-types was carried out using the Dice similarity waste foraged food, we used the distance of each colony to the coefficient and the unweighted-pair group method with nearest human waste disposal site. This is based on the reduced arithmetic averages (optimization of 1.25% and position home range of storks during chick raising and incubation and tolerance of 1.25%). the assumption that storks are less likely to undertake long Multi locus sequence typing (MLST) was performed according foraging flights during this period as compared to other stages. to the protocol and primers specified on the E. coli MLST website2 This hypothesis is supported by satellite transmitter data from (Wirth et al., 2006). Sequences were analyzed using Vector NTI radio-tagged adult storks from colonies in South-central Spain advance 11 (InforMax, Inc., Bethesda, MD, United States). The that show distinct foraging either in natural habitat or at human allelic profile and the sequence types (STs) were obtained through waste disposal sites (Bécares et al., 2019). In addition, the the electronic database at the E. coli MLST website. distance between a stork’s nest and solid human waste landfills Whole-genome sequencing (WGS) was performed with has been shown to correlate with the frequency and intensity E. coli isolates which revealed a new allele sequence or new with which the storks forage on waste (Djerdali et al., 2016; allele combination after the Sanger sequencing. WGS was Gilbert et al., 2016). performed on a MiSeq Instrument (Illumina, San Diego, CA, For this, the coordinates for each colony/nest sampled, in United States) using the Nextera DNA Flex Library Prep Kit parallel to the coordinates of all known human waste disposal (Illumina) and paired-end sequenced with a MiSeq Reagent sites, were projected onto a scaled flat reference system in kit v3 (Illumina) with an average insertion size of 300 bp. the geographic information system QGIS (QGIS desktop 2.8.1), 1 http://www.eucast.org/ 2 3 http://mlst.warwick.ac.uk/mlst/dbs/Ecoli http://enterobase.warwick.ac.uk/ Frontiers in Microbiology | www.frontiersin.org 5 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli FIGURE 2 | Phenotypic and genotypic characteristics of the cephalosporin-resistant E. coli isolates: dendrogram showing the genotypic relationships of the different XbaI-types of the isolates. Further characteristics are also shown: year of isolation phylogroup (Phyl. U, unidentified phylogroup), virulence-associated genes (VG) and cephalosporin and colistin resistance genes (CR), sequence type (ST); clonal complex (CC) and sampling location. The antimicrobial agents tested are also displayed and the black boxes below their abbreviated designations are indications for a non-wild-type phenotype. Amp, ampicillin (WT ≤ 8 mg/L); Ctx, cefotaxime (WT ≤ 0.25 mg/L); Caz, ceftazidime (WT ≤ 0.5 mg/L); Fox, cefoxitin (WT ≤ 8 mg/L); Fep, cefepime (WT ≤ 0.125 mg/L); Nal, nalidixic acid (WT ≤ 16 mg/L); Cip, ciprofloxacin (WT ≤ 0.064 mg/L); Gen, gentamicin (WT ≤ 2 mg/L); Str, streptomycin (WT ≤ 16 mg/L); Kan, kanamycin (WT ≤ 8 mg/L); Chl, chloramphenicol (WT ≤ 16 mg/L); Ff, florfenicol (WT ≤ 16 mg/L); Tm, trimethoprim (WT ≤ 2 mg/L); Sul, sulfamethoxazole (WT ≤ 64 mg/L); Tet, tetracycline (WT ≤ 8 mg/L); Col, colistin (WT ≤ 2 mg/L). and the direct flight distance to the nearest human waste disposal within the colony as random factor, to determine the effect of site (known to be used by storks) was calculated. sampling year, sex, number of siblings in the nest, presence of We calculated the body condition of white stork nestlings livestock (absence, extensive small ruminant, and cattle grazing) and adults sampled in the field according to the SMI proposed and the distance to human waste disposal sites on the prevalence by Peig and Green (2009). This index can be computed as: of CR E. coli, in white stork nestlings. All analyses were carried M = Mi (L0 /Li )bsma , where Mi and Li are the body mass out using SPSS statistical software, version 24.0 (IBM , SPSS Inc., R and the structural size measurement (tarsus length) of each Chicago, IL, United States) and significance was set at p ≤ 0.05. individual respectively; bsma is the scaling exponent estimated by the standardized major axis (SMA); regression of M on L, L0 00 is the arithmetic mean value for the study population; M RESULTS is the predicted body mass for individual i when the structural size body measure is standardized to L0 . The scaling exponent Prevalence of CR E. coli, AST and “bsma” has been calculated indirectly by dividing the slope Resistance Genes from an ordinary least squares (OLS) regression (bols) by the Overall CR E. coli prevalence in cloacal swab samples was 8.8% Pearson’s correlation coefficient r. Normal distribution of the (41 out of 467). Five (7.1%) of the isolates from recently deposited continuous variable body condition index M was confirmed droppings (n = 70) were identified as CR E. coli, but, in contrast using the Kolmogórov–Smirnov test prior to further analysis. We to cloacal swab samples, we cannot unequivocally associate the compared body condition between ESBL and APEC “infected” droppings to specific individuals. For this reason, statistical and “uninfected” nestlings using Student’s t-test and the effect analysis concerning CR E. coli was only carried out on results of distance of colonies to solid human waste landfills on SMI from cloacal swab samples. as a proxy of body condition using a one-way ANOVA. We Extended-spectrum β-lactamase and/or presumptive pAmpC employed a generalized linear mixed model (GLMM) with a phenotypes were confirmed for all 46 CR isolates. These 46 binary response and a logit regression, and the nest of origin isolates were also multidrug-resistant (resistant, according to Frontiers in Microbiology | www.frontiersin.org 6 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli ECOFFs, to at least three antimicrobial families). Furthermore, ST10 was the most prevalent type (6 isolates; 13%), followed 54% (25/46) exhibited a non-wild-type phenotype to more by ST155, ST602, and ST2309 (3 isolates; 6.5%, each) and than seven antimicrobial classes. MIC values determined by ST48, ST93, ST131, ST457, and ST1844 (2 isolates; 4.3%, that all isolates exhibited a non-wild-type phenotype to each). ST8877 was firstly identified in this study. Grouping cephalosporins (100% resistance to cefotaxime and 96% STs by clonal complexes (CC) showed that CC10 was the to ceftazidime), with 20 isolates yielding amplicons for most prevalent including 22% of the isolates, followed by blaCTX−M−1 , nine blaCMY−2 , six blaCTX−M−14 , four blaSHV−12 , CC38, CC155, CC168, and CC446 in the 3% of the population three blaCTX−M−15 , two blaCTX−M−32 , one blaCTX−M−1 respectively (Figure 2). together with blaCMY−2 , and one blaCTX−M−1 together with blaSHV−12 . Additionally, 100% of the isolates were Detection of Virulence Genes Associated non-wild-type to ampicillin, 85% to sulfamethoxazole, 80% to ciprofloxacin and nalidixic acid, 67% to tetracycline, 65% to APEC to trimethoprim, 54% to streptomycin, 33% to gentamicin, Out of 46 isolates, 70, 70, 70, 57, and 52% yielded amplicons 22% to kanamycin, 9% to chloramphenicol, and 7% to for ompT, hlyF, iutA, iss, and iroN, respectively. Accordingly, 25 florfenicol (Figure 2). isolates (54%) were considered APEC, since they had four to five In addition, four isolates from recently deposited droppings of the genes mentioned above (Figure 2). collected at nests located at the colony closest to a landfill (Alcázar de San Juan, Table 1) were phenotypically resistant to colistin Conjugation and Transformation (MIC ≥ 4) and were confirmed to carry the mcr-1 gene (id. 173, Experiments 176, 178, and 184). Among the 46 CR isolates, a subset of 42 representing each XbaI-macrorestriction profile were selected for conjugation and Phylogeny, PFGE, and MLST transformation experiments. Of them, 15 were able to transfer The phylotyping analysis identified six different phylogenetic the CR genes by conjugation. Additionally, 33 transferred the groups among the 46 isolates (Figure 2). Of them, 14 belonged CR genes to the BL21 electrocompetent strain. S1-nuclease to phylogroup B1 (30%), 12 to group A (26%), eight to group E PFGE confirmed the presence of a unique plasmid in all (17%), four to group B2 (9%), four to group F (9%), and one to selected isolates except in two of them (518 and 598), which group C (2%). No phylogroup could be determined for three of contained more than one plasmid. Sizes of plasmids varied the isolates (7%). between 40 and 150 kb approximately (Table 2). PCR-based Among the 46 CR E. coli isolates, 42 different replicon typing showed that IncI1 was the most common macro-restriction profiles were detected. However, during replicon and was associated to blaCTX−M−1 , blaCTX−M−14 , 2013, a cluster of isolates sharing a similar PFGE profile (100% blaCMY−2 , and blaSHV−12 genes. IncI1 was present in 25 of similarity) and identical MLST type (ST2309) was identified. the isolates, followed by IncFIB and IncN. The replicons These isolates (506, 626, and 635) were obtained from two IncFIA, IncFIC, IncA/C, IncK, and IncL/M were also detected different colonies located within a radius of about 250 km, in (Table 2). Four isolates presented between two and five Madrid and Cáceres. replicons on the same plasmid (517, 631, 176, and 184), The MLST analysis also revealed a high degree of diversity. and none of the tested replicons were detected in seven Overall, 30 different STs were found among the 46 isolates of the transformants or transconjugants (434, 514, 554, 508, (mean of number of isolates per ST, 1.53; range, 1–6) (Figure 2). 173, 626, and 634). TABLE 2 | Characteristics of plasmids harboring ESBL and/or pAmpC genes from 42 cephalosporin-resistant E. coli isolates. Gene and isolate Conjugation Transformation Inc type(s) Plasmid result result size (kb) I1 FIB N K FIA FIC A/C L/M blaCTX−M−1 516 TC1b + 100 524 TC3a + 90 530.13 TF4a + 60 586 TC5a TF5a + 100 598 – – 597 TC7a TF7a + 100 610 TF9a + 100 615 TF10b + 90 619 TF11a + 100 627 TF12a + 100 (Continued) Frontiers in Microbiology | www.frontiersin.org 7 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli TABLE 2 | Continued Gene and isolate Conjugation Transformation Inc type(s) Plasmid result result size (kb) I1 FIB N K FIA FIC A/C L/M 706 TF14a + 100 433 TF15a + 110 434 TC16b TF16a 110 495 TC17a TF17a + 50 514 TF18a 60 520 TF19a + 100 522 TF38a + 110 530.14 TF20a + 40 554 TC21a TF21a 50 blaCTX−M−32 517 TF2a + + + 130 593 TF6a + 100 blaCTX−M−14 626 TF30a 110 547 TF31a + 110 582 TC32a TF32a + 80 584 TC33a TF33a + 90 blaCTX−M−15 631 TF13a + + 150 634 TC39b 60 518 – – blaCMY−2 599 TF22a + 120 508 TF23a 100 565 TC24a TF24a + 80 173 TF25a 90 176 TF26a + + + + + 90 178 TF27a + 90 184 TC28b + + + 40 215 TF29b + 40 blaSHV−12 587 TC34a + 90 600 TC35c + 90 605 TF36a + 100 606 TF8b + 100 *blaCTX−M−1 + blaCMY−2 519 TF37a + 100 *blaCTX−M−1 + blaSHV−12 465 TC40a + 110 Transconjugants (TC) and transformants (TF) used for PCR-based replicon typing method are in boldface. Two isolates (518 and 598) presented more than one plasmid in the TC and/or TF isolates), therefore they are not included in the table. ∗ Only blaCTX−M−1 was transferred to the transformant and transconjugant isolate, respectively. Risk Factors for the Acquisition of CR the prevalence of CR E. coli [GLMM F(1,359) = 8.641, p = 0.004], E. coli by White Storks with nestlings from colonies close to landfills being significantly Cephalosporin resistant E. coli prevalence did not vary more likely to carry CR E. coli (Figure 1 and Table 3). Carriage of significantly between nestling (8.8%, 39 out of 441) and CR E. coli [Student’s t-test F(1,359) = 2.946, p = 0.258] or APEC adult storks (7.7%, 2 out of 26). Sampling year [F(1,359) = 2.538, [Student’s t-test F(1,359) = 1.921, p = 0.164] had no significant p = 0.112], Sex [F(1,359) = 0.517, p = 0.473], number of siblings effect on the body condition of white stork nestlings. However, in the nest [F(1,359) = 1.645, p = 0.179], and livestock grazing white stork nestlings from colonies close to human waste disposal close to the colony [F(2,359) = 1.78, p = 0.17] did not affect CR sites were in significantly better body condition than nestlings E. coli carriage in white stork nestlings. However, distance of the from colonies far from human waste disposal sites [ANOVA colony to human waste disposal sites had a significant effect on F(7,341) = 8.469, p < 0.001]. Frontiers in Microbiology | www.frontiersin.org 8 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli TABLE 3 | Results of generalized mixed linear model (GLMM) for cephalosporin resistant (CR) E. coli carriage in white stork nestlings in Spain. Model Predictor Test model Description F Standard t p effects Error CR E. coli Livestock presence 0.304 None 0.468 1.121 0.418 0.677 Ext sheep −1.08 0.758 –1.424 0.155 Ext cattle 0 – – – Sex 0.170 F 0.319 0.444 0.719 0.473 M 0 – – – Sampling year 0.112 2013 −0.921 0.578 –1.593 0.112 2014 0 – – – Distance landfill (m) 0.004 Continuous 0.000 0.000 2.909 0.004 Number of siblings in nest 0.179 Single chick 1.729 0.921 1.877 0.061 Two siblings 1.54 0.775 1.988 0.048 Three siblings 0.816 0.661 1.234 0.218 Four siblings 0 – – – Statistically significant results are highlighted in bold. DISCUSSION (Table 1 and Figure 1). According to our results, extensive livestock farming in natural habitats (present in the proximity White storks are free-living colonial birds that frequently use of at least five of six natural habitat colonies) appears to be humanized habitats. In the recent past, they have adapted to less of a risk factor for the acquisition of CR E. coli by white foraging at solid urban waste disposal sites and introduced storks. However, nestlings from one colony located on a farm American crayfish (Procambarus clarkii) in rice fields and small that raises cattle and where storks and cattle share the pastures, water bodies as predictable food sources during winter. This has also situated at a flight distance of 11 km to the nearest landfill led to a significant modification of migratory behavior of a large (Prado Herrero, Table 1), most commonly carried CR E. coli part of the adult stork population of Western Europe as well as, with the blaCTX−M−1 gene, which is commonly described in in the case of solid urban waste disposal sites, to the exposure cattle in Europe (Schmid et al., 2013). Storks frequently collect to numerous contaminants and pathogens (Flack et al., 2016; cattle dung and place it in the nest just prior to, and a few Gilbert et al., 2016). weeks after hatching, presumably to help chicks maintain body As storks cohabit in the proximities of humans and food temperature (Tortosa and Villafuerte, 1999). This behavior as well producing animals, they are able to acquire bacteria from these as foraging of the adults on contaminated pastures (especially on and transport them to other locations in their annual or regular dung beetles) is a likely route of exposure of the nestlings to CR movements (Tryjanowski et al., 2006). The present study takes E. coli excreted by cattle. advantage of the fact that even though during winter most Some studies suggest that wildlife also play an important storks forage on human waste; during breeding the nesting site role in the global dissemination of antimicrobial resistance and colony determine the foraging ecology and allow for the genes, transferring mobile genetic elements between bacterial characterization of risk factors for the exposure of white storks communities (Allen et al., 2010). In our study, the presence of to CR E. coli (Alonso et al., 1991; Tortosa et al., 2002; Bécares multidrug-resistant E. coli demonstrated the potential capacity et al., 2019). Our results suggest that the stork nestlings more of white stork to disseminate and transport ESBL and/or AmpC likely fed human waste by their parents (using distance to rubbish encoding genes located in conjugative plasmids. These isolates dump as a proxy) were more likely to carry CR E. coli. CR E. coli were all resistant to third generation cephalosporins and most carriage did not affect body condition in “infected” nestlings. of them were also resistant to fluoroquinolones. These two Hence, the present study has demonstrated the presence of CR families of antimicrobials are nominated of critical importance E. coli in white storks feeding on human waste and their capacity in human health since they are widely used for the treatment of to disseminate and transport the resistant bacteria. In particular, human infections by Gram-negative bacteria (European Centre similar clones with the same resistance genes have been detected for Disease Prevention and Control [ECDC], 2018). In addition, in nestlings from colonies in Madrid and Caceres, located within four of the isolates, obtained in 2011 from recently deposited a 250 km distance. These results suggest a common source of droppings, co-harbored blaCMY−2 and mcr-1, although on contamination, together with the capacity of the birds to spread different plasmids. They were also resistant to multiple antibiotics the same clones during their movements. In this context, a including third-generation cephalosporins, fluoroquinolones, colony that is now at a more than 50 km flight distance from colistin, and aminoglycosides, among others, making these the closest human waste disposal site, was formerly situated isolates resistant to almost all last-line antimicrobials available. less than 1 km from a different disposal site that was sealed in Interestingly, all mcr-1 carrying isolates presented different PFGE 2005, but satellite transmitter data from two adults evidenced profiles and belonged to the CC10, two were ST10, one was ST48 occasional visits to this landfill even during the breeding season and one was ST1286. This high risk CC has previously been Frontiers in Microbiology | www.frontiersin.org 9 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli reported as the most commonly associated with mcr-1 globally, waste cannot be excluded completely. In fact satellite transmitter not only in humans but also in animals and in environmental data from an adult stork from this colony shows extensive use samples (Caltagirone et al., 2017; Matamoros et al., 2017; García- of solid urban waste disposal sites outside the breeding period Meniño et al., 2018; Lalaoui et al., 2019). All STs found within (Bécares et al., 2019). this study, except for ST8877, have previously been identified in Significant differences were found between location of the third-generation CR isolates of animal origin (Dierikx et al., 2013; colonies and presence of CR genes. For instance, in the colony Solà-Ginés et al., 2015; Alcalá et al., 2016; Batalha de Jesus et al., located in Caceres, the most prevalent gene was blaCTX−M−1 2019). Of note, CR E. coli CC10 has been reported as one of the associated to 90 and 100 kb plasmids, whereas in the colony most prevalent lineages on farms, food, and waste water plants, located in Alcázar de San Juan (Ciudad Real) the most prevalent and the second most abundant in humans in Spain (Ojer-Usoz was blaCMY−2 harbored in two types of plasmids (40 and 90 kb). et al., 2017). Additionally, ST131, ST69, and ST10 are the most The former was also the colony in which nestlings carried the prevalent ExPEC lineages found in humans (Manges et al., 2019), greatest variety of CR genes (5) as compared to the other colonies. and all of them have also been identified in this study. This fact The most common replicons found in the study were IncI1 also highlights the importance of wildlife as potential reservoirs, (60%), followed by IncFIB (12%), and IncN (10%). To date, not only of antimicrobial resistance genes, but also of human these three replicons are the most commonly found in plasmids pathogenic bacteria belonging to high-risk clones. carrying CR genes in E. coli isolated from poultry (Wang et al., Several studies have suggested a relationship between the 2013; Solà-Ginés et al., 2015). Almost all of the CR genes different E. coli phylogroups and the virulence of the isolates containing IncI1 replicons were located in plasmids of the same (Picard et al., 1999; Clermont et al., 2011). Accordingly, sizes, between 90 and 110 kb; specifically, 100 kb in the case non-virulent isolates mainly belong to A, B1, and C phylogenetic of blaCTX−M−1 . However, in the case of two isolates, isolation groups, whereas the most virulent phylogroups are associated to of the plasmid carrying the CR gene could not be achieved. B2 followed by D (Russo and Johnson, 2000). In this study, most During transformation and conjugation experiments, co-transfer of the isolates belonged to phylogroups A and B1, and only four of plasmids of different molecular weights was observed. The isolates were B2. On the contrary, analyzing phylogroups and entry of two or more plasmids into a single transformation virulence genes in this strain collection, we have observed that procedure is not uncommon; Weston et al. (1979) demonstrated almost all B1 isolates had the APEC virulence-associated genes, that 20% of the transformants could be double transformants whereas only one B2 strain had the APEC genes. This may be due (Weston et al., 1979). This type of transformation can be to the presence of virulence factors associated to mobile genetic frequent if it is performed with high amounts of plasmid DNA elements such as in the case of the previously described ColV (Goldsmith et al., 2007). plasmid (Johnson and Nolan, 2009), which contained several virulence genes within the same plasmid (hlyF, ompT, iss, and cvaC). Therefore, the acquisition of such type of plasmid may CONCLUSION confer B1 isolates with more virulence factors than expected. Carriage of E. coli isolates harboring APEC-associated genes in In conclusion, results from this study confirm that exposure the cloaca of white stork nestlings was apparently not related to of white storks to human waste constitutes a risk factor for solid urban waste disposal site origin of food or any of the other the acquisition of CR genes as the carriage of CR E. coli factors; neither did it affect body condition. was significantly higher in colonies located close to solid The present study showed a high variability of CR genes urban waste disposal sites. As all individuals included in this among the isolates, blaCTX−M−1 being the most prevalent study are free-living birds, they have not been treated with (48%), followed by blaCMY−2 and blaCTX−M−14 (20 and 13%, antimicrobials. Since they are not subjected to any direct respectively). These blaCTX−M types are the most frequent antibiotic pressure and they cohabit in the proximities of humans, variants in ESBL producers in animals and food of animal the presence of these resistance traits is a reflection of the origin, while blaCTX−M−15 has been mostly described in human anthropic pressure of human activities and particularly exposure medicine (Valverde et al., 2015) and occasionally on farms to human waste and farming practices. In fact, outside the (Cameron-Veas et al., 2015). This study has demonstrated breeding season adult and juvenile storks have been observed the presence of the blaCTX−M−15 gene in three isolates of to use human waste disposal sites indistinctive of the habitat white stork harbored in plasmids of different sizes and in one of the colony of origin (Bécares et al., 2019). The distinctive occasion contained in an IncFIB replicon, the most common pattern of prevalence of CR genes in white stork nestlings incompatibility group for blaCTX−M−15 isolated from humans in association to foraging behavior of their parents during in Spain (Carattoli, 2009). Additionally, the remaining two the breeding season, in addition to recent findings that show blaCTX−M−15 isolates were B2-ST131. They were obtained from that a large proportion of storks are exposed to human waste two different colonies, one of which is closely associated to a from solid urban waste landfills during wintering, suggests village with part of the nests on urban structures, and confirmed that persistence of CR genes in bacteria of the digestive consumption of human residues. The other colony is in a natural tract of white storks is relatively short. This makes white environment with no confirmed consumption of human residues storks excellent sentinels for local antibiotic pressure in their during the breeding season, nevertheless this colony is situated at foraging habitat. Nevertheless, due to their great mobility and 30.4 km flight distance from Madrid, and thus exposure to human migratory habits, the white stork can also act as carriers of CR Frontiers in Microbiology | www.frontiersin.org 10 July 2020 | Volume 11 | Article 1397 Höfle et al. Storks, Waste and CR E. coli E. coli, contributing to the global dissemination of antimicrobial The VHIR-HUVH was supported by Plan Nacional de I+D+i resistance genes and plasmids. 2013–2016, Instituto de Salud Carlos III and the Spanish Ministry of Health (FIS PI15/00604) and the Subdirección General de Redes y Centros de Investigación Cooperativa, DATA AVAILABILITY STATEMENT MINECO, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0003) – co-financed by European The raw data supporting the conclusions of this article will be Development Regional Fund “A way to achieve Europe. made available by the authors, without undue reservation, to any AM-M is a Ph.D. student registered with the Universidad qualified researcher. Autònoma de Barcelona. The contract of LM-G was supported by INIA and the European Social Fund. JP-P was funded by a grant from the Complutense University of Madrid ETHICS STATEMENT (CT45/15-CT46/15). FT-M was supported by the Isabel Maria Lopez Martinez Memorial Scholarship at the University of Ethical review and approval was not required for the animal Saskatchewan (Canada). Trapping activities of adult birds were study because handling of the storks included in the study partially supported in the framework of SEO/BirdLife Migra had been approved for the specific ongoing studies in each program and financed by Fundación Iberdrola España and colony, and our study was based on fecal droppings or Storch Schweiz, by grant CGL2012-32544 from the MINECO, cloacal swabs only. and by grant 511/2012 from the Organismo Autónomo de Parques Nacionales. AUTHOR CONTRIBUTIONS MC, AR, NM, and UH designed the study. UH, MC, JM, JD, ACKNOWLEDGMENTS JP-P, JA, FT-M, and JB sampled and collected relevant biological data from the stork colonies and isolated the isolates for further We are indebted to Cabañeros National Park and the urban characterization. MS-G and LM-G carried out the identification, residues treatment plant of Alcázar de San Juan for permission phenotypic, and molecular characterization of isolates and of access to white stork colonies. We gratefully acknowledge the plasmids. JJ and AM-M performed MLST and WGS. UH and help of A. Herrera-Dueñas; the owners and workers of Prado MC performed the statistical analysis. UH, MS-G, and LM-G Herrero Farm; J. Prieto in Alcalá de Henares; the forest officers drafted the manuscript. All authors revised the draft manuscript in Pinilla and the ringing group Monticola, in particular R. and contributed to writing the final version of the manuscript. Moréno-Opo and V. de la Torre in “La Torrecilla” for help with sample collection in the Community of Madrid, and S. Palacios and J. Ayala for field work in Seville and Doñana, FUNDING as well as the contribution of the following bird ringers in sample collection in colonies in Malpartida de Cáceres: J. M. This work was partially supported by the grants Abad, T. Álvarez, M. Giraldo, C. Domínguez, M. Iglesias y M. AGL2013-47852-R and RTI2018-095586-B-C22 from the Cardalliaguet, and León: I. Rodríguez, B. Fuertes, D. Miguélez, Ministerio de Economía y Competitividad (MINECO), project I. Roa, F. de la Calzada, J. García, F. Ortega, D. Prada, and M. RTA2011-00111-C03 funded by the Instituto Nacional de Cendoya (GIA-León). An earlier version of this article constitutes Investigación y Tecnología Agraria y Alimentaria (INIA) one of the chapters of the Ph.D. thesis of one of the authors and by the CERCA program from Generalitat de Catalunya. (MS-G), published by Solà-Ginés (2018). REFERENCES Báez, J., Hernández-García, M., Guamparito, C., Díaz, S., Olave, A., Guerrero, K., et al. (2015). Molecular characterization and genetic diversity of Ahlstrom, C. A., Bonnedahl, J., Woksepp, H., Hernandez, J., Reed, J. 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Antimicrob. Agents 41, 296–297. Resistance in Escherichia coli of Different Origins : Broilers, Flies and White Storks. Dissertation, Universidad Autónoma de Barcelona, Bellaterra. Conflict of Interest: The authors declare that the research was conducted in the Solà-Ginés, M., Cameron-Veas, K., Badiola, I., Dolz, R., Majó, N., Dahbi, G., absence of any commercial or financial relationships that could be construed as a et al. (2015). Diversity of multi-drug resistant avian pathogenic Escherichia coli potential conflict of interest. (APEC) causing outbreaks of colibacillosis in broilers during 2012 in Spain. PLoS One 10:e0143191. doi: 10.1371/journal.pone.0143191 Copyright © 2020 Höfle, Jose Gonzalez-Lopez, Camacho, Solà-Ginés, Moreno- Szczepanska, B., Kaminski, P., Andrzejewska, M., Spica, D., Kartanas, E., Ulrich, Mingorance, Manuel Hernández, De La Puente, Pineda-Pampliega, Aguirre, Torres- W., et al. (2015). Prevalence, virulence, and antimicrobial resistance of Medina, Ramis, Majó, Blas and Migura-Garcia. This is an open-access article Campylobacter jejuni and Campylobacter coli in white stork Ciconia in Poland. distributed under the terms of the Creative Commons Attribution License (CC BY). Foodborne Pathog. Dis. 12, 24–31. The use, distribution or reproduction in other forums is permitted, provided the Tortosa, F. S., Caballero, J. M., and Reyes-López, J. (2002). Effect of rubbish dumps original author(s) and the copyright owner(s) are credited and that the original on breeding success in the White Stork in Southern Spain. Waterbirds 25, publication in this journal is cited, in accordance with accepted academic practice. No 39–43. use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 13 July 2020 | Volume 11 | Article 1397