Whole Genome Sequencing and Characteristics of mcr-1–Harboring Plasmids of Porcine Escherichia coli Isolates Belonging to the High-Risk Clone O25b:H4-ST131 Clade B

fmicb-11-00387 March 21, 2020 Time: 9:8 # 1 ORIGINAL RESEARCH published: 24 March 2020 doi: 10.3389/fmicb.2020.00387 Edited by: Benjamin Andrew Evans, University of East Anglia, United Kingdom Reviewed by: Catherine M. Logue, University of Georgia, United States Chitrita Debroy, Pennsylvania State University (PSU), United States *Correspondence: Jorge Blanco [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Antimicrobials, Resistance, and Chemotherapy, a section of the journal Frontiers in Microbiology Received: 17 December 2019 Accepted: 24 February 2020 Published: 24 March 2020 Citation: Flament-Simon S-C, de Toro M, Mora A, García V, García-Meniño I, Díaz-Jiménez D, Herrera A and Blanco J (2020) Whole Genome Sequencing and Characteristics of mcr-1–Harboring Plasmids of Porcine Escherichia coli Isolates Belonging to the High-Risk Clone O25b:H4-ST131 Clade B. Front. Microbiol. 11:387. doi: 10.3389/fmicb.2020.00387 Whole Genome Sequencing and Characteristics of mcr-1–Harboring Plasmids of Porcine Escherichia coli Isolates Belonging to the High-Risk Clone O25b:H4-ST131 Clade B Saskia-Camille Flament-Simon1,2†, María de Toro3†, Azucena Mora1,2, Vanesa García1,2, Isidro García-Meniño1,2, Dafne Díaz-Jiménez1,2, Alexandra Herrera1and Jorge Blanco1,2* 1Laboratorio de Referencia de E. coli (LREC), Departamento de Microbiología y Parasitología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain, 2Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago, Spain, 3Plataforma de Genómica y Bioinformática, Centro de Investigación Biomédica de La Rioja (CBIR), Logroño, Spain Porcine Escherichia coli ST131 isolates are scarcely documented. Here, whole genome sequencing and core genome (CG) and plasmidome analysis of seven isolates collected from diarrheic piglets and four from pork meat were performed. All of the 11 ST131 isolates belonged to serotype O25b:H4 and clade B and showed fimH22 allele or mutational derivatives. The 11 porcine isolates possessed virulence traits that classified the isolates as avian pathogenic, uropathogenic, and extraintestinal pathogenic E. coli– like (APEC-, UPEC-, and ExPEC-like) and constituted virotype D. The CG was performed for all porcine isolates in addition to 73 ST131 reference isolates from different origins. Within clade B, the CG showed nine subclusters, allowing us to describe five new subclades (B6, B6-like, B7, B8, and B9). There was an association between subclade B6, PST43, virotype D2, and food origin, whereas subclade B7 included PST9 isolates with virotype D5 from diarrheic piglets (p= 0.007). The distance between human and porcine isolates from subclades B6 and B7 had an average of 20 and 15 SNP/Mb, respectively. [F2:A-:B1]-IncF, ColE1-like, and IncX plasmids were the most prevalent. Besides, IncF plasmids harbored a ColV region frequent among APEC isolates. Antimicrobial resistance genes conferring resistance to penicillin, tetracycline, quinolones, and colistin were the most common. The mcr-1.1 gene was detected in 5 of 11 porcine isolates, integrated into the chromosome of one isolate and into plasmids in the remainder isolates (two MOBH11/IncHI2-ST4, one MOBP3/IncX4, and one MOBF12/IncF [F2:A-:B1] supposedly cointegrated with an IncHI2). The surrounding environments of the mcr-1 cassette showed variability. However, there were conserved structures within the same plasmid family. In conclusion, CG analysis defined five new subclades. The ST131 porcine isolates belonged to new subclades B6 and B7. Moreover, porcine and clinical human isolates were strongly related. The 11 porcine ST131 isolates harbored a wide variety of plasmids, virulence, and resistance genes. Frontiers in Microbiology | www.frontiersin.org 1March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 2 Flament-Simon et al. Porcine ST131 Escherichia coli Furthermore, epidemic plasmids IncX4 and IncHI2 are responsible for the acquisition of mcr-1.1 gene. We hypothesize that the APEC-IncF plasmid acquired the mcr-1.1 gene via cointegrating an IncHI2 plasmid, which is worrying due to combination of virulence and resistance attributes in a single mobile genetic element. Keywords: Escherichia coli, ST131, swine, colistin, mcr-1.1, core genome, plasmidome INTRODUCTION Escherichia coli exists as part of the commensal microbiota in the mammalian digestive tract, as well as a zoonotic pathogen responsible for intestinal and extraintestinal infections in both humans and animals (Poirel et al., 2018). Treatment of multidrug-resistant (MDR) E. coli infections has become a serious clinical issue, especially with the emergence of high-risk clones, such as clone ST131 (Nicolas-Chanoine et al., 2008, 2014). ST131 clone is one of the most prevalent extraintestinal pathogenic E. coli clones (ExPEC). Although it remains unclear why this clone is prevailing over the others, antimicrobial resistance and virulence factors are suspected contributors (Dobrindt, 2005;Stoesser et al., 2016b). Even though ST131 clone is prevalent in human samples, it remains less frequent from animals and especially rare from porcine source (Reid et al., 2019). However, it has already been identified in many animal species, including in Antarctic pinnipeds (Mora et al., 2018). The majority of animal isolates belong to clade B (associated with fimH22), whereas in those that cause extraintestinal infections in humans predominate clade C (associated with fimH30) and especially those belonging to subclades C1-nM27, C1-M27, and C2 (H30Rx). This could be the reason of the underrepresentation of ST131 clade B isolates in the literature and sequence databases (Reid et al., 2019). Worryingly, resistance to last-resort antibiotics such as carbapenems and polymyxins has already been reported in the pandemic E. coli clone ST131 (Trobos et al., 2009;Schink et al., 2013;Hasman et al., 2015;Ewers et al., 2016;Kuo et al., 2016; Sonnevend et al., 2016;de Toro et al., 2017;Ortiz de la Tabla et al., 2017;García-Meniño et al., 2018;Liu H. et al., 2018;Ellaby et al., 2019;Hojabri et al., 2019;Reid et al., 2019). Colistin (polymyxin E) is associated with nephrotoxicity and neurotoxicity. However, it has been widely used in veterinary medicine. Livestock, and particularly porcine farming, has been singled out as reservoir for colistin resistance (García-Meniño et al., 2019) and foodborne pathogens (FBPs) that could have devastating health and economic consequences (Sekse et al., 2017). Intrinsic resistance to colistin has been related with two chromosomally encoded systems, the PhoPQ component and the pmrCAB operon (Olaitan et al., 2014;Poirel et al., 2018). However, the newly discovered plasmid-borne mobile colistin resistance (mcr) gene is responsible for a transferable mechanism of resistance (Liu Y.-Y. et al., 2016). Since the description of the mcr-1 gene by Liu Y.-Y. et al. (2016), several amino acid variants have been described, encoded in mcr-2 to mcr-9 genes (AbuOun et al., 2017;Borowiak et al., 2017;Carattoli et al., 2017;Yin et al., 2017;Wang et al., 2018;Yang et al., 2018;Carroll et al., 2019). Besides, several subvariants for some of them have been reported, that is, 13 nucleotide and protein variants in the mcr-1 family (designated mcr-1.1 to mcr-1.13) (Partridge et al., 2018). Colistin plasmid-borne resistance has widely spread geographically (Kempf et al., 2016). It has been described in numerous genera of Enterobacteriaceae including Escherichia, Moraxella,Klebsiella,Salmonella,Enterobacter,Cronobacter, Shigella,Kluyvera,Citrobacter, and Raoultella (Campos et al., 2016;Liu B.-T. et al., 2016;Liu Y.-Y. et al., 2016;Olaitan et al., 2016;Pham Thanh et al., 2016;Stoesser et al., 2016a;Zeng et al., 2016;Zhao and Zong, 2016;AbuOun et al., 2017;Li et al., 2017c; Luo et al., 2017). Although mcr genes have been found in a large diversity of clones, Matamoros et al. (2017) and García-Meniño et al. (2019) established the ST10 and ST155 E. coli clones as potential reservoirs of the mcr-1 gene. The mcr gene has been detected in many plasmid types, including IncI2, IncHI2, IncP, IncX4, IncY, IncF, and ColE10-like ones (Madec and Haenni, 2018) from different origins (Sun et al., 2018). Nevertheless, Matamoros et al. (2017) and García-Meniño et al. (2019) found that the majority of the mcr-carrier plasmids belonged mainly to four plasmid incompatibility groups: IncX4, IncI2, IncHI2, and ColE10-like. In contrast, it remains rare to find colistin resistance genes chromosomally encoded, in accordance to Li et al. (2018) studies, where the prevalence of chromosomal mcr carrier isolates was estimated in 4% of the analyzed isolates. Broadly, the mcr-1 cassette is described as an approximately 2,600 base pair (bp) fragment containing the mcr-1 gene followed by a phosphoesterase (Poirel et al., 2016). It has been proposed that the ISApl1 insertion sequence (IS) mediates the transmission of mcr-1 by forming circular intermediates, which can translocate (Tegetmeyer et al., 2008;Chandler and Siguier, 2013;Snesrud et al., 2016;Zurfluh et al., 2016a,b;Li et al., 2017a). Genomic tools allow a compressive characterization of FBPs and the identification of clonal groups of bacteria that represent public health hazards (Kovac et al., 2017). However, epidemiological surveillance of epidemic plasmids related to antimicrobial resistances or/and virulence genes spreading in the bacterial population is still complicated because of the intrinsic plasticity of plasmids (Orlek et al., 2017). In this study, we have performed the whole genome sequencing (WGS) analysis of 11 ST131 E. coli isolates from porcine samples. Our objectives were (I) perform a core genome (CG) analysis to establish the phylogenetic relationship of our isolates within clade B of ST131 E. coli isolates, (II) to determine and describe the genetic location of the resistance and virulence genes, (III) to investigate the role of mobile genetic elements (MGEs) in the dissemination of those genes, and (IV) to explore the genetic environment of the mcr-1 gene. As far as we know, Frontiers in Microbiology | www.frontiersin.org 2March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 3 Flament-Simon et al. Porcine ST131 Escherichia coli this study would be the first one in which the whole genome of an extended collection of ST131 clade B isolates from porcine origin, collected from diseased animals and food, has been performed. MATERIALS AND METHODS Epidemiological Background of the E. coli Collection In the present study, we performed WGS analysis of a collection of 11 resistant E. coli O25b:H4-B2-ST131 clade B isolates, seven from piglets with diarrhea and four from pork meat. The isolates belong to extensive epidemiological studies accomplished in Spain. The seven isolates from diarrheic piglets were isolated during period 2006–2016 (García-Meniño et al., 2018) and the four from pork meat during the years 2011 and 2012 (Herrera Estévez, 2015). Antimicrobial Susceptibility Testing Antimicrobial susceptibility was determined by minimal inhibitory concentrations by using the MicroScan WalkAwayR - automated system (BECKMAN COULTER, Inc., Brea, CA, United States) according to the manufacturer’s instructions. The antibiotics tested were ticarcillin, aztreonam, ceftazidime, cefepime, ampicillin–sulbactam, piperacillin–tazobactam, imipenem, meropenem, amikacin, gentamicin, tobramycin, levofloxacin, ciprofloxacin, trimethoprim–sulfamethoxazole, fosfomycin, colistin, minocycline, and tigecycline. Additionally, resistance to ampicillin, cefotaxime, chloramphenicol, and nalidixic acid was determined by disk diffusion assays (Becton Dickinson, Sparks, MD, United States). All results were interpreted according to the CLSI guidelines (Wayne, 2017). Multidrug resistance status was attributed to those isolates resistant to at least one agent of three or more different antimicrobial categories, including resistance to β-lactamase inhibitors (Magiorakos et al., 2012). Serotyping, Phylogenetic Grouping, Multilocus Sequence Typing, CH Typing, and Virulence Genotyping ST131 isolates were characterized with regard to O:H serotypes, phylogenetic groups, clonotypes (fumC and fimH genes), sequence type by multilocus sequence typing (MLST) (according to the Achtman scheme and by the Pasteur Institute scheme), and 34 extraintestinal virulence-associated genes encoding adhesins (fimH,fimAvMT78,papAH,papC,papEF,papGII,papGIII, sfa/focDE,afa/draBC,yfcV), toxins (sat,cnf1,hlyA,hlyF,cdtB, tsh,vat), siderophores-iron uptake (iucD,iutA,iroN,fyuA,chuA), capsule (kpsM II,kpsM II-K2,kpsM II-K5,neuC-K1,kpsM III), and miscellaneous [cvaC,iss,traT,ibeA,malX (PAI), usp, ompT] as described previously (Clermont et al., 2013;Dahbi et al., 2014;Mamani et al., 2019). Based on the definitions given by Johnson et al. (2008, 2015) and Spurbeck et al. (2012), the isolates that genetically satisfied the following criteria (I) positive for ≥2 of 5 markers, including papAH and/or papC, sfa/focDE,afa/draBC,kpsM II, and iutA; (II) positive for three or more of four markers, including chuA,fyuA,vat, and yfcV; and (III) positive for ≥4 of 5 markers, including hlyF,iutA, iroN,iss, and ompT, were presumptively designated as ExPEC, uropathogenic E. coli (UPEC), and avian pathogenic E. coli (APEC)–like isolates, respectively, taking into account that their site of isolation (meat and feces) and animal species differ from the original definitions. The virotypes (A–F) of the ST131 isolates were established according to the scheme described by Dahbi et al. (2014). Primers used in this study for polymerase chain reaction (PCR) amplification of virulence genes are indicated in Supplementary Table S1. WGS, Assembly, and Primary Analysis Total DNA was extracted with the QIAmp DNA Mini Kit (Qiagen GmbH, Qiagen Strasse 1, Hilden, Germany). Libraries were prepared by using the TruSeq DNA PCR-Free protocol (Illumina, San Diego, California, United States) at the Genomics and Bioinformatics Core Facility (Centre for Biomedical Research of La Rioja). Paired-end 100-bp reads on fragments of 550-bp insert size were sequenced in an Illumina HiSeq 1500. Genomes were reconstructed by using PLACNETw (Vielva et al., 2017). Identification of Open Reading Frames (ORFs) and genome annotation of the assembled genetic elements was performed by using Prokka (Seemann, 2014). Genomes were in silico typed by the following databases: SerotypeFinder (Joensen et al., 2015), MLSTtyper (Larsen et al., 2012), and CHtyper (Camacho et al., 2009). Three different databases were used for the identification of antibiotic resistance genes: ResFinder (Zankari et al., 2012), CARD Resistance Gene Identifier (McArthur et al., 2013), and ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) (Gupta et al., 2014). Besides, the PointFinder database (Zankari et al., 2017) was used to determine point mutations. The VirulenceFinder (Ren et al., 2017) and the VFDB (Chen et al., 2005) databases were used to explore virulence factors. The genetic environment of the mcr detected genes was manually revisited and compared to previously reported ones with CLC Sequence Viewer (version 8.9; Qiagen) and EasyFig tools (Sullivan et al., 2011). CG and Phylogenetic Analysis For phylogenetic analysis, we used the 11 genomes of the porcine E. coli sequenced in this study plus 73 full-genomes references from the ST131 clone retrieved from the Enterobase1and the NCBI Bioproject Database.2The CG was defined as the collection of genes present at least once in all the ST131 genomes analyzed, with more than 90% similarity and 90% coverage, as defined by Lanza et al. (2014). All 84 genomes used in this study and their accession number are available on Supplementary Table S2. The analysis included genomes representative of all clades and subclades described by Ben Zakour et al. (2016) and Matamoros et al. (2017). Plasmid Analysis Plasmid reconstruction from WGS data was performed by PLACNETw method (Vielva et al., 2017). In PLACNETw representation, most plasmids can be recognized by their 1http://enterobase.warwick.ac.uk/species/index/ecoli 2https://www.ncbi.nlm.nih.gov/bioproject/ Frontiers in Microbiology | www.frontiersin.org 3March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 4 Flament-Simon et al. Porcine ST131 Escherichia coli replication initiation proteins (RIPs) and/or RELaxase proteins (REL), both of them considered as plasmid markers. Incompatibility groups (Inc) and pMLST subtypes were in silico determined with PlasmidFinder and pMLST (Carattoli et al., 2014), respectively, via the CGE online services.3Besides, reconstructed plasmids were subtyped according to the exact relaxase family subtype by phylogenetic comparison with previously defined relaxases subfamilies (Alvarado et al., 2012). Reconstructed plasmids and references belonging to the same Inc groups were compared by using BRIG (Alikhan et al., 2011) and EasyFig tools (Sullivan et al., 2011). All reference plasmids used in this study were recovered from NCBI database4 (Supplementary Table S3). Statistics Normality test was performed (Shapiro–Wilk test). A nonparametric Mann–Whitney–Wilcoxon test on paired data was conducted. A significance level of 0.05 was used for all tests. All statistical analyses were carried out with XLSTAT software.5 RESULTS Pasteur Sequence Types and fimH Alleles All of the 11 ST131 isolates belonged to serotype O25b:H4 and the phylogenetic group B2. The seven isolates from piglets with diarrhea showed the Pasteur sequence type (PST) 9, whereas the four isolates from pork meat showed PST43 (Table 1). Also all of the 11 ST131 isolates belonged to clade B and showed the fimH22 allele or mutational derivatives of the fimH22 allele (fimH161, fimH298, fimH326, fimH332, fimH336, fimH338, and fimH374), resulting in clonotypes CH40-22 (3 isolates), CH40-161 (1), CH40-298 (2), CH40-326 (1), CH40-332 (1), CH40-336 (1), CH40-338 (1), and CH40-374 (1). Virotypes The 11 ST131 isolates harbored between 17 and 20 virulence gene markers screened by PCR. All isolates harbored the following genes: iroN, iss,hlyF, ompT, papEF, papG III, kpsM-II-K5, iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, and usp. However, some genes were variables: papC and papAH (10 isolates), cnf 1 (six isolates), hlyA (five isolates), and cdtB (four isolates). All isolates belonged to virotype D (Table 1). Six of seven isolates recovered from piglets with diarrhea with PST9 showed the virotype D5 (ibeA,papG III, cnf1,hlyA, and kpsM-II-K5), whereas the four PST43 isolates from meat showed the virotype D2 (ibeA,papG III, cdtB, and kpsM-II-K5). All the 11 porcine ST131 isolates exhibited a virulence profile that satisfied the APEC, UPEC, and ExPEC status and because of their origin were classified as APEC, UPEC, and ExPEC like isolates (Table 1 and Supplementary Table S1). 3http://genomicepidemiology.org/ 4https://www.ncbi.nlm.nih.gov/pubmed 5https://www.xlstat.com Resistance Phenotype The resistance profile of the 11 ST131 porcine isolates was determined, showing that all but one exhibited an MDR phenotype. Amoxicillin/clavulanic acid resistance was present in 10 isolates, quinolone resistance in seven, colistin resistance in five isolates (with mcr-1 gene), cotrimoxazole resistance in four isolates, chloramphenicol resistance in two isolates, aminoglycoside resistance in two isolates, and tetracycline resistance in one isolate (Table 1). Whole Genome Sequencing The draft genomes of the 11 ST131 porcine isolates yielded 32 to 78 contigs larger than 1 Kb, with assembly sizes ranging from 4.940 to 5.451 Mb (Supplementary Table S4). The WGS data were in complete agreement with our previous characterization performed with conventional methods. CGand Phylogenetic Analysis A total of 84 E. coli genomes were taken from different clades and subclades of ST131 in order to calculate the ST131 CG. The main objectives were in regard to insights of ST131 lineage and the relationship between isolates of different origins, with special attention to clade B of ST131 lineage. Because of that, the CG analysis was dominated by clade B (n= 49, 58%), and the remainder isolates belonged to clade C (n= 26, 31%), clade A (n= 8, 10%), and clade I (n= 1, 1%). The final collection represented sequences from human (n= 51, 61%), porcine (n= 19, 23%), avian (n= 8, 10%), and wild animals (Antarctic pinnipeds) (n= 6, 7%) sources. The CG encompasses 2.98 Mb (length = 2,983,333 ±7,338 pb), which comprised 3,100 total CDS. The single nucleotide polymorphisms (SNPs) analysis of the CG performed within ST131 lineage revealed a well-defined clade structure, similar to those previously described, including the three defined clades: A, B, and C. In total, 14,838 SNPs were present in the alignment. Furthermore, there was a distance of 7,742 to 8,565 SNPs between isolates from clade A and clade C; 6,508 to 8,675 between isolates from clade A and clade B; and 2,214 to 4,280 between isolates from clade B and clade C. The distance within clades varies greatly: being 36 to 1,782 SNPs in clade A, 8 to 2,051 SNPs in clade B, and 1 to 723 SNPs in clade C (Figure 1 and Supplementary Table S2). All isolates of porcine origin analyzed in this study were grouped in clade B. As shown in Figure 1, clade B was characterized by the presence of a mixture of human, livestock, and food origin genomes and showed the greatest intragroup diversity. We identified 11 clusters or subclades (B0, B1, B2, B3, B4, B5, B6, B6-like, B7, B8, and B9), including three subclades (B3, B6, and B7) with isolates of both human and animal origin. In this study, we described five new subclades, named B6, B6-like, B7, B8, and B9. There was a distance of 167 to 232 SNPs between isolates from B6 and B6-like and 172 to 304 SNPs between isolates from B6 and B7. Further, B7 showed 119 to 145 SNPs distance with B1. Those subclades appeared as the closer within each other in clade B (Supplementary Table S2). The 11 ST131 porcine isolates from this study were grouped in the new subclades B6 (LREC_160/PST43, LREC_161/PST43, LREC_168/PST43, LREC_176/PST43 from pork meat and Frontiers in Microbiology | www.frontiersin.org 4March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 5 Flament-Simon et al. Porcine ST131 Escherichia coli TABLE 1 | Features, molecular typing, virulence, and resistance profiles of ST131 Escherichia coli isolates from porcine origin. Isolate Origin (year) Serotype PhylogroupaClonotypebMLSTcVirulence profiled,eVirotypefResistance phenotypeg MDR +Ih LREC_153 (FV11838) Diarrhea (2008) O25:H4 B2 CH40-332 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cnf1,hlyA D5 AMC, AMP, AMP/SAM, CZ, GEN, NAL, TI, TOB, TMP/SMX + LREC_154 (FV9067) Diarrhea (2006) O25:H4 B2 CH40-22 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp D-ntiAMC, AMP, AMP/SAM, CHL, COL, NAL, TI + LREC_155 (FV14441) Diarrhea (2010) O25:H4 B2 CH40-374 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cnf1,hlyA D5 AMC, AMP, AMP/SAM, TI − LREC_157 (FV14983) Diarrhea (2010) O25:H4 B2 CH40-161 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cnf1,hlyA D5 AMC, AMP, AMP/SAM, NAL, TI + LREC_158 (FV15156) Diarrhea (2010) O25:H4 B2 CH40-326 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cnf1,hlyA D5 AMC, AMP, AMP/SAM, NAL, TI + LREC_159 (FV12310) Diarrhea (2009) O25:H4 B2 CH40-338 ST131/PST9 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cnf1 hlyA D5 AMC, AMP, AMP/SAM, COL, GEN, MI, NAL, TI, TOB, TMP/SMX + LREC_160 (C153-3A) Meat (2012) O25:H4 B2 CH40-298 ST131/PST43 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cdtB D2 AMC, AMP, AMP/SAM, CZ, COL, PI/TZP, TI + LREC_161 (C187-6A) Meat (2012) O25:H4 B2 CH40-22 ST131/PST43 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cdtB D2 AMC, AMP, AMP/SAM, COL, TI, TMP/SMX + LREC_162 (FV14984) Diarrhea (2010) O25:H4 B2 CH40-336 ST131/PST9 iroN, iss,hlyF, ompT, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA,usp,cnf1 D5 AMC, AMP, AMP/SAM, CHL, NAL, TI + LREC_168 (C41-4A) Meat (2011) O25:H4 B2 CH40-298 ST131/PST43 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cdtB, tsh D2 AMC, AMP, AMP/SAM, NAL, TI + LREC_176 (C84-4A) Meat (2011) O25:H4 B2 CH40-22 ST131/PST43 iroN, iss,hlyF, ompT, papC, papAH, papEF (GIII), kpsM-II (K5), iucD, iutA, chuA, fyuA, yfcV, cvaC, traT, malX,ibeA, usp,cdtB D2 AMP/SAM, COL, TI, TMP/SMX + (a) Phylogroups by Clermont et al. (2013); (b) CH, clonotype (fumC-fimH alleles); (c) ST, sequence type according to Achtman scheme; PST, sequence type according to the Pasteur scheme; (d) Extraintestinal virulence genes found by PCR; (e) All isolates exhibited the ExPEC, UPEC and APEC status; (f) The virotype was determined by PCR, based on the presence or absence of 13 virulence genes (Dahbi et al., 2014); (g) AMP, ampicillin; AMC, amoxicillin–clavulanic acid; AMP/SAM, ampicillin–sulbactam; CZ, ceftazidime; CHL, chloramphenicol; COL, colistin; GEN, gentamicin, MI, minocycline; NAL, nalidixic acid; TI, ticarcillin; TMP/SMX, trimethoprim–sulfamethoxazole; TOB, tobramycin; PI/TZP, piperacillin–tazobactam; (h) multidrug drug resistance (MDR); being resistant to at least one agent of three or more different antimicrobial categories including resistance to β-lactamase inhibitors (I); (i) nt, non-typable. Frontiers in Microbiology | www.frontiersin.org 5March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 6 Flament-Simon et al. Porcine ST131 Escherichia coli FIGURE 1 | Phylogenetic analysis by core genome of the Escherichia coli sequenced in this study (n= 11) plus other references (n= 73) from ST131 clone. virotype D2) and B7 (LREC_153/PST9, LREC_154/PST9, LREC_155/PST9, LREC_157/PST9, LREC_158/PST9, LREC_159/PST9, LREC_162/PST9 from piglets with diarrhea and virotype D5). The new subclade B6-like includes two isolates from porcine origin. Otherwise, new subclades B8 and B9 only include ST131 isolates from Antarctic pinnipeds. Notably, there were three (H1410, 19BA, H1698) isolates from human origin in subclade B6 and two (EcHUCA4, E61BA) in subclade B7. The distance between human isolates and porcine isolates from subclades B6 and B7 had an average of 60 SNPs and of 46 SNPs (i.e., 20 and 15 SNP/Mb), respectively. Plasmidome Analysis The 11 ST131 porcine isolates showed high heterogeneity in their plasmid content, harboring between two and seven different plasmids (Table 2 and Supplementary Figure S1). We find members of eight of the 17 main MOB plasmid groups found in γ−proteobacteria, ordered by prevalence: MOBF12 (n= 13), MOBP51 (n= 8), MOBP3 (n= 3), MOBV2 (n= 2), MOBH11 (n= 2), MOBP12 (n= 2), MOBP131 (n= 1), and MOBQ12 (n= 1). Finally, we described six plasmids (16%) in which any MOB protein was found (non-mobilizable-infective plasmids). Twenty-two plasmids were considered large (>33 kb) and presumptively conjugative, and sixteen were small multicopy plasmids (<7 kb). In summary, 38 plasmids were described in the 11 genomes analyzed. The plasmids belonged to the following relaxase families (MOB) and incompatibility groups (Inc): MOBP51/ColE1-like (n= 8), MOBF12/IncF [F2:A-:B1] (n= 8), MOBF12/IncF [F2:A-:B1] plus and presumptively cointegrated IncHI2-ST4/ST-nt (not typable) (n= 3), MOBF12/IncF [F10:A- :B1] (n= 2), MOBP12/IncI1-ST27/ST171 (n= 2), MOBP3/IncX4 (n= 2), MOBV2/nd (not detectable) (n= 2), MOBH11/IncHI2ST4 (n= 1), MOBH11/IncHI2-ST4 plus and presumptively cointegrated IncQ1 (n= 1), MOBP131/IncL/M plus and presumptively cointegrated IncQ1 (n= 1), MOBP3/IncX1 (n= 1), and MOBQ12/nd (n= 1). We also localized six plasmids that could not be affiliated with any categories. All 13 MOBF12/IncF plasmids were carriers of virulence genes. The genes iroBCDEN, iss,mchF, iutA, and iucABCD were constant (Table 2). All the MOBF12/IncF plasmids found in our study were structurally compared by BRIG software, using the pJIE186_2 (NC_020271) plasmid as reference (Supplementary Figure S2). The plasmids analyzed showed extensive sequence similarity over at least 100 kb, which includes not only backbone genes. The comparison emphasizes that all of them share an 80kb conserved region comprising the virulence genes described previously as well as cvaABC,sitABC,ompT, and hlyF. The eight MOBP51/ColE1-like plasmids identified in this study exhibited a wide size range (1.7–6.8 kb) and were Frontiers in Microbiology | www.frontiersin.org 6March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 7 Flament-Simon et al. Porcine ST131 Escherichia coli TABLE 2 | Plasmid content of the ST131 Escherichia coli isolates from porcine origin and location of resistance and virulence genes. Isolate Genetic elementa No. of Contigs Size (bp) No. of copiesb Relaxase proteinc Replication proteinc Inc group (pMLST)d Virulence geneseResistence genesf, point mutations and efflux/transporter genes LREC_153 chr_LREC153 40 4,904,938 1 ndhnd nagpapACDEFHJKX, hlyABD, cnf1, chuA PBP, AmpH, gyrA_S83L, mdfA pLREC153_1 8 330,910 1 MOBF12, MOBH11 RptA1 IncF [F2:A- :B1][IncHI2-ST4]i iroBCDEN, iss, mchF, iutA, iucABCD pLREC153_2 16 102,056 1 MOBP131 RptZ, RptC IncL/M [IncQ1]iblaTEM−1A/blaTEM−54/blaTEM−150, AAC(30)-IIa, AAC(30)-IIc, APH(30)-Ia, APH(30’)-Ib, APH(60)-Id, aadA1, strA, strB, sul1, sul2, sul3, dfrA1, dfrA15, tet(A), tet(C), qacEdelta1 pLREC153_3 1 6,851 5 MOBP51 nd ColE1-like pLREC153_4 1* 2,461 4 MOBV2 RepL nd LREC_154 chr_LREC154 111 4,946,997 1 nd nd na papBCDEFHIJKX, iss, iroBCDEN, iucABCD, iutA PBP, AmpH, gyrA_S83L, mdfA, mcr-1.1 pLREC154_1 2 124,513 1 MOBF12 RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD pLREC154_2 27 56,729 3 MOBP3 RptF IncX4 blaTEM−1C/blaTEM−40/blaTEM−135, tet(A), tet(C), tet(M), tet(R) LREC_155 chr_LREC155 60 4,947,621 1 nd nd na papACDEFHJKX, hlyABD, cnf1, chuA, mchC, iha, iroBCDEN, iucABCD, iutA PBP, AmpH, mdfA pLREC155_1 25 316,521 1 MOBF12 RptA1 IncF [F2:A- :B1][IncHI2-ST-nt]i iroBCDEN, iss,cma, cba, mchF, iutA, iucABCD pLREC155_2 23 208,964 1 MOBP12 RptZ IncI1-ST27 blaTEM−1C/blaTEM−40/blaTEM−135,tet(A), tet(C), tet(R) LREC_157 chr_LREC157 79 4,896,484 1 nd nd na papACDEFHJKX, hlyABD, cnf1, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, gyrA_S83L, mdfA pLREC157_1 12 137,775 1 MOBF12 RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135 pLREC157_2 1 46,172 1 MOBP3 RptF IncX1 pLREC157_3 1 6,851 3–4 MOBP51 nd ColE1-like pLREC157_4 1* 2,047 5 MOBV2 nd (RepA partial hit) nd LREC_158 chr_LREC158 58 4,853,444 1 nd nd na papACDEFHJKX, hlyABD, cnf1, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, blaTEM−1C/blaTEM−40/blaTEM−135, gyrA_S83L, mdfA pLREC158_1 5 135,569 1 MOBF12 RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD tet(A), tet(C), tet(R) pLREC158_2 1* 6,648 4 MOBP51 nd ColE1-like pLREC158_3 1* 1,554 5 nd RptL2 nd (Cryptic_1) LREC_159 chr_LREC159 97 4,869,944 1 nd nd na papACDEFHJKX, hlyABD, cnf1, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, gyrA_S83L, mdfA pLREC159_1 10 248,461 1 MOBH11 RptC IncHI2-ST4 [IncQ1]iAPH(30)-Ia, APH(300)-Ib, APH(6)-Id, strA, strB, sul2, sul3, mcr-1.1 pLREC159_2 9 177,070 1 MOBF12 RptA1 IncF[F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135, aadA1, AAC(3)-IIa, AAC(3)-IIc, sul1, sul2, dfrA1, drfA15,tet(A), tet(C), tet(R), qacEdelta1 pLREC159_3 1 68,523 1 MOBP12 RptZ Incl1-ST171 pLREC159_4 1 2,014 2 MOBQ12 nd nd aadA2, ANT(300)-Ia, linG, lnu(F) pLREC159_5 1* 1,553 4 nd RptL2 (HTH36 family) nd (Cryptic_1) (Continued) Frontiers in Microbiology | www.frontiersin.org 7March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 8 Flament-Simon et al. Porcine ST131 Escherichia coli TABLE 2 | Continued Isolate Genetic elementa No. of Contigs Size (bp) No. of copiesb Relaxase proteinc Replication proteinc Inc group (pMLST)d Virulence geneseResistence genesf, point mutations and efflux/transporter genes pLREC159_6 1* 1,507 3 nd nd (RepA partial hit) nd (Cryptic_2) LREC_160 chr_LREC160 107 5,049,024 1 nd nd nd papCDEFJK, sfaX, mchCF, iha, iss, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, mdfA pLREC160_1 18 145,187 1 MOBF12 RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135, tet(A), tet(C), tet(R) pLREC160_2 5 40,221 2 MOBP3 RptF IncX4 blaTEM−1C/blaTEM−40/blaTEM−135, mcr-1.1 pLREC160_3 1 5,630 3 MOBP51 nd ColE1-like pLREC160_4 1 5,006 4–5 MOBP51 nd ColE1-like pLREC160_5 1 4,515 3–4 MOBP51 nd ColE1-like pLREC160_6 1 1,765 3–4 MOBP51 nd ColE1-like pLREC160_7 1* 1,551 4 nd RptL2 nd (Cryptic_1) LREC_161 chr_LREC161 64 5,072,645 1 nd nd na papCDEFJK, sfaX, iss, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, mdfA pLREC161_1 8 330,357 1 MOBF12 RptA1 IncF [F2:A- :B1][IncHI2-ST-nt]i iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135, mcr-1.1, tet(A), tet(C), tet(R) pLREC161_2 15 33,473 2 nd RptF nd blaTEM−1C, aadA2, aadA17, ANT(300)-Ia, sul3, drfA12, linG, lnu(F), mef(B) pLREC161_3 1 6,826 3 MOBP51 nd ColE1-like pLREC161_4 1* 1,553 4 nd RptL2 (HTH36 family) nd (Cryptic_1) LREC_162 chr_LREC162 46 4,720,511 1 nd nd na chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, gyrA_S83L, mdfA pLREC162_1+2J29 216,025 1 MOBF12 (2) J RptA1 IncF [F10:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135 LREC_168 chr_LREC168 113 5,117,208 1 nd nd na papCDEFJK, sfaX, iss, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, gyrA_S83L, mdfA pLREC168_1+2J13 144,861 1 MOBF12 (2) J RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD blaTEM−1C/blaTEM−40/blaTEM−135, tet(A), tet(C), tet(R) LREC_176 chr_LREC176 145 5,039,239 1 nd nd na papDFJKX, sfaX, iss, chuA, iroBCDEN, iucABCD, iutA PBP, AmpH, mdfA pLREC176_1 15 278,079 1 MOBH11 RepFIB_RepA (x2) IncHI2-ST4 blaTEM−1A/blaTEM−150, aadA1, aadA2, APH(6)-Id, APH(300)-Ib, strA, strB, sul1, sul2, sul3, drfA1, drfA15, mcr-1.1, tet(A), tet(C), tet(R), cmlA1, cmlA6, catA1, qacEdelta1, qacHR pLREC176_2 9 132,494 1 MOBF12 RptA1 IncF [F2:A-:B1] iroBCDEN, iss, mchF, iutA, iucABCD aChr, chromosome; p, plasmid. bApproximative number of plasmid copies based on sequencing coverage identified by PLACNET. cRelaxase and replication proteins were identified by the use of PLACNET databases. dIncompatibility groups were determined according to the PBRT scheme and pMLST subtypes according to the allele scheme in http://pubmlst.org/plasmid/. eVirulence genes were detected through VirulenceFinder and VFDB databases (minimum coverage of 90%, minimum identity of 75%); all genomes presented in their chromosome the following genes: aslA, cheBWY, chuSTUVWXY, csgBDEFG, entABCEFS, fdeC, fepABCDG, fes, fimABCDEFGHI, flgG, flhAC, fliGIMP, fyuA, gad, gspCDEFGHIJKLM, ibeA, irp1, irp2, kpsDM, OmpA, yagVWXYZ/ecpEDCBA, ybtAEPQSTUX, ykgK/ecpR. fAntimicrobial resistance genes were detected by using ARG-ANNOT, CARD and ResFinder databases (minimum coverage of 93%, minimum identity of 75%); all genomes presented in their chromosome the following genes (efflux pumps and transport modulators): acrAEFS, bacRS, cpxA, CRP, emrABEKRY, eptA, evgAS, gadWX, H-NS, kdpE, marA, mdtABCEFGHKNOP, msbA, pmrF, tolC, ugd, yojI. gna, not-applicable; hnd, not detected. iCointegrated plasmid. jTwo plasmids that could not be separated (two MOBF12).*Closed plasmids. Frontiers in Microbiology | www.frontiersin.org 8March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 9 Flament-Simon et al. Porcine ST131 Escherichia coli analyzed using the ColE1 plasmid (J01566.1) as reference (Supplementary Figure S3). The plasmids pLREC153_3, pLREC157_3, pLREC158_2, and pLREC161_3 (all of them >6.8 Kb) showed the highest homology to the reference plasmid. All of them carried the colicin E1 cea and immunity (imm) genes and the entry exclusion (exc1-exc2) system genes. On the other hand, plasmids from isolate LREC_160 were different among them and with the others, exhibiting a wide size range (1.8–5.6 Kb), and only pLREC160_4 carried the colicin E1 gene. Three MOBP3/IncX plasmids were present among our isolates. We found pLREC157_2 that belong to the IncX1 subcluster and had a similar backbone than p2ESCUM (NC_011739.1) but codes for a completely different RIP (Supplementary Figure S4), whereas pLREC154_2 and pLREC160_2 belong to the IncX4 subcluster and were analyzed using pSH696_34 (JX258654.1) as a reference (Supplementary Figure S5). Because pLREC160_2 carried the mcr.1-1 gene, some previously reported IncX4 reference plasmids harboring the mcr gene were added. Compared to pSH696_34, plasmid pLREC160_2 and the other mcr carrier plasmids (pESTMCR and pICBEC7Pmcr) lacked two important backbone genes: the conjugative coupling protein gene (traG) and the replication protein encoding region (rep). Structural comparison of the two MOBP12/IncI1-ST171 plasmids and the single MOBP131/IncL/M plasmid found within our isolates was also performed using pEK204 (EU935740) and pEC743-OXA48 (CP015071.1) as references, respectively. Both showed a high coverage of homology with the reference (Supplementary Figures S6, S7). We also described five small (1.5 kb) multicopy plasmids in which any relaxase protein was found, categorized as cryptic. Those cryptic no-MOB plasmids were analyzed by using pEC10D (NC_017650.1) as reference (Supplementary Figure S8). BRIG comparison showed that four plasmids were highly similar to pEC10D and codes for the same replication protein (cryptic 1, from the HTH36 superfamily replication proteins), whereas pLREC159_6 codes for a different replication protein and was called cryptic 2 (Table 2). Finally, we analyzed the MOBV2 plasmids found in the isolates LREC_153 and LREC_157. A BLAST research was performed to identify a reference plasmid, being pEC0674 (MF684783.1) as the closest match. Two MOBV2-like–related plasmids were also included for structural comparison (Supplementary Figure S9). Although no significant similarity was found between these latter two references and the pEC0674, the pLREC153_4 showed similarity with the replication protein, a hypothetical one and the Rec protein from the reference plasmid, whereas the pLREC157_4 only showed similarity to the latter two proteins. Antimicrobial Resistance Genes Many antimicrobial resistance genes (ARGs) were found in the 11 ST131 porcine isolates (Table 2), including acquired resistance genes, point mutations, and efflux/transporter genes. Besides constitutive genes (PBP and AmpH, which were present in all isolates), ARGs conferring resistance to penicillin (variants of blaTEM gene, accounting: blaTEM−1C,n= 10; blaTEM−40,n= 10; blaTEM−135,n= 10; blaTEM−150,n= 2; blaTEM−1A,n= 2; blaTEM−54,n= 1), tetracycline (variants of tet gene, accounting: tetA, n= 9; tetC, n= 9; tetR, n= 8; tetM, n= 1), quinolones (gyrA_S83L, n= 7), and colistin (mcr1.1, n= 5) were the most common. In addition, we described genes conferring resistance to sulfonamides (variants of sul gene, accounting: sul2, n= 4; sul3, n= 4; sul1, n= 3), trimethoprim (variants of drf gene, accounting: dfrA1, n= 3; dfrA15, n= 3; dfrA12, n= 1), aminoglycosides (different ACCs, APHs, and ANTs genes, accounting: APH-300-Ib, n= 3; APH-60-Id, n= 3; aadA1, n= 3; ANT-300-Ia, n= 3; strA, n= 3; strB, n= 3; APH-30-Ia, n= 2; aadA2, n= 2; AAC-30-IIa, n= 2; AAC-30-IIc, n= 2; aadA17, n= 1), phenicols (variants of cmlA gene, catA1, and mdf A gene, accounting: mdfA,n= 11; cmlA1, n= 1; cmlA6, n= 1; catA1, n= 1), and to ammonium quaternary biocide (qacEdelta1, n= 3; qacHR, n= 1) (Table 2). The phenotypic resistance profile in the studied genomes was due to genes carried by plasmids, except for quinolone resistance, which in our study is mediated by a Ser83Leu point mutation found in the chromosomal gyrA gene. In the case of LREC_158 isolate, it exhibited the blaTEM genes (−1c, −40, and −135) variants set into a chromosomal assembled contig. However, these genes were located into IncL/M, IncX4, IncI1, IncF, and IncHI2 in the other analyzed isolates (Table 2). As shown in Table 1, all but one isolate exhibited an MDR profile. Large conjugative MDR plasmids were identified in four of the 10 isolates. These were typed as MOBH11/IncHI2-ST4 (pLREC159_1 and pLREC176_1), MOBF12/IncF (pLREC159_2, pLREC161_1), and MOBP131/IncL/M (pLREC153_2) and a nontypable one (pLREC161_2). Most common antibiotic resistance profiles encoded by these plasmids are ampicillin/penicillin, aminoglycosides, tetracyclines, sulfonamides, cotrimoxazole, and polymyxins. Finally, we described some drug efflux genes in pLREC159_4 and pLREC161_2 (Table 2). Class 1 integrons were present in pLREC153_2 (IntI-attI [-sul2 +aph-(300)-Ib +aph-(6)-Id +dfrA1 +aadA1]-qacE1sul1), pLREC159_2 (IntI-attI [dfrA1 +aadA1]-qacE1-sul1), and pLREC176_1 (IntI-attI [dfrA1 +aadA1]-qacE1-sul1). No class 2 or 3 integrons were detected in our genomes. Colistin Resistance Vehicles and Genetic Environment Colistin resistance by mcr gene was found in five of the analyzed isolates. Therefore, the mcr-carrying plasmids were investigated in greater depth. The mcr-1.1 gene was located in two MOBH11/IncHI2-ST4, a MOBP3/IncX4 and a MOBF12/IncF [F2:A-:B1], which was theoretically cointegrated with an IncHI2ST-nt plasmid. Besides, the LREC_154 isolate showed the mcr1.1 in a chromosomal location (Table 2). In this study, any point mutation in the chromosomally encoded genes pmrA or pmrB was found. The complete sequence of pLREC160_2 (MOBP3/IncX4) was 40,221 bp in size and contains 69 predicted ORFs. A structural comparison against other reference IncX4 plasmids is shown in Figure 2, where pICBEC7Pmcr (34,992 pb, NZ_CP017246.1) was used as the internal reference. The structure of the mcr-1 carriers IncX4 plasmid remains stable if we exclude one ORF encoding for an IS91 family transposase present Frontiers in Microbiology | www.frontiersin.org 9March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 16 Flament-Simon et al. Porcine ST131 Escherichia coli However, pLREC159_1 (IncHI2) presumptively harbored the ISApl1 upstream, whereas any copy of ISApl1 was present around the mcr-1 gene in pLREC160_2 (IncX4). Nevertheless, pap2 gene and ISs IS26 and IS1924 were present in pLREC160_2. Zhong et al. (2018) suggested that for IncX4 plasmids IS26/26 structures could be involved in mcr-1 gene mobilization. The BRIG comparison showed that the mcr-1–harboring IncX4 plasmids were genetically homologous between each other as previously described (Sun et al., 2017), suggesting that mobilization of the mcr-1 cassette was quite stable within this type of plasmids. Furthermore, pLREC159_1 and pLREC176_1 were almost identical in structure around the mcr-1 gene and were related to the presence of tellurium resistance genes, as well as for pLREC161_1. This heavy metal resistance lends HI2 plasmids to success in wastewater environments (Reid et al., 2019). Moreover, a highly relevant finding of the present study was the appearance of chromosomally encoded colistin resistance. We found a presumptive ISApl1–mcr-1–orf–ISApl1-like structure. This genetic arrangement was described as the most prevalent by Li et al. (2018) in bacterial chromosomes. It has been proposed that the lack of one or both of the ISApl1-elements implies stabilization of the mcr-1 gene (non-transposable) (Snesrud et al., 2016, 2018). Thus, chromosomally encoded colistin resistance might mean a more stable inheritance in ST131 porcine lineage. Nevertheless, it also implies less mobilization among isolates. Some studies have proved that stopping using colistin in positive mcr-1 carriers porcine farms has resulted in the disappearance of the resistance (Randall et al., 2018). However, because of the MDR profile of our ST131 isolates, this might not be that easy, due to coselection by other antimicrobials and host fitness adaptation. The fitness effects of carrying the mcr-1 gene were evaluated by Wu et al. (2018). They proved that IncI2 and IncX4 mcr-1–positive plasmids conferred fitness advantage for its host. In contrast, IncHI2 plasmids imposed a slight fitness cost and competitive disadvantage. To sum up, it seems that epidemic plasmid types IncX4 and IncHI2, which have been reported as the more prevalent carriers of mcr-1 gene, are responsible for the acquisition of the colistin resistance in the high-risk clone ST131 of E. coli in our isolates from porcine origin. Worryingly, the mcr-1 gene has also integrated an IncF plasmid from ST131. The clinical significance of this category of plasmids is therefore highlighted because they are responsible for the epidemic dissemination of ESBL resistance worldwide (Johnson et al., 2016). We hypothesize that IncF plasmids such as pLREC161_1 had acquired the mcr gene via cointegrating an IncHI2 mcr-1–harboring plasmid. The mcr-carrying IncF plasmid pMR0516mcr that has been described by McGann et al. (2016) also shared 89 kb with pHNSHP452 (mcr-1–carrying IncHI2 plasmid), and possibly the same phenomenon had occurred. The impact that humans have on the environment and other species is evident. A multidisciplinary point of view, taking into account the environment, animals, and human for the surveillance of health, is given by the concept of “One Health” (Destoumieux-Garzón et al., 2018). This philosophy fits perfectly the actual global situation. Measures to reinforce prevention and proper management for overall antimicrobials might be the right strategy to fight against antibiotic resistance. Further studies to understand why some plasmids evolve to become genetically stable are necessary to be able to anticipate the evolutionary dynamic of high risk clones. As a limitation, it is worth mentioning that short reads obtained by WGS do not always allow a perfect genome reconstruction due to the high amount of repeated sequences in the bacterial genomes. Long read sequencing technologies will probably bring new perspectives in future researches. CONCLUSION Clade B of ST131 showed a huge genetic diversity, and five new subclades were defined (B6, B6-like, B7, B8, and B9). The majority of ST131 porcine isolates belong to new subclades B6 and B7. An association between the phylogeny, the virotype, and the origin of porcine isolates was established. Some porcine and human clinical isolates were highly related. Most porcine ST131 isolates are MDR (91%) and carry many ARGs. Colistin resistance was introduced through MGEs and had been able to stabilize chromosomally. IncX4 and IncHI2 epidemic mcr-1–harboring plasmids are responsible for acquired colistin resistance encoded by mcr-1.1 gene. The surrounding environment of the mcr-1 cassette is variable but within the same family of plasmids, insertion, and stabilization had common structures. The plasmidome of ST131 clade B is distinct from other clades within ST131 clone and contains an impressive variety of different plasmids. Characteristically ST131 porcine isolates have APEC [F2:A-:B1]–IncF plasmids. ColE1like plasmids and IncX plasmids are also frequently observed. This behavior could be related to the clonal success due to its ability to easily rearrange DNA by horizontal gene transfer. DATA AVAILABILITY STATEMENT The datasets of the 11 LREC genomes for this study can be found in the NCBI sequence databases as part of BioProject SUB5714329. With accession codes SAMN11936814 to SAMN11937940. Accession in NCBI is PRJNA546088. AUTHOR CONTRIBUTIONS S-CF-S, MT, AM, VG, IG-M, DD-J, and AH undertook the laboratory work. MT and JB conceived the concept for the manuscript and designed the experiments. All authors provided the critical input and contributed to the writing of the manuscript and approved the final version. FUNDING This study was supported by projects: PI16/01477 from Plan Estatal de I+D+I 2013–2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento Frontiers in Microbiology | www.frontiersin.org 16 March 2020 | Volume 11 | Article 387 fmicb-11-00387 March 21, 2020 Time: 9:8 # 17 Flament-Simon et al. Porcine ST131 Escherichia coli de la Investigación and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Spanish Agencia Estatal de Investigación (AEI) from the Spanish Ministerio de Economía y Competitividad (MINECO) and FEDER; and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria of Xunta de Galicia and FEDER. ACKNOWLEDGMENTS S-CF-S acknowledges the FPU programme for her grant (FPU15/02644) from the Secretaría General de Universidades, Spanish Ministerio de Educación, Cultura y Deporte. IG-M and VG acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for his predoctoral grant (ED481A-2015/149) and her postdoctoral grant (ED481B2018/018), respectively. AM acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant PRX16/00023 for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016. She also expresses her gratitude to Dr. Mark Achtman and his group for their invaluable help with Enterobase during the stay at the Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.00387/full#supplementary-material REFERENCES AbuOun, M., Stubberfield, E. J., Duggett, N. A., Kirchner, M., Dormer, L., Nunez-Garcia, J., et al. (2017). mcr-1 and mcr-2 variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015. J. Antimicrob. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 20 March 2020 | Volume 11 | Article 387