ORIGINAL RESEARCH published: 17 January 2020 doi: 10.3389/fmicb.2019.03072 mcr-Colistin Resistance Genes Mobilized by IncX4, IncHI2, and IncI2 Plasmids in Escherichia coli of Pigs and White Stork in Spain Lourdes Migura-Garcia 1,2* , Juan J. González-López 3 , Jaime Martinez-Urtaza 4 , J. R. Aguirre Sánchez 5,6 , A. Moreno-Mingorance 3 , A. Perez de Rozas 1 , Ursula Höfle 6 , Y. Ramiro 6 and Narjol Gonzalez-Escalona 7 1 Centre de Recerca en Sanitat Animal, Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spain, 2 Research and Control of Emerging and Re-emerging Swine Diseases in Europe, OIE Collaborating Centre, CReSA, IRTA, Barcelona, Spain, 3 Servei de Microbiologia, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, Edited by: 4 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth, United Kingdom, 5 Centro Daniel Yero, de Investigación en Alimentación y Desarrollo, Culiacán, Mexico, 6 Health and Biotechnology (SaBio) Group, Instituto Autonomous University of Barcelona, de Investigación en Recursos Cinégéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain, 7 Food and Drug Administration, Spain Silver Spring, MD, United States Reviewed by: Roger Stephan, University of Zurich, Switzerland Colistin has become the last-line antimicrobial for the treatment of multidrug resistant Magdalena Maria Zaja̧c, (MDR) Enterobacterales in human medicine. To date, several colistin resistance genes National Veterinary Research Institute have been described. Of them mcr-1 is disseminated worldwide in Escherichia coli (NVRI), Poland Min Yue, of human and animal origin. The aim of this study was to characterize mcr-mediated Zhejiang University, China resistance plasmids from E. coli of animal origin in Spain. From our strain collection, *Correspondence: 70 E. coli of pig origin collected between 2005 and 2014 (10 per year, except for Lourdes Migura-Garcia lourdes.migura@irta.cat years 2009–2010–2013) were randomly selected and screened for the presence of mcr-genes. Additionally, 20 E. coli isolated in 2011 from white storks (Ciconia ciconia) Specialty section: from the same urban household waste landfill associated colony were also included. This article was submitted to Evolutionary and Genomic Whole genome sequencing of mcr-positive isolates was carried out on a MiSeq Microbiology, (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina a section of the journal Frontiers in Microbiology technologies were performed in a selection of isolates to close the genomes and Received: 04 October 2019 plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory Accepted: 19 December 2019 concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments Published: 17 January 2020 were carried out to evaluate transferability of the mcr-genes. A total of 19 mcr-1 and Citation: one mcr-4 positive isolates were detected, 15 from pigs distributed during the study Migura-Garcia L, González-López JJ, period, and five from storks collected in 2011. No other mcr-variants were found. The Martinez-Urtaza J, MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected Aguirre Sánchez JR, Moreno-Mingorance A, among the mcr-1 positive E. coli isolates, with only ST-10 shared between pigs and Perez de Rozas A, Höfle U, Ramiro Y white storks. Except for one isolate, all were genotypic and phenotypically MDR, and and Gonzalez-Escalona N (2020) five of them also harbored cephalosporin resistance genes (blaCTX−M−14 , blaSHV−12 , mcr-Colistin Resistance Genes Mobilized by IncX4, IncHI2, and IncI2 and three blaCMY−2 ). mcr-1 genes were mobilizable by conjugation, associated with Plasmids in Escherichia coli of Pigs IncX4, IncHI2, and IncI2 plasmids. In our study, mcr-1 genes have been circulating and White Stork in Spain. Front. Microbiol. 10:3072. in pig farms since 2005 harbored by a variety of E. coli clones. Its persistence may doi: 10.3389/fmicb.2019.03072 be driven by co-selection since plasmids containing mcr-1 also exhibit resistance to Frontiers in Microbiology | www.frontiersin.org 1 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of mcr-1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes. Keywords: Escherichia coli, colistin, mcr-plasmids, MinION nanopore, pigs, storks, Spain INTRODUCTION as a reliable continuous food source. Spain holds the largest breeding population of storks in Europe, and due to availability Antimicrobials have been used for many decades for the of waste has also become a wintering area for storks from treatment of infectious diseases. This continued use, and in northern and central Europe. During the nesting period, storks some cases, overuse has increased the emergence of resistant are spatially bound to the surrounding areas. In some cases, these bacteria (Wall et al., 2016). The case of colistin is of particular areas are waste landfills that represent a source of contamination interest. Colistin was introduced in human medicine in the (Gomez et al., 2016). early 1950s. Due to its toxicity when applied systemically, it The aim of this study was to characterize the mechanisms was restricted to ophthalmic and topical use (Koch-Weser et al., involved in conferring resistance to colistin in isolates of 1970; Falagas et al., 2009). However, the emergence of pan- animal origin in Spain. In particular, E. coli causing post- resistant enterobacteria causing infections in hospital settings has weaning diarrhea between consecutive batches of animals in revived the use of colistin as the last-line treatment option. On pig farms, and in commensal E. coli isolated from white storks the contrary, colistin has been prescribed in animal husbandry during the breeding season. Additionally, combining Illumina for the treatment of infections caused by Enterobacterales since and Nanopore technology, we have completely sequenced the the 1960s, especially in pig production. Furthermore, several chromosome and closed whole plasmids of a set of isolates studies carried in different European countries have identified the annotating all the genes, including those associated with used of medicated feed mills supplemented with colistin for the antimicrobial resistance. prevention and/or treatment of post-weaning diarrhea (Trauffler et al., 2014; Cameron-Veas et al., 2016; Sjolund et al., 2016). Resistance to colistin was attributed to chromosomal mutations that resulted in the modification in the lipid A of the MATERIALS AND METHODS LPS, reducing the binding of colistin (Rhouma et al., 2017). It was not until 2015 (Liu et al., 2016), when a plasmid-mediated Study Design mechanism of colistin resistance (mcr-1) was described for From our strain collection, 70 E. coli of pig origin were randomly the first time, that the concern of its use reached the scientific selected for screening the presence of colistin resistance genes. community. The mcr-1 was first isolated in China from an For each year, they represented 10 isolates obtained from Escherichia coli of pig origin and was harbored in a conjugative diagnostic samples of post-weaning diarrhea collected from five plasmid, facilitating its transfer to other enterobacteria. The different farms in consecutive batches of animals (one isolate potential spread of plasmid mediated mcr-genes from animals per batch) between 2005 and 2014 (10 per year). Isolates from to humans rang the alarms of the Authorities, and the European years 2009, 2010, and 2013 could not be found. Additionally, Medicine Agency (EMA) reviewed the use of colistin in the 20 E. coli obtained from white storks (C. ciconia) in 2011 from European Union. Back in 2015, the use of colistin in swine the same urban household waste landfill associated colony were production in Spain was estimated in 50 mg/PCU (population also screened for the presence of mcr-genes. They represented correction units) and a reduction target to 5 mg/PCU was set up fecal droppings collected at the side of different nesting platforms for the next following years (EMA, 2016). within the same colony. Ten of them were isolated in MacConkey Currently, different variants of genes encoding colistin agar and the other ten in MacConkey agar supplemented with resistance have been identified (Xavier et al., 2016; Borowiak cefotaxime (4 mg/L). et al., 2017; Carattoli et al., 2017; Yin et al., 2017). However, This study uses strains obtained from fecal samples of pigs mcr-1 has been isolated from enterobacteria of different origins, with post-weaning diarrhea collected by the farm veterinarian humans, livestock, wildlife, and the environment (Prim et al., and sent for diagnostics to our laboratory. Handling and 2016; Caltagirone et al., 2017; El Garch et al., 2018). Furthermore, sampling of the storks was carried out following all applicable in 90% of the cases, mcr-1 has been associated to IncX4, IncHI2, international, national, and/or institutional guidelines for the and IncI2 mobilizable plasmids (Matamoros et al., 2017; Elbediwi care and ethical use of animals, specifically directive 2010/63/EU et al., 2019) facilitating its worldwide distribution (Li et al., 2017). and Spanish laws 9/2003 and 32/2007, and Royal decrees In addition, the emissions of antimicrobial resistant bacteria 178/2004 and 1201/2005. derived from human activities could have a negative effect in the environment and particularly in wildlife (Arnold et al., 2016). Detection of mcr Genes This is especially true for species, such as the white stork (Ciconia All isolates were tested by PCR methods for the presence of ciconia), that have adapted to using solid urban waste landfills the mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes as previously Frontiers in Microbiology | www.frontiersin.org 2 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals described (Rebelo et al., 2018). No other mechanisms of sequencing libraries were prepared using the rapid barcoding resistance were tested. sequencing kit RBK004 and run in a FLO-MIN106 (R9.4.1) flow cell, according to manufacturer’s instructions (Nanopore), Antimicrobial Susceptibility Testing for 48 h, at 50–130× average coverage. The sequencing library All mcr-positive isolates were susceptibility tested using a contained DNA fragmented randomly by a transposase present in minimum inhibitory concentration (MIC) broth microdilution the Fragmentation Mix of the RBK004 kit, rendering fragments (VetMIC GN-mo, National Veterinary Institute, Uppsala, >30 kb. The run was base called live using default settings Sweden). Antimicrobials tested were ampicillin (1–128 mg/L), (Minknow v18.05.5, Albacore). The genomes for each strain were cefotaxime (0.016–2 mg/L), ceftazidime (0.25–16 mg/L), nalidixic obtained by de novo assembly, using Nanopore data and default acid (1–128 mg/L), ciprofloxacin (0.008–1 mg/L), gentamicin settings within CANU program v1.6 (Koren et al., 2017). (0.12–16 mg/L), streptomycin (2–256 mg/L), kanamycin (8– A second assembly was generated using a SPAdes v3.11.1 to 16 mg/L), chloramphenicol (2–64 mg/L), florfenicol (4– (Bankevich et al., 2012) hybrid assembly (with default settings) 32 mg/L), trimethoprim (1–128 mg/L), sulfamethoxazole (8– using both Nanopore and MiSeq data generated for each strain. 1,024 mg/L), tetracycline (1–128 mg/L), and colistin (0.5– The final assembly (FA) was performed by comparing the SPAdes 4 mg/L). Isolates were considered to be susceptible or resistant hybrid and CANU assemblies using Mauve (Darling et al., 2004) based on epidemiological cut-off values defined by EUCAST1 . and filling in the missing regions in the SPAdes assembly with the CANU assembly. The FA sequences were annotated using Mating Experiments the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP)2 (Tatusova et al., 2016). Antimicrobial resistance genes To evaluate the transferability of the genes conferring resistant were annotated using the Comprehensive Antibiotic Resistance to colistin, filter-mating experiments were performed with all Database (CARD v3) (Jia et al., 2017). Plasmid identification was isolates as donors and E. coli HB101 rifampicin resistant as carried out using PlasmidFinder 2.1 (Carattoli et al., 2014). recipient. Conjugations were carried out as described before We performed a Basic Local Alignment Search Tool (BLAST) (Bielak et al., 2011). Briefly, 500 uL of the mixture of each donor (Altschul et al., 1990) to detect high similar published plasmids and recipient were placed in a sterile paper filter on a blood agar to pGN-295 sequenced herein, and we included pSLK172- plate. After overnight incubation, the filter was diluted in PBS 1 (CP017632.1), pRDB9 (MH924589), pS38 (KX129782.1), and 100 uL were spread on LB agar plates containing rifampicin 180-PT54 (CP015833.1), pCHL5009T-88k (CP032939.1), and (100 mg/L) and colistin (2 mg/L). NRZ14408 (LT599829). We used BLAST Ring Generator (BRIG) to display circular comparisons between the 11 E. coli plasmids Whole Genome Sequencing, Contigs (five identified in this study and six downloaded from NCBI with Assembly and Annotation high similarity to our closed plasmids). For this purpose, plasmid mcr-positive isolates were selected for WGS studies. DNA was pGN-295 was used as reference with an upper identity threshold extracted using QIAGEN DNeasy Ultraclean Microbial Kit R of 90% and a lower identity threshold of 70% and a nucleotide (Germany) following the manufacture’s recommendations. Short search using BLASTn (Supplementary Table 3). sequencing reads for each strain was generated using MiSeq Illumina sequencing with the MiSeq V3 kit using 2 × 250 Nucleotide Sequence Accession base pair paired-end chemistry (Illumina, San Diego, CA, United States) according to manufacturer’s instructions, at 160– Numbers 180× coverage. The libraries for the MiSeq were constructed The draft genome sequences of the 20 E. coli strains used in using 100 ng of genomic DNA using Nextera DNA Flex our study are available in GenBank under the accession numbers kit (Illumina), according to manufacturer’s instructions. The listed in the Supplementary Table 1. genomes for each strain were de novo assembled using CLC Genomics Workbench v9.5.2 (QIAGEN) using defaults settings Phylotyping and Phylogenetic Analyses except that the minimum contig size threshold was set to The Clermont et al. (2013) phylotyping scheme was performed 500 bp in length. by multiplex PCR for detecting phylotypes A, B1, B2, C, D, and F Based on results from the MiSeq assemble, five isolates as described before (Clermont et al., 2013). harboring mcr-1 and IncHI2 replicons in the same contig, and The initial identification of the strains was performed using therefore, with the two genes presumably located in the same an in silico E. coli MLST approach, based on the information plasmid were selected for further studies. They represented available at EnteroBase for E. coli website3 and using Ridom isolates of different origin collected during the study period SeqSphere + software v2.4.0 (Münster, Germany)4 to perform the and with the same replicon family. The whole genomes were in silico search. Seven housekeeping genes (dnaE, gyrB, recA, dtdS, sequenced and closed by using a combination of long reads and pntA, pyrC, and tnaA), described previously for E. coli were used the short reads generated earlier. The long reads sequences for for determining the STs (Wirth et al., 2006). each strain were generated through nanopore sequencing using a MinION device (Nanopore, Oxford, United Kingdom). The 2 http://www.ncbi.nlm.nih.gov/genome/annotation_prok 3 https://enterobase.warwick.ac.uk/species/index/ecoli 1 4 http://www.eucast.org/ http://www.ridom.com/seqsphere Frontiers in Microbiology | www.frontiersin.org 3 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals The phylogenetic relationship of the strains was assessed by Characterization of E. coli Bearing mcr-1 a custom core genome multilocus sequence typing (cgMLST) Plasmids analysis using Ridom SeqSphere + software v2.4.0. We used the Among pig isolates, phylotyping identified seven isolates cgMLST scheme reported earlier (Lorenz et al., 2017; Gonzalez- belonging to group A, five to group B1, two to group D and one Escalona and Kase, 2019). The genome of O157:H7 strain Sakai to E. E. coli of white stork origin belonged to group A (n = 3), B1 (NC_002695.1) was used as the reference for the cgMLST. A total (n = 1), and E (n = 1). B2 was never detected (Table 2). of 4,651 genes were used as templates for the analysis of the WGS identified a high variety of serotypes among the E. coli strains from this study. We also added nine other genomes different isolates, with five of them of pig origin typically of known E. coli strains to establish their phylogenetic context associated with post-weaning diarrhea, three of serotype O138, (Supplementary Table 2). A Neighbor-Joining (NJ) tree using the one O8, O15, and O157. appropriate genetic distances was built after the cgMLST analysis. There was a polyclonal expansion of E. coli bearing mcr- 1 plasmids, with 20 isolates belonging to 14 different MLST In silico Virulence Genes Detection types. Some of these ST types, such as ST42, ST118, ST3205, Each de novo assembled genome was screened in silico for the ST763, and ST351 are not very frequent according to the presence of 102 virulence genes reported for E. coli as described MLST database (see text footnote 3). ST10 was detected in elsewhere (Gonzalez-Escalona and Kase, 2019). three isolates of pig origin distributed over the years. This was the only ST type shared between E. coli of pig and white RESULTS stork origin. Phylogenetic studies demonstrated the presence of the same clonal linages of E. coli causing post-weaning Presence of mcr-Genes diarrhea between different batches of animals in two different Out of 70 E. coli isolates of pig origin, 14 were positive for occasions; isolates GN-444 and GN-445 in 2005 and GN-3020 the presence of the mcr-1. Additionally one isolate collected in and GN-3021 in 2012 (Table 2). Additionally, two isolates 2007 was positive for mcr-4.2. They were distributed between obtained from white stork droppings (171 and 173) were clonally the study-period (Table 1). No other mcr-variants were detected. related (Figure 1). Additionally, five isolates of white stork origin obtained from WGS also detected a wide variety of virulence genes, ranging 2011 were also positive, four contained mcr-1, and one contained from 2 to 12 (Table 2), with no apparent association between the mcr-1.2 variant. number of virulence genes, phylogroup or ST type. Antimicrobial Susceptibility Testing and Plasmid Characterization and Genetic Presence of Resistance Genes Context of the mcr-Genes The 20 isolates exhibited a MIC to colistin ≥4 mg/L. All All mcr-plasmids (mcr-1 and mcr-4) successfully transferred E. coli were phenotypically resistant to tetracycline confirmed the colistin resistant gene by conjugation to the recipient by the presence of tet(A), tet(B), tet(C), tet(M) or the strain, demonstrating their ability to move the mcr-genes combination of some of these genes, and except for one isolate between isolates. of pig origin, they were also resistant to ampicillin (Table 1) For all isolates, Illumina sequencing allowed to detect mcr- conferred by TEM-1A or TEM-1B. Furthermore, co-resistance 1 genes in the same contigs as replicons of the families IncX4 was observed for streptomycin, kanamycin and sulfonamides (n = 12), IncHI2 (n = 7), and IncI2 (n = 1). In the case of mcr-4.2, (67%) with an average of six different genes coding resistance it was also associated to IncX4 (Table 2). Additionally, isolates for aminoglycosides. Sixteen isolates exhibited resistance to contained a wide variety of replicons of different incompatibility florfenicol conferred by the presence of a unique gene or the groups presumably in different plasmids, ranging from 3 to 12. combination of different genes. Nalidixic acid and ciprofloxacin IncF and ColRNAI were the more represented (n = 15) followed resistance was detected in 14 isolates (56%). The presence by IncFIB (n = 11), IncI1 (n = 10), Col(MG8282) (n = 9) and of single point mutations in quinolone resistance-determining finally, IncHI2A, IncHI2, and col156 (n = 8, for each of them). regions (QRDR) in chromosomal gyrA, parC, or parE genes The sizes of the five IncHI2 plasmids sequenced in this study was confirmed for these isolates. Plasmid mediated quinolone varied between 235,260 and 356,700 bp (Table 2) and were all resistance genes were not detected. Except for isolate GN-2933, pMLST ST-4. In general, the architecture of the IncHI2 plasmids they were all multidrug resistant (MDR) (phenotypically resistant appeared to present high level of similarity with all of them having to at least three different families of antimicrobials). Phenotypic the conjugative transfer system, HigB-HigA toxin-antitoxin results correlated with the presence of the different resistance system for plasmid maintenance, macrolide resistance efflux genes for each antimicrobial family. As illustrated in Table 1, pump mef (B) and a tellurium resistant operon (Figure 2). Except the number of resistance genes varied depending of the isolate. for p176, they also harbored a mercury resistance operon, which Resistance to cephalosporins coded by CTX-M-14 and SHV-12 also included the genes for cobalt-zinc-cadmium resistance. was observed in two isolates of pig origin and was confirmed by In particular, pGN-115A contained the copper resistance gene the MIC results (MICs for cefotaxime and ceftazidime >2 and pcoE. Plasmid p176 also possessed a MazEF toxin-antitoxin >16 mg/L, respectively). CMY-2 was confirmed in three isolates system. Additionally, these high molecular weight plasmids also of white stork origin. yielded resistance genes for different families of antimicrobials, Frontiers in Microbiology | www.frontiersin.org 4 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals TABLE 1 | Resistance genes for the different antimicrobials families found in isolates bearing mcr-plasmids. ID Year Origin Colistin Aminoglycoside Sulfonamide Trimethoprim Tetracycline Phenicol Beta- MLS lactam GN-295 2005 Pig mcr-1.1 aac(3)-IIa, aadA1, sul1, sul2, sul3 dfrA1 tet(A) catA1, cml, TEM-1A aadA2, strA, strB cmlA1 GN-444 2005 mcr-1.1 aac(3)-IIa, aac(3)-IVa, sul2, sul3 dfrA12 tet(B), tet(M) catA1, cmlA1, TEM-1A mef(B) aadA1, aadA2, floR aph(30 )-Ia, aph(4)-Ia, strA, strB GN-445 2005 mcr-1.1 aac(3)-IIa, aac(3)-IVa, sul2, sul3 dfrA12 tet(B), tet(M) catA1, cmlA1, TEM-1A mef(B) aadA1, aadA2, floR aph(30 )-Ia, aph(4)-Ia, strA, strB GN-609 2006 mcr-1.1 aac(3)-IVa, aadA1, sul1, sul2 dfrA1 tet(A), tet(M) TEM-1A mph(B) aph(4)-Ia, strA, strB GN-1035 2007 mcr-1.1 aadA1, aadA2, sul1, sul3 dfrA1 tet(A) cmlA1 TEM-1B aph(30 )-Ia GN-1036 2007 mcr-1.1 aac(3)-IIa, aadA1, sul1, sul2, sul3 dfrA1 tet(A) catA1, cml, TEM-1A aadA2, aph(30 )-Ic, strA, cmlA1 strB GN-1044 2007 mcr-1.1 aac(3)-IIa, aadA1, sul1, sul2, sul3 dfrA1 tet(A), tet(B) catA1, cml, TEM-1A Inu(G), aadA2, aph(30 )-Ia, strA, cmlA1 mdf(A) strB GN-1058 2007 mcr-4.2 aac(3)-IId, aac(3)-IVa, sul3 dfrA12 tet(A), tet(M) catA2, cmlA1 TEM-1B lnu(F) aadA1, aadA2, aph(4)-Ia, strA, strB GN-1639 2008 mcr-1.1 aac(3)-IVa, aadA1, sul3 dfrA12 tet(A), tet(M) cmlA1, floR TEM-1B aadA2, aph(4)-Ia, strA, strB GN-2933 2011 mcr-1.1 tet(A) GN-3020 2012 mcr-1.1 aac(3)-IVa, aadA1, sul3 dfrA12 tet(C) cmlA1, floR TEM-1B aadA2, aph(4)-Ia, strA, strB GN-3021 2012 mcr-1.1 aac(3)-IVa, aadA1, sul3 dfrA12 tet(C) cmlA1, floR TEM-1B aadA2, aph(4)-Ia, strA, strB GN-3022 2012 mcr-1.1 aadA1, aadA1, sul1 dfrA1 tet(B), tet(M) catA1, catB3 TEM-1B mph(E), aph(30 )-Ic, aac(60 )Ib-cr msr(E) GN-102A 2014 mcr-1.1 aac(3)-IVa, aadA1, sul1, sul2, sul3 dfrA1, dfrA5 tet(A) floR TEM-1B, aph(30 )-Ia, aph(4)-Ia, CTX-M- strA, strB 14 GN-115A 2014 mcr-1.1 aadA1, aadA2 sul3 dfrA1 tet(A) catA1, floR SHV-12 171 2011 Stork mcr-1.1 tet(A) TEM-1B 172 2011 mcr-1.1 aac(3)-IId, aadA2, strA, sul1, sul2 dfrA12 tet(A) catA1 TEM-1B mph(A) strB 173 2011 mcr-1.1 tet(A) TEM-1B, CMY-2 176 2011 mcr-1.2 aac(3)-Iva, aadA1, strB, sul3 dfrA1 tet(B) catA1 TEM-1B, aph(4)-Ia, CMY-2 178 2011 mcr-1.1 aac(3)-IIa, aadA1, sul1, sul2, sul3 dfrA1, dfrA12 tet(A) catA1, cmlA1 TEM-1A, aadA2, aph(30 )-Ic, strA, TEM-1B, strB CMY-2 In gray isolates belonging to the same farm, but consecutive batches. MLS: Macrolides, lincosamides, streptogramines, ID: Identification. including aminoglycosides, beta-lactams, tetracycline, phenicols, Furthermore, there were three different mcr-1 context or sulfonamides, and trimethoprim (Table 2). Interestingly, pGN- arrangements found in all these families of plasmids (Table 2). 295 with the largest molecular weight, presented and extra-DNA mcr-1 was flanked upstream and downstream by the ISApl1 region highly similar to a previously sequenced plasmid named elements, loss of the downstream ISApl1 element, and the pCHL5009T-88k and detected in and E. coli of human origin complete loss of ISApl1 elements surrounding the mcr-1. in New Zealand (E. coli NRZ14408). In that region, an IncY Regarding mcr-4.2, following the gene was an ORF coding for a replicon was detected. hypothetical protein and a relA gene. Frontiers in Microbiology | www.frontiersin.org 5 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals TABLE 2 | Serotype, phylogroup, MLST, plasmid replicon associated to mcr-genes, flanking regions and virulence genes. ID Origin Serotype phylo MLST, CC mcr- size GC ORF mcr-1 context Virulence genes plasmid content GN-295 Pig Ounk:H32 A ST-10, CC10 IncHI2 356,700 47.2 447 ISApl1-mcr-1-hp-ISApl1 gad, iss GN-444 O25:H28 B1 ST-156, CC156 IncX4 Unknown hp-mcr-1-hp gad, iss, lpfA GN-445 O25:H28 B1 ST-156, CC156 IncX4 Unknown hp-mcr-1-hp gad, iss, lpfA GN-609 O98:H12 A ST-10, CC10 IncX4 Unknown hp-mcr-1-hp aaiC, capU, celb, gad, iha, iss, katP GN-1035 O15:H45 E ST-118 IncX4 Unknown hp-mcr-1-hp air, astA, celb, eilA, gad, stb GN-1036 O8:H20 A ST-3205 IncHI2 265,340 47.2 344 ISApl1-mcr-1-hp-ISApl1 astA, capU, gad, stb GN-1044 O138:H A ST-100, CC165 IncHI2 Unknown ISApl1-hp-mcr-1-hp astA, capU, gad, iha, K88ab, ltcA, stb GN-1058 O123:H23 B1 ST-224 IncX4 Unknown mcr-4.2-hp-relE astA, gad, lpfA GN-1639 O20:H20 A ST-542 IncX4 Unknown hp-mcr-1-hp astA, gad GN-2933 O157:H19 B1 ST-763 IncI2 Unknown hp-mcr-1-hp astA, gad, lpfA, stb GN-3020 O138:H14 D ST-42 IncX4 Unknown hp-mcr-1-hp air, astA, cba, cma, fedA, fedF, iha, iss, lpfA, ltcA, sta1, stb GN-3021 O138:H14 D ST-42 IncX4 Unknown hp-mcr-1-hp air, astA, cba, cma, fedA, fedF, iha, iss, lpfA, ltcA, sta1, stb GN-3022 O29:H51 B1 ST-156, CC156 IncX4 Unknown hp-mcr-1-hp2 gad, iss, lpfA, tsh GN-102A O11:H8 A ST-448, CC488 IncHI2 Unknown hp-hha-mcr-1-hp celb, gad, iroN, iss, lpfA, mchF GN-115A O98:H12 A ST-10, CC10 IncHI2 252,383 46.1 319 ISApl1-mcr-1-hp aaiC, capU, celb, gad, iha, iss, katP 171 Stork O5:H4 A ST-93, CC168 IncX4 Unknown hp-mcr-1-hp capU, cma, gad, iroN, iss, tsh 172 O86:H51 E ST-1011 IncX4 Unknown hp-mcr-1-hp eilA, gad, iss 173 O5:H4 A ST-93, CC168 IncX4 Unknown hp-mcr-1-hp capU, cma, gad, iroN, iss, tsh 176 O107:H10 A ST-10, CC10 IncHI2 235,260 46.1 309 putative kinase-mcr-1.2-hp gad, lpfA, astA 178 O18ac:H7 B1 ST-351 IncHI2 264,642 47.2 344 ISApl1-mcr-1-hp-ISApl1 cma, gad, iroN, iss, lpfA In gray isolates belonging to the same farm, but consecutive batches. DISCUSSION animals. However, a vertical transmission from sows to their litter cannot be disregarded. Plasmid mediated colistin resistance has been described Although we do not have antimicrobial consumption data for worldwide in a variety of Enterobacterales of different origins, these farms, it is interesting to notice that in pig production environment, food producing animals, wildlife and humans in Spain, before the finding of the mcr-genes, colistin was (Zurfluh et al., 2016; Guenther et al., 2017; Ovejero et al., the first choice antimicrobial for the treatment of E. coli 2017; Barlaam et al., 2019; Elbediwi et al., 2019; Lalaoui et al., causing post-weaning diarrhea (Moreno, 2014). The majority 2019; Mendes Oliveira et al., 2019; Nang et al., 2019; Zajac ˛ of these treatments were prescribed empirically instead of et al., 2019). In particular, in Spain, mcr-1 has been described based on antimicrobial susceptibility testing. This fact highlights circulating in pig farms for many years (Quesada et al., 2016; the importance of performing phenotypic tests to provide Garcia-Menino et al., 2018), and our results demonstrate its a successful therapy for the treatment of disease and to presence in pig farms in Spain as early as 2005. Although acquire epidemiological surveillance data of the phenotypes other mcr-gene variants have also been reported in Spain circulating in the farms. Generally, National Programs in (Carattoli et al., 2017), studies by Garcia-Menino et al. (2018) Europe only include commensal E. coli to monitor antimicrobial screening large number of E. coli isolates from diagnostic resistance from food producing animals (Garcia-Migura et al., cases of post-weaning diarrhea detected mcr-1 as the most 2014), and little is known about epidemiology and resistant common variant followed by mcr-4. Our study also confirmed mechanisms present in diagnostic samples. Additionally, except similar results, although more recently described mcr-genes for one, all of the isolates herein were MDR, impairing have not been tested herein. Furthermore, in two different a successful treatment of post-weaning diarrhea and most occasions, carry over of the same clones causing diarrhea probable facilitating the proliferation of the pathogen, since between batches of animals within the same farm was observed. the antimicrobials would eliminate beneficial microbiota that In addition to the consumption of antimicrobials, internal could colonize the same niche and compete for the nutrients biosecurity including cleaning and disinfection can play an (Soler et al., 2018). important role in the maintenance of antimicrobial resistant Wildlife is generally not treated with antibiotics but has been bacteria in the farms (Raasch et al., 2018), facilitating its shown to acquire commensal microbiota carrying resistance transmission and persistence between different batches of genes when in contact with anthropized environments and Frontiers in Microbiology | www.frontiersin.org 6 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals FIGURE 1 | Neighbor joining phylogenetic tree generated by a cgMLST analysis of the strains sequenced in this study and 10 other known E. coli strains (Supplementary Table 2). The genome of E. coli O157:H7 strain Sakai (NC_002695) was used as reference. The final cgMLST analysis was based on 2514 shared loci among those strains. The sequenced strains in this study are in red. Next to the name of the strain is their ST separated by a comma. livestock (Gomez et al., 2016; Darwich et al., 2019). To the range of hosts harboring mcr-plasmids. The unique ST type best of our knowledge, this would be the first report of ST10 shared between isolates of both origins was ST10. ST10 is E. coli isolates mcr-1 positive described in white storks. The widely disseminated in humans and animals and frequently colony from which these originated is located on the premises associated not only with the presence of cephalosporin resistance of a solid urban waste disposal site. Acquisition of these genes genes (Ojer-Usoz et al., 2017), but currently also with the is probably through foraging of feed at the landfill. Several occurrence of mcr-1 (Matamoros et al., 2017; Elbediwi et al., studies have reported the presence of mcr-1 in migratory birds 2019). In fact, two ST10 isolates of pig and white stork (Liakopoulos et al., 2016; Mohsin et al., 2016; Ruzauskas and origin from our study exhibited co-resistance to colistin and Vaskeviciute, 2016; Tarabai et al., 2019), including co-resistance cephalosporins, both critically important antimicrobials to treat to colistin and cephalosporins. In fact, three of our isolates severe human infections caused by MDR Gram-negative in of white stork origin contained mcr-1 and blaCMY−2 genes hospital settings (World Health Organization [WHO], 2017). within the same host. The fact that a large proportion of the Furthermore, ST10 has been identified as a high-risk lineage European white stork population is migratory may facilitate causing human extraintestinal infections (Manges et al., 2019). the spread of the mcr-1 gene between different continents and In Spain mcr-1 ST10 has also been described in clinical emphasizes the importance of studies monitoring wildlife as isolates causing severe infections (Lalaoui et al., 2019). The potential reservoirs of resistance genes with an impact in public fact that food-producing animals, and wildlife associated health along their migration routes. Furthermore, as especially to human settlements such as white storks that frequently juvenile storks with different geographical origins may share the nest on structures within villages and cities, could be a same areas during wintering in Africa, this could potentially lead reservoir and source of potential pathogens with a MDR to cross contamination between storks from different regions profile is worrisome, and emphasizes the need to coordinate (Flack et al., 2016). efforts from human and veterinary sectors within a One The diversity of clones in terms of serotype, phylotype Health approach. and ST type encountered among isolates of both, pigs and Despite the clonal diversity observed among E. coli isolates white stork origin was very high, demonstrating a wide in this study, only three plasmid incompatibility groups, IncX4, Frontiers in Microbiology | www.frontiersin.org 7 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals FIGURE 2 | BRIG visualization of multiple E. coli plasmids comparisons. The solid innermost ring (black) correspond to the reference plasmid (pGN-295). After this ring plasmid p176 is shown (light purple). The third ring correspond to p178 (light blue), then pGN-1036 (navy blue) and pGN-115A (green). Additionally high identity plasmids to pGN-295 were download and added to the comparisons. The fifth (mustard) correspond to CP015833 plasmid, next CP07632.1 (aqua), KX129782 (purple), LT599829 (red), MH924589 (brown), pCHL5009T-88k (yellow), and finally we add the reference again (gray). The label in the outer ring represent the annotation on the genes associated to virulence, antibiotic resistance, stress resistance and replicons. IncHI2, and IncI2 could be directly associated to the presence the BLAST of our sequenced IncHI2 plasmids, retrieved of the mcr-1 gene. These replicons are well described in the similar backbones of small molecular weight plasmids with literature for their fidelity toward mcr-1 genes (Matamoros et al., the absence of mcr-1. Furthermore, a region of pGN-295 2017; Sun et al., 2017). Zurfluh et al. (2017) described IncI2 containing an IncY replicon matched a plasmid obtained from mcr-1 positive plasmids carried by E. coli strains belonging to a patient in New Zealand, pCHL5009T-88k (CP032939.1). It distinct STs than the one in our study, confirming the high is impossible to find an epidemiological connection between transmissibility of plasmids containing mcr-1 gene. Interestingly, plasmids found so far apart. However, these results suggest that Frontiers in Microbiology | www.frontiersin.org 8 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals some plasmids might be highly adapted to their host and able to Additionally, to the best of our knowledge this is the first report evolve through recombination processes. They are dynamic and of mcr-1 detected in commensal E. coli isolated from white plastic structures prone to merge and generate mega-plasmids, stork as far back as 2011. Anthropogenic pressure is highly providing an advantage for the bacterium. associated to the presence of these resistance mechanisms. The Mating experiments demonstrated the capability of all the study also showed the benefits of applying an advanced strategy isolates to mobilize the resistant gene to a recipient strain, combining Illumina and Nanopore sequencing technologies to and WGS data corroborated the presence of the conjugative close the genomes and plasmids as a key element to gain elements. It is worth noticing that closed IncHI2 plasmids from a basic understanding on genetic variation and dynamics of pigs and white storks also carried a variety of resistance genes plasmid transfer. Using this innovative approach along with for different families of antimicrobials (aminoglycosides, comprehensive analysis within a One Health context, we might beta-lactams, tetracycline, sulfonamides, phenicols, and be able to design strategies to minimize the emergence and trimethoprim). This means that even withdrawing the use persistence of resistance mechanisms within food producing of colistin in pig farms, other common antimicrobials used animals and improve our knowledge about the spread of these during the rearing cycle in pig production such as tetracycline mechanisms driven by forces such as migratory movements of or beta-lactams could co-select for the presence of colistin wild birds. Hence, efforts should be made to mitigate the effect resistance, persisting for long periods in food producing animals. of the anthropogenic pressure and the released of antimicrobial Interestingly, these five IncHI2-ST4 plasmids also contained resistance genes into the environment. genes encoding resistance for heavy metals (mercury, cobalt, zinc, cadmium, copper and tellurium) and small multidrug resistance efflux transporter (QacE) conferring resistance to DATA AVAILABILITY STATEMENT quaternary ammonium compounds (QAC). QAC have been commonly used as disinfectants with different applications in The draft genome sequences of the 20 E. coli strains used in health care and the food-industry (Kaskova et al., 2007; Quinn our study are available in GenBank under the accession numbers et al., 2015). Resistance to disinfectants presumably confers listed in the Supplementary Table 1. these clones the capacity to survive under extreme conditions. Furthermore, since mazEF and hipBA toxin–antitoxin systems were detected, these isolates are bound to the plasmid, and ETHICS STATEMENT losing the plasmid will trigger the pathway of cellular death (Engelberg-Kulka et al., 2005). Strategies to target these systems Ethical review and approval was not required for the animal study appear to be a promising therapy to defeat these MDR pathogens because this study is performed with E. coli of fecal samples of due to the bactericidal effect (Rownicki et al., 2018). animal origin stored in our freezer. The mcr-1 flanking areas described herein have also been reported in studies elsewhere for the three-replicon families (Hadjadj et al., 2017; Zurfluh et al., 2017; Duggett et al., 2018; AUTHOR CONTRIBUTIONS ˛ et al., 2019). The ISApl1 flanking the mcr-1 gene appears to Zajac LM-G designed the study. AP, YR, and UH contributed to the be a very efficient mechanism for “traveling” around, facilitating collection of samples. NG-E, JG-L, JM-U, JA, AM-M, and LM-G mcr-1 transposition between a limited number of incompatibility performed the sequencing, analyses, and interpretation. LM-G families of plasmids (Matamoros et al., 2017). Same genetic drafted the first manuscript. All authors contributed with the final context has been identified in IncHI2 plasmids herein together writing, reviewing, and editing. with the loss of the ISApl1 element downstream the mcr-1 gene or the total absence of ISApl1, anchoring the mcr-1 gene to the plasmid. These differences in the surrounding regions of FUNDING mcr-1 probably indicate different stages in the evolution of the plasmid once integrating the transposon, becoming more stable This work was partially supported by the grant RTI2018-095586- while losing the ISApl1 elements (Wang et al., 2018). In the B-C22 from the Ministerio de Economía y Competitividad case of mcr-4, recent studies have also detected similar flanking (Gobierno de España), by Plan Nacional de I+D+i 2013–2016, regions to the one described here in E. coli causing post-weaning Instituto de Salud Carlos III and the Spanish Ministry of Health diarrhea presumably in Italy (MG800338). Again, it is difficult to (FIS PI15/00604), and by the CERCA program from Generalitat elucidate an epidemiological relation, unless providers of animals de Catalunya. Contract of LM-G was supported by INIA and the were shared at some point between European countries. More European Social Fund. likely, mcr-genes have found a successful plasmid expansion by transposition and conjugation to perpetuate its transmission. To conclude, we confirmed the polyclonal expansion of IncX4 SUPPLEMENTARY MATERIAL and IncHI2 plasmids bearing mcr-1 in E. coli causing post- weaning diarrhea in pig farms in Spain since at least 2005. The Supplementary Material for this article can be found online This highlights the role of food producing animals as reservoirs at: https://www.frontiersin.org/articles/10.3389/fmicb.2019. of antimicrobial resistant genes with impact in human health. 03072/full#supplementary-material Frontiers in Microbiology | www.frontiersin.org 9 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals REFERENCES Engelberg-Kulka, H., Hazan, R., and Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. J. Cell Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic Sci. 118(Pt 19), 4327–4332. doi: 10.1242/jcs.02619 local alignment search tool. J. Mol. Biol. 215, 403–410. doi: 10.1016/s0022- Falagas, M. E., Vouloumanou, E. K., and Rafailidis, P. I. (2009). Systemic colistin 2836(05)80360-2 use in children without cystic fibrosis: a systematic review of the literature. Int. J. Arnold, K. E., Williams, N. J., and Bennett, M. (2016). ‘Disperse abroad in the land’: Antimicrob. Agents 33, 503.e1–503.e13. doi: 10.1016/j.ijantimicag.2008.10.021 the role of wildlife in the dissemination of antimicrobial resistance. Biol. Lett. Flack, A., Fiedler, W., Blas, J., Pokrovsky, I., Kaatz, M., Mitropolsky, M., et al. 12:20160137. doi: 10.1098/rsbl.2016.0137 (2016). Costs of migratory decisions: a comparison across eight white stork Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., populations. Sci. Adv. 2:e1500931. doi: 10.1126/sciadv.1500931 et al. (2012). SPAdes: a new genome assembly algorithm and its applications Garcia-Menino, I., Garcia, V., Mora, A., Diaz-Jimenez, D., Flament-Simon, S. C., to single-cell sequencing. J. Comput. Biol. 19, 455–477. doi: 10.1089/cmb.2012. Alonso, M. P., et al. (2018). Swine enteric colibacillosis in Spain: pathogenic 0021 potential of mcr-1 ST10 and ST131 E. coli isolates. Front. Microbiol. 9:2659. Barlaam, A., Parisi, A., Spinelli, E., Caruso, M., Taranto, P. D., and Normanno, doi: 10.3389/fmicb.2018.02659 G. (2019). Global emergence of colistin-resistant Escherichia coli in food chains Garcia-Migura, L., Hendriksen, R. S., Fraile, L., and Aarestrup, F. M. (2014). and associated food safety implications: a review. J. Food Prot. 82, 1440–1448. Antimicrobial resistance of zoonotic and commensal bacteria in Europe: the doi: 10.4315/0362-028x.jfp-19-116 missing link between consumption and resistance in veterinary medicine. Vet. Bielak, E., Bergenholtz, R. D., Jorgensen, M. S., Sorensen, S. J., Hansen, L. H., Microbiol. 170, 1–9. doi: 10.1016/j.vetmic.2014.01.013 and Hasman, H. (2011). Investigation of diversity of plasmids carrying the Gomez, P., Lozano, C., Camacho, M. C., Lima-Barbero, J. F., Hernandez, J. M., blaTEM-52 gene. J. Antimicrob. Chemother. 66, 2465–2474. doi: 10.1093/jac/ Zarazaga, M., et al. (2016). Detection of MRSA ST3061-t843-mecC and ST398- dkr331 t011-mecA in white stork nestlings exposed to human residues. J. Antimicrob. Borowiak, M., Fischer, J., Hammerl, J. A., Hendriksen, R. S., Szabo, I., Chemother. 71, 53–57. doi: 10.1093/jac/dkv314 and Malorny, B. (2017). Identification of a novel transposon-associated Gonzalez-Escalona, N., and Kase, J. A. (2019). Virulence gene profiles and phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in phylogeny of Shiga toxin-positive Escherichia coli strains isolated from FDA d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B. regulated foods during 2010-2017. PLoS One 14:e0214620. doi: 10.1371/journal. J. Antimicrob. Chemother. 72, 3317–3324. doi: 10.1093/jac/dkx327 pone.0214620 Caltagirone, M., Nucleo, E., Spalla, M., Zara, F., Novazzi, F., Marchetti, V. M., et al. Guenther, S., Falgenhauer, L., Semmler, T., Imirzalioglu, C., Chakraborty, T., (2017). Occurrence of extended spectrum beta-Lactamases, KPC-Type, and Roesler, U., et al. (2017). Environmental emission of multiresistant Escherichia MCR-1.2-producing Enterobacteriaceae from wells, river water, and wastewater coli carrying the colistin resistance gene mcr-1 from German swine farms. treatment plants in Oltrepo Pavese area, Northern Italy. Front. Microbiol. J. Antimicrob. Chemother. 72, 1289–1292. doi: 10.1093/jac/dkw58510.1093/jac/ 8:2232. doi: 10.3389/fmicb.2017.02232 dkw585 Cameron-Veas, K., Moreno, M. A., Fraile, L., and Migura-Garcia, L. (2016). Hadjadj, L., Riziki, T., Zhu, Y., Li, J., Diene, S. M., and Rolain, J. M. (2017). Study Shedding of cephalosporin resistant Escherichia coli in pigs from conventional of mcr-1 gene-mediated colistin resistance in Enterobacteriaceae isolated from farms after early treatment with antimicrobials. Vet. J. 211, 21–25. doi: 10.1016/ humans and animals in different countries. Genes (Basel) 8:394. doi: 10.3390/ j.tvjl.2016.02.017 genes8120394 Carattoli, A., Villa, L., Feudi, C., Curcio, L., Orsini, S., Luppi, A., et al. (2017). Novel Jia, B., Raphenya, A. R., Alcock, B., Waglechner, N., Guo, P., Tsang, K. K., plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia et al. (2017). CARD 2017: expansion and model-centric curation of the coli, Italy 2013, Spain and Belgium, 2015 to 2016. Euro Surveill. 22:30589. comprehensive antibiotic resistance database. Nucleic Acids Res. 45, D566– doi: 10.2807/1560-7917.es.2017.22.31.30589 D573. doi: 10.1093/nar/gkw1004 Carattoli, A., Zankari, E., Garcia-Fernandez, A., Voldby Larsen, M., Lund, O., Villa, Kaskova, A., Ondrasovicova, O., Vargova, M., Ondrasovic, M., and Venglovsky, J. L., et al. (2014). In silico detection and typing of plasmids using PlasmidFinder (2007). Application of peracetic acid and quarternary ammonium disinfectants and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 58, as a part of sanitary treatment in a poultry house and poultry processing plant. 3895–3903. doi: 10.1128/aac.02412-14 Zoonoses Public Health 54, 125–130. doi: 10.1111/j.1863-2378.2007.00987.x Clermont, O., Christenson, J. K., Denamur, E., and Gordon, D. M. (2013). The Koch-Weser, J., Sidel, V. W., Federman, E. B., Kanarek, P., Finer, D. C., and Clermont Escherichia coli phylo-typing method revisited: improvement of Eaton, A. E. (1970). Adverse effects of sodium colistimethate. Manifestations specificity and detection of new phylo-groups. Environ. Microbiol. Rep. 5, and specific reaction rates during 317 courses of therapy. Ann. Intern. Med. 72, 58–65. doi: 10.1111/1758-2229.12019 857–868. doi: 10.7326/0003-4819-72-6-857 Darling, A. C., Mau, B., Blattner, F. R., and Perna, N. T. (2004). Mauve: multiple Koren, S., Walenz, B. P., Berlin, K., Miller, J. R., Bergman, N. H., and Phillippy, alignment of conserved genomic sequence with rearrangements. Genome Res. A. M. (2017). Canu: scalable and accurate long-read assembly via adaptive 14, 1394–1403. doi: 10.1101/gr.2289704 k-mer weighting and repeat separation. Genome Res. 27, 722–736. doi: 10.1101/ Darwich, L., Vidal, A., Seminati, C., Albamonte, A., Casado, A., Lopez, F., et al. gr.215087.116 (2019). High prevalence and diversity of extended-spectrum beta-lactamase and Lalaoui, R., Djukovic, A., Bakour, S., Sanz, J., Gonzalez-Barbera, E. M., Salavert, M., emergence of OXA-48 producing Enterobacterales in wildlife in Catalonia. PLoS et al. (2019). Detection of plasmid-mediated colistin resistance, mcr-1 gene, in One 14:e0210686. doi: 10.1371/journal.pone.0210686 Escherichia coli isolated from high-risk patients with acute leukemia in Spain. Duggett, N. A., Randall, L. P., Horton, R. A., Lemma, F., Kirchner, M., Nunez- J. Infect. Chemother. 25, 605–609. doi: 10.1016/j.jiac.2019.03.007 Garcia, J., et al. (2018). Molecular epidemiology of isolates with multiple mcr Li, R., Xie, M., Zhang, J., Yang, Z., Liu, L., Liu, X., et al. (2017). Genetic plasmids from a pig farm in Great Britain: the effects of colistin withdrawal characterization of mcr-1-bearing plasmids to depict molecular mechanisms in the short and long term. J. Antimicrob. Chemother. 73, 3025–3033. doi: underlying dissemination of the colistin resistance determinant. J. Antimicrob. 10.1093/jac/dky292 Chemother. 72, 393–401. doi: 10.1093/jac/dkw411 El Garch, F., de Jong, A., Bertrand, X., Hocquet, D., and Sauget, M. (2018). Liakopoulos, A., Mevius, D. J., Olsen, B., and Bonnedahl, J. (2016). The colistin mcr-1-like detection in commensal Escherichia coli and Salmonella spp. from resistance mcr-1 gene is going wild. J. Antimicrob. Chemother. 71, 2335–2336. food-producing animals at slaughter in Europe. Vet. Microbiol. 213, 42–46. doi: 10.1093/jac/dkw262 doi: 10.1016/j.vetmic.2017.11.014 Liu, Y. Y., Wang, Y., Walsh, T. R., Yi, L. X., Zhang, R., Spencer, J., et al. (2016). Elbediwi, M., Li, Y., Paudyal, N., Pan, H., Li, X., Xie, S., et al. (2019). Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in Global burden of colistin-resistant bacteria: mobilized colistin resistance genes animals and human beings in China: a microbiological and molecular biological study (1980-2018). Microorganisms 7:461. doi: 10.3390/microorganisms710 study. Lancet Infect. Dis. 16, 161–168. doi: 10.1016/s1473-3099(15)00424-7 0461 Lorenz, S. C., Gonzalez-Escalona, N., Kotewicz, M. L., Fischer, M., and Kase, J. A. EMA (2016). Sales of Veterinary Antimicrobial Agents in 29 European Countries in (2017). Genome sequencing and comparative genomics of enterohemorrhagic 2014. Report No 6. Brussels: EU Publications. Escherichia coli O145:H25 and O145:H28 reveal distinct evolutionary paths Frontiers in Microbiology | www.frontiersin.org 10 January 2020 | Volume 10 | Article 3072 Migura-Garcia et al. mcr-Genes in Animals and marked variations in traits associated with virulence & colonization. BMC farrow-to-finish pig herds in Belgium, France, Germany and Sweden. Prev. Vet. Microbiol. 17:183. doi: 10.1186/s12866-017-1094-3 Med. 130, 41–50. doi: 10.1016/j.prevetmed.2016.06.003 Manges, A. R., Geum, H. M., Guo, A., Edens, T. J., Fibke, C. D., and Pitout, J. D. D. Soler, C., Goossens, T., Bermejo, A., Migura-Garcia, L., Cusco, A., Francino, O., (2019). Global extraintestinal pathogenic Escherichia coli (ExPEC) lineages. et al. (2018). Digestive microbiota is different in pigs receiving antimicrobials Clin. Microbiol. Rev. 32:e00135-18, doi: 10.1128/cmr.00135-18 or a feed additive during the nursery period. PLoS One 13:e0197353. doi: 10. Matamoros, S., van Hattem, J. M., Arcilla, M. S., Willemse, N., Melles, D. C., 1371/journal.pone.0197353 Penders, J., et al. (2017). Global phylogenetic analysis of Escherichia coli and Sun, J., Xu, Y., Gao, R., Lin, J., Wei, W., Srinivas, S., et al. (2017). Deciphering plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid MCR-2 colistin resistance. MBio 8:e00625-17. doi: 10.1128/mBio.00625-17 restriction. Sci. Rep. 7:15364. doi: 10.1038/s41598-017-15539-7 Tarabai, H., Valcek, A., Jamborova, I., Vazhov, S. V., Karyakin, I. V., Raab, R., Mendes Oliveira, V. R., Paiva, M. C., and Lima, W. G. (2019). Plasmid-mediated et al. (2019). Plasmid-Mediated mcr-1 colistin resistance in Escherichia coli colistin resistance in Latin America and Caribbean: a systematic review. Travel from a black kite in Russia. Antimicrob. Agents Chemother. 63:e01266-19. doi: Med. Infect. Dis. 31, 101459. doi: 10.1016/j.tmaid.2019.07.015 10.1128/aac.01266-19 Mohsin, M., Raza, S., Roschanski, N., Schaufler, K., and Guenther, S. (2016). Tatusova, T., DiCuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, E. P., First description of plasmid-mediated colistin-resistant extended-spectrum Zaslavsky, L., et al. (2016). NCBI prokaryotic genome annotation pipeline. beta-lactamase-producing Escherichia coli in a wild migratory bird from Nucleic Acids Res. 44, 6614–6624. doi: 10.1093/nar/gkw569 Asia. Int. J. Antimicrob. Agents 48, 463–464. doi: 10.1016/j.ijantimicag.2016. Trauffler, M., Griesbacher, A., Fuchs, K., and Kofer, J. (2014). Antimicrobial drug 07.001 use in Austrian pig farms: plausibility check of electronic on-farm records and Moreno, M. A. (2014). Survey of quantitative antimicrobial consumption per estimation of consumption. Vet. Rec. 175, 402. doi: 10.1136/vr.102520 production stage in farrow-to-finish pig farms in Spain. Vet. Rec. Open Wang, R., van Dorp, L., Shaw, L. P., Bradley, P., Wang, Q., Wang, X., et al. (2018). 1:e000002. doi: 10.1136/vropen-2013-000002 The global distribution and spread of the mobilized colistin resistance gene Nang, S. C., Li, J., and Velkov, T. (2019). The rise and spread of mcr plasmid- mcr-1. Nat. Commun. 9:1179. doi: 10.1038/s41467-018-03205-z mediated polymyxin resistance. Crit. Rev. Microbiol. 45, 131–161. doi: 10.1080/ Wall, B. A., Mateus, A., Marshall, L., and Pfeiffer, D. U. (2016). Drivers, Dynamics 1040841x.2018.1492902 and Epidemiology of Antimicrobial Resistance in Animal Production. Rome: Ojer-Usoz, E., Gonzalez, D., and Vitas, A. I. (2017). Clonal Diversity of ESBL- Food and Agriculture Organization. producing Escherichia coli isolated from environmental, human and food Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L. H., et al. (2006). Sex samples. Int. J. Environ. Res. Public Health 14:676. doi: 10.3390/ijerph1407 and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 0676 60, 1136–1151. doi: 10.1111/j.1365-2958.2006.05172.x Ovejero, C. M., Delgado-Blas, J. F., Calero-Caceres, W., Muniesa, M., and World Health Organization [WHO] (2017). Critically Important Antimicrobials for Gonzalez-Zorn, B. (2017). Spread of mcr-1-carrying Enterobacteriaceae in Human Medicine, 5th Edn. Geneva: World Health Organization. sewage water from Spain. J. Antimicrob. Chemother. 72, 1050–1053. doi: 10. Xavier, B. B., Lammens, C., Ruhal, R., Kumar-Singh, S., Butaye, P., Goossens, 1093/jac/dkw533 H., et al. (2016). Identification of a novel plasmid-mediated colistin-resistance Prim, N., Rivera, A., Rodriguez-Navarro, J., Espanol, M., Turbau, M., Coll, P., et al. gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 21:30280. (2016). Detection of mcr-1 colistin resistance gene in polyclonal Escherichia doi: 10.2807/1560-7917.es.2016.21.27.30280 coli isolates in Barcelona, Spain, 2012 to 2015. Euro Surveill. 21:30183. doi: Yin, W., Li, H., Shen, Y., Liu, Z., Wang, S., Shen, Z., et al. (2017). Novel plasmid- 10.2807/1560-7917.es.2016.21.13.30183 mediated Colistin resistance gene mcr-3 in Escherichia coli. MBio 8:e00543-17. Quesada, A., Ugarte-Ruiz, M., Iglesias, M. R., Porrero, M. C., Martinez, R., Florez- doi: 10.1128/mBio.00543-17 Cuadrado, D., et al. (2016). Detection of plasmid mediated colistin resistance Zajac, ˛ M., Sztromwasser, P., Bortolaia, V., Leekitcharoenphon, P., Cavaco, L. M., (MCR-1) in Escherichia coli and Salmonella enterica isolated from poultry and Ziȩtek-Barszcz, A., et al. (2019). Occurrence and characterization of mcr- swine in Spain. Res. Vet. Sci. 105, 134–135. doi: 10.1016/j.rvsc.2016.02.003 1-positive Escherichia coli isolated from food-producing animals in Poland, Quinn, M. M., Henneberger, P. K., Braun, B., Delclos, G. L., Fagan, K., 2011–2016. Front. Microbiol. 10:1753. doi: 10.3389/fmicb.2019.01753 Huang, V., et al. (2015). Cleaning and disinfecting environmental surfaces in Zurfluh, K., Klumpp, J., Nüesch-Inderbinen, M., and Stephan, R. (2016). Full- health care: toward an integrated framework for infection and occupational length nucleotide sequences of mcr-1-harboring plasmids isolated from illness prevention. Am. J. Infect. Control 43, 424–434. doi: 10.1016/j.ajic.2015. extended-spectrum-β-lactamase-producing Escherichia coli isolates of different 01.029 origins. Antimicrob. Agents Chemother. 60, 5589–5591. doi: 10.1128/aac. Raasch, S., Postma, M., Dewulf, J., Stark, K. D. C., and Grosse Beilage, E. (2018). 00935-16 Association between antimicrobial usage, biosecurity measures as well as farm Zurfluh, K., Nuesch-Inderbinen, M., Klumpp, J., Poirel, L., Nordmann, P., and performance in German farrow-to-finish farms. Porcine Health Manag. 4, 30. Stephan, R. (2017). Key features of mcr-1-bearing plasmids from Escherichia doi: 10.1186/s40813-018-0106-5 coli isolated from humans and food. Antimicrob. Resist. Infect. Control 6, 91. Rebelo, A. R., Bortolaia, V., Kjeldgaard, J. S., Pedersen, S. K., Leekitcharoenphon, doi: 10.1186/s13756-017-0250-8 P., Hansen, I. M., et al. (2018). Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for Conflict of Interest: The authors declare that the research was conducted in the surveillance purposes. Euro Surveill. 23:17–00672. doi: 10.2807/1560-7917.es. absence of any commercial or financial relationships that could be construed as a 2018.23.6.17-00672 potential conflict of interest. Rhouma, M., Fairbrother, J. M., Beaudry, F., and Letellier, A. (2017). Post weaning diarrhea in pigs: risk factors and non-colistin-based control strategies. Acta Vet. The handling Editor declared a shared affiliation, though no other collaboration, Scand. 59:31. doi: 10.1186/s13028-017-0299-7 with the authors JG-L and AM-M at time of review. Rownicki, M., Pienko, T., Czarnecki, J., Kolanowska, M., Bartosik, D., and Trylska, J. (2018). Artificial activation of Escherichia coli mazEF and hipBA toxin- Copyright © 2020 Migura-Garcia, González-López, Martinez-Urtaza, Aguirre antitoxin systems by antisense peptide nucleic acids as an antibacterial strategy. Sánchez, Moreno-Mingorance, Perez de Rozas, Höfle, Ramiro and Gonzalez- Front. Microbiol. 9:2870. doi: 10.3389/fmicb.2018.02870 Escalona. This is an open-access article distributed under the terms of the Creative Ruzauskas, M., and Vaskeviciute, L. (2016). Detection of the mcr-1 gene in Commons Attribution License (CC BY). The use, distribution or reproduction in Escherichia coli prevalent in the migratory bird species Larus argentatus. other forums is permitted, provided the original author(s) and the copyright owner(s) J. Antimicrob. Chemother. 71, 2333–2334. doi: 10.1093/jac/dkw245 are credited and that the original publication in this journal is cited, in accordance Sjolund, M., Postma, M., Collineau, L., Losken, S., Backhans, A., Belloc, C., with accepted academic practice. No use, distribution or reproduction is permitted et al. (2016). Quantitative and qualitative antimicrobial usage patterns in which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 11 January 2020 | Volume 10 | Article 3072