F4- and F18-Positive Enterotoxigenic Escherichia coli Isolates from Diarrhea of Postweaning Pigs: Genomic Characterization

1 Genomic characterization of Enterotoxigenic E. coli F4 and F18 positive strains from post-1 weaning diarrhea in pigs 2 Vanesa Garcíaa,b, Michela Gambinoa, Karl Pedersenc, Sven Haugegaardd, John Elmerdahl Olsena 3 and Ana Herrero-Fresnoa,# 4 aDepartment of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, 5 University of Copenhagen, Frederiksberg, Denmark 6 bLaboratorio de Referencia de Escherichia coli (LREC), Departamento de Microbioloxía e 7 Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela (USC), Lugo, 8 Spain 9 cStatens Veterinärmedicinska Anstalt, Uppsala, Sweden 10 dSEGES Danish Pig Research Centre, Denmark. 11 #Corresponding author, Ana Herrero-Fresno, 12 Email: [email protected] 13 14 Running title: Genome analysis of E. coli from post-weaning diarrhea 15 Keywords: pigs, diarrhea, Enterotoxigenic E. coli, whole genome sequencing, bioinformatics 16 17 18 19 20 AEM Accepted Manuscript Posted Online 18 September 2020 Appl. Environ. Microbiol. doi:10.1128/AEM.01913-20 Copyright © 2020 American Society for Microbiology. All Rights Reserved. on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 2 ABSTRACT 21 This study aimed to characterize in silico Enterotoxigenic E. coli F4- and F18-positive-isolates 22 (n=90) causing swine post-weaning diarrhea, including pathogenic potential, phylogenetic 23 relationship, antimicrobial and biocide resistance (R), prophage content and metal tolerance rates. 24 F4-strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 MLST-types. F18-25 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23 and ST42. The 26 highest rates of antimicrobial-R were found against streptomycin, sulfamethoxazole, tetracycline, 27 trimethoprim and ampicillin. No resistance was found towards ciprofloxacin, cefotaxime, ceftiofur 28 and colistin. Genes conferring tolerance to copper (showing highest diversity), cadmium, silver and 29 zinc were predicted in all genomes. Enterotoxins (ltcA; 100 % F4, 62 % F18, astA; 100 % F4, 38.1 30 % F18, sta;18.8 % F4, 38.1 % F18, stb; 100 % F4, 76.2 % F18) and fimbriae encoding genes typed 31 as F4ac and F18ac were detected in all the strains, in addition to up to 16 other virulence genes in 32 individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each 33 strain. High diversity of plasmid replicons was shown; IncFII, IncFIB and IncFIC were prevalent 34 among F4-isolates while IncI1, and IncX1 were dominant among F18-strains. Interestingly, F4-35 isolates from the early 90´s belonged to the same clonal group detected for most of the F4-strains 36 from 2018-2019 (ONT:H10-A-ST100-CH27-0). The low number of SNPs differences between the 37 oldest and recent F4-ST100-isolates suggests a relative stable genome. Overall, the isolates 38 analyzed in this study showed remarkable different genetic traits depending on the fimbria type. 39 IMPORTANCE 40 Diarrhea in the post-weaning period due to Enterotoxigenic E. coli (ETEC) is an economically 41 relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by Zinc-42 oxide administration (discontinued by 2022). Besides, a breeding program has been implemented 43 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 3 aiming to reduce this illness. Treatment with antimicrobials contributes to the problem of 44 antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand 45 the genetic population structure and variation among isolates diarrhea-associated by whole genome 46 sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds 47 and showed high similarity to ETEC-ST100-isolates from China, USA and Spain. High rates of 48 AMR and high diversity of virulence genes were detected. Characterization of ETEC diarrhea-49 related is important for understanding the disease epidemiology and pathogenesis, and for 50 implementation of new strategies aiming to reduce the impact of the disease in pig production. 51 52 53 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 4 INTRODUCTION 54 Post-weaning diarrhea (PWD) affects pigs after weaning, leading to significant economic costs for 55 the pig industry due to weight loss, mortality, as well as cost of prevention (i.e. vaccination), 56 treatment and handling (1, 2). In addition to sudden death or profuse diarrhea, the disease is 57 accompanied by growth retardation in surviving piglets (1, 3). During acute outbreaks, mortality 58 due to PWD may reach 20-30% over a 1 to 2 months’ time span among infected pigs (3). Besides, 59 as it is one of the most common reasons for use of antimicrobials in pig industry worldwide, PWD 60 significantly contributes to the problem of antimicrobial resistance (AMR) development (2, 4, 5). 61 Enterotoxigenic E. coli (ETEC) is the main etiological agent involved in PWD worldwide. The 62 ETEC pathotypes in pigs are characterized by the expression of specific fimbrial adhesins, which 63 mediate bacterial colonization of the gut mucosal surface. The most commonly detected types of 64 fimbriae are F4 (previously termed K88) and F18 (F107, 2134P and 8813). Both include different 65 antigenic variants; three for F4 (ab, ac and ad) with F4ac being the most prevalent, and two types 66 for F18 (ab and ac), with F18ac being the one associated with PWD (6, 7). Intestinal adhesion and 67 subsequent colonization by ETEC depends on F4 or F18 specific receptors, the presence of which 68 therefore is essential for ETEC to cause disease (8). ETEC F4 is usually related to PWD of recently 69 weaned piglets occurring 2-3 days after weaning (classical PWD), while F18 is commonly found 70 associated to diarrhea 2-6 weeks after weaning. The age-dependent expression of F4 and F18 71 receptors in the small intestine might explain why ETEC F4 infection mainly takes place right after 72 weaning as well as during the neonatal period, while ETEC F18 infection mainly occurs later in the 73 post-weaning period (5). 74 Once ETEC bacteria have adhered and colonized the small intestine, they can produce 75 enterotoxin(s) leading to diarrhea. Both ETEC F4 and F18 are reported to encode two classes of 76 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 5 enterotoxins; heat labile (LT) and heat stable (Sta, Stb, and EAST1 -enteroaggregative heat-stable 77 toxin 1) enterotoxins, which induce secretory diarrhea in the pigs (1, 2, 9-11). The predominant 78 serogroup of ETEC associated with classical PWD in pigs worldwide is O149-F4 (1). 79 In Denmark, and other countries, zinc oxide (ZnO) in therapeutic concentrations has been used 80 during the last decades to prevent PWD in the first 14 days after weaning. ZnO has been found to 81 improve growth performance and reduce scours (ETEC induced) in weaning piglets (12). Moreover, 82 ZnO reduces bacterial adhesion and inflammatory cytokine expression, and prevents the disruption 83 of membrane integrity caused by ETEC (13). However, due to the environmental toxicity and 84 potential co-selection for AMR, use of ZnO has been banned in pig production in EU by 2022 85 (https://www.ema.europa.eu/en/medicines/veterinary/referrals/zinc-oxide). Other strategies that 86 haven been used to tackle PWD caused by ETEC include a breeding program (DanBred) which has 87 been implemented in some Danish farms since 2003 and vaccines. The former consists in breeding 88 pigs which do not express the F4ac specific receptors on the intestinal mucosa aiming at reducing 89 occurrence of diarrhea due to ETEC F4 (5). The live attenuated vaccine Coliprotec® F4/F18 90 (https://www.ema.europa.eu/en/medicines/veterinary/EPAR/coliprotec-f4f18) has been developed 91 to diminish the incidence of PWD caused by both ETEC F4 and F18 bacteria. Besides these 92 preventive strategies, neomycin, apramycin, spectinomycin, tetracycline, amoxicillin and 93 sulphadiazine-trimethoprim are the antimicrobials commonly used to treat PWD in Denmark (14). 94 In the present work, we characterized a collection of Danish ETEC F4 and F18 positive strains 95 through whole genome sequencing in order to analyze the pathogenic potential of the strains 96 through identification of relevant virulence factors and to determine the occurrence of AMR and 97 biocide and metal resistances. Further, the analysis allowed us to understand the population genetic 98 structure and variation among strains that are associated with PWD. Moreover, we also analyzed the 99 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 6 phylogenetic relationship between the Danish strains under study and swine ETEC strains from 100 other countries of the world. 101 RESULTS 102 Phylogroups, sequence types, clonotypes and serotypes 103 Most of the ETEC isolates belonged to the phylogroup A (74 isolates, 82.2 %), and the remaining 104 were assigned to four different phylogroups; B1 (one isolate, 1.1 %), C (nine isolates, 10 %), D 105 (five isolates, 5.5 %) and E (one isolate, 1.1 %). The strains displayed eight different MLST-types 106 (STs) (ST10, ST23, ST42, ST48, ST90, ST100, ST155 and ST3524), with ST100 accounting for 56 107 (62.2 %) isolates. Based on fumC-fimH allele combinations (CH), eight clonotypes were identified, 108 with CH27-0 as the most prevalent type (56 isolates, 62.2 %), corresponding to strains assigned to 109 ST100 (Supplementary Table S2). 110 The SerotypeFinder tool detected six O serogroups (O6, O8, O29, O141, O147 and O149) and eight 111 different H antigens (H4, H10, H12, H14, H16, H17, H19 and H30). It was not able to predict the O 112 and H antigen in 14 and 8 isolates, respectively. Overall, 13 O:H combinations (serotypes) were 113 found (Supplementary Table S2), with O149:H10 being the most common serotype identified (49 114 isolates, 54.4 %) followed by O6:H16 (11 isolates, 12.2 %). 115 The association between clonal groups (defined by serotype, phylogroup, ST and clonotype) and 116 fimbrial type detected among the strains is shown in Table 1. Two main clonal groups were 117 identified among the 69 F4 positive isolates, O149/ONT:H10/HNT-A-ST100-CH27-0 and O6:H16-118 A-ST48-CH11-34, representing 81.2 % (56 strains) and 15.9 % (11 strains) of the isolates, 119 respectively. The remaining F4 positive isolates belonged to the O8:H19-C-ST90-CH4-54 clonal 120 group (two isolates, 2.9 %) (Supplementary Table S2). Interestingly, five out of the six F4 positive 121 isolates isolated in the early 90 ´s also belonged to the ONT:H10-A-ST100-CH27-0 clonal group 122 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 7 shown for most of the F4 isolates collected during the period 2018-2019 (Supplementary Table S2). 123 In contrast to the homogeneity observed among F4 isolates, the 21 F18 positive isolates showed a 124 higher diversity, and were assigned to six different clonal groups, of which O141/ONT:H4-A-ST10-125 CH11-24 (33.3 %; seven isolates) and O8:H17-C-ST23-CH4-54 (28.6 %; six isolates) were the 126 predominant ones. The remaining F18 positive isolates were assigned to the O147/ONT:H14-D-127 ST42-CH-28-65 (five isolates, 23.8 %), O29:H12-B1-ST155-CH4-121, ONT:H19-C-ST90-CH4-0 128 (E. coli Nysø) and O8:H31-E-ST3524-CH23-31 (one isolate each, 4.7 %) clonal groups. F18 129 positive strains from the same herd belonged to the same clonal group including seven, six and five 130 isolates from herd D, herd B, and herd C, respectively (Supplementary Table S2). 131 Phylogeny analysis of ETEC isolates 132 The raw-read mapping of all 90 genomes to the reference E. coli K12 genome showed that 133 3,475,685 out of 4,641,652 (74.8 %) nucleotide positions in the reference genome were present in 134 all the analyzed genomes. A total of 42,172 variable nucleotide positions were detected in this core 135 genome (Supplementary Table S3). 136 Isolates clustered into four major clades (I, II, III and IV) and grouped according to their ST (Figure 137 1). Clade I included seven ST10-F18 isolates recovered from the same herd and showed 1 to 14 138 SNP differences. Isolates belonging to ST48 and encoding F4 fimbriae (eleven isolates) grouped in 139 clade II and showed from 6 (between isolates from the same herd) to 633 SNP differences. Clade III 140 encompassed isolates assigned to different STs (ST155, ST23, ST42, ST3524 and ST90) and all 141 except two ST90 isolates were F18 positive. This cluster was divided into two well-defined 142 subclades: subclade A (including five ST42 isolates, from the same herd with SNP differences 143 ranging from 4 to 18 and one ST3524 isolate) and subclade B containing six ST23 isolates (SNP 144 differences between 3 and 21), three ST90 (two of them were F4 positive) strains (SNPs differences 145 between 104 and 556) and a single ST155 isolate. Lastly, clade IV consisted of F4 positive isolates 146 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 8 belonging to ST100 (56 isolates) and was split into two subclades, with the number of different 147 SNPs spanning from 16 to 866 between all genomes and from 4 to 15 between isolates belonging to 148 the same herd. SNP differences between the five F4- ST100 isolates from the 90´s and F4-ST100 149 strains recovered during 2018-2019 ranged from 130 to 699. 150 We investigated the relationship between our strains and ETEC isolates from other countries 151 (Figure 2, Supplementary Table 11). Phylogenetic analysis based on SNP, indicated that the Danish 152 ST100 isolates are closely related to ST100 isolates from China (SNP differences between 120 and 153 443), Spain (178 to 708) and USA (196 to 466). The lowest number of SNP differences among 154 ST10 isolates were detected versus ST10 strains from USA (300 and 301), followed by Spain (1779 155 to 1852) and China (2023 to 2025). Regarding ST48, the single isolate from China included for 156 comparisons showed SNP differences between 4163 and 4291 with regards to the Danish ST48 157 isolates. Finally, our ST90 strains were more similar to the ST90 isolate from China (SNP 158 differences between 80 and 268) than to those ST90 strains from USA (SNP differences ranging 159 from 388 to 476). 160 Antimicrobial resistance phenotypes and genotypes 161 The antimicrobial susceptibility testing revealed that 85 (94.4 %) of the isolates were resistant to at 162 least one of the antimicrobials investigated, 60 (66.7 %) were MDR and only five were susceptible 163 to all antimicrobials. High rates of resistance were found against streptomycin (68.9 % of the 164 isolates), sulfamethoxazole (67.8 %), tetracycline (56.7 %), spectinomycin (55.6 %), trimethoprim 165 (53.3 %) and ampicillin (48.3 %). Importantly, none of the isolates was resistant to ceftiofur, 166 cefotaxime, colistin or ciprofloxacin, which are considered highly critical drugs in human medicine 167 (15) (Table 2). In addition, one and eight F4 positive isolates showed resistance to 168 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 9 amoxicillin/clavulanic acid and nalidixic acid, respectively, while none of the F18 isolates tested 169 positive for these drugs (Table 2). 170 Results from Kappa statistical analysis showed that there was an almost perfect agreement between 171 phenotypic resistance and the in silico prediction of resistance genotype (Table 3), which identified 172 a total of 39 different AMR genes (Supplementary Table S2). The genes blaTEM-1B, tet(A) and dfrA1 173 were the most commonly detected among ampicillin, tetracycline and trimethoprim resistant 174 isolates, respectively. Similarly, sul1 and sul2 were the predominant genes responsible for 175 sulphonamide resistance. A total of 16 different genes encoding for aminoglycoside modifying 176 enzymes were identified, with the aph (phosphotransferases: aph(3')-Ia, aph(3')-Ib, aph(3'')-Ib, 177 aph(4)-Ia, aph(6)-Id) and aadA (nucleotidyltranferases: aadA1, aadA2, aadA5, aadA11, aadA12, 178 aadA17, aadA22, aadA24) genes as the most common, consistent with the high resistance to 179 spectinomycin and streptomycin, respectively. Regarding phenicols, catA1, cmlA1 and floR genes 180 were found among the resistant isolates (Table 3, Supplementary Table S2). Susceptibility to 181 macrolides and lincosamides was not phenotypically tested, however, genes conferring resistance to 182 both classes of drugs were detected (Table 3, Supplementary Table S2). 183 Notably, none of the isolates was found to harbor genes that suggested extended-spectrum beta-184 lactamase (ESBL) production or transferrable colistin resistance (mcr-class genes). 185 The ResFinder bioinformatics tool also allows the identification of chromosomal mutations related 186 with antimicrobial resistance. Eight isolates, phenotypically resistant to nalidixic acid, showed a 187 single chromosomal mutation in the gyrA (S83L) gene. The substitution V161G, associated with 188 colistin resistance, was also detected in the pmrB gene in five isolates; however, these isolates were 189 phenotypically susceptible to the antimicrobial. In addition, one isolate had a nucleotide change in 190 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 16 isolates from four different countries suggest that the ETEC ST100 isolates are closely related, 330 since the SNP differences were relatively low among them. Interestingly, a relatively low number of 331 SNPs (130-699) were identified between the F4 positive isolates from the 90´s and those recovered 332 during 2018-2019, indicating that very similar clones have been circulating for at least 30 years. To 333 confirm this hypothesis a larger number of F4 strains from previous years (from the 90 ´s up to 334 nowadays) should be analyzed. The clone O6:H16-ST48, which included eleven isolates recovered 335 from five different herds and differing in 6 to 633 SNPs, was the second most prevalent among F4 336 positive isolates. Notably, O6 is one of the most important ETEC serogroups involved in human 337 diarrhea globally, particularly among children under the age of five in developing countries (38), 338 and it is not classically associated with PWD. A recent study based on the genomic characterization 339 of 40 ETEC O6:H16/HNT human isolates collected during 1975–2016 showed significant genomic 340 diversity among them, but none was assigned to ST48 (38). The occurrence of ETEC O6:H16 341 among pig ETEC isolates may indicate a zoonotic potential, however, the strains did not harbor 342 known fimbriae genes involved in adhesion to the human intestine. 343 The World Health Organization has defined AMR a global health issue in both humans and 344 animals, and has recommended surveillance for AMR bacteria in food-producing animals, such as 345 pigs, as they represent a possible source and disseminator of AMR to humans (39). ETEC from pigs 346 are not considered zoonotic (key virulence factors required to cause disease differ between pigs and 347 human (40), but treatment with antimicrobials against PWD may select for AMR in commensal 348 intestinal bacteria, and such bacteria may transfer critical resistances to humans via the food chain. 349 To investigate this aspect, we determined both, phenotypically and in silico, the AMR levels among 350 the ETEC isolates. The highest rates of AMR were found against aminoglycosides and 351 sulfamethoxazole, the use of which has increased in livestock in Denmark during last years (41). 352 Resistance to tetracycline, trimethoprim and ampicillin, which are among the frequent 353 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 17 antimicrobials used for treatment of PWD in Denmark, were broadly detected. Notably, levels of 354 tetracycline and ampicillin resistance were similar to those detected in a previous study on 355 pathogenic E. coli from pigs in Denmark (42). All the isolates were susceptible to ciprofloxacin, 356 cefotaxime, ceftiofur and colistin, which are critically important antimicrobials for human medicine, 357 (15) but treatment with others antimicrobial agents may allow their co-selection. The absence of 358 these resistances is possibly linked to the restricted use of these drug classes in the pig industry, 359 where fluoroquinolones, cephalosporins and colistin all bear a penalty of factor 10 in the herd level 360 registration scheme of use of antimicrobials (the Yellow card scheme) in the Danish pig production 361 (41). Similar findings were described in a recent study in Denmark, where the highest proportions 362 of AMR among ETEC isolates were found for ampicillin (60.7 %), sulphamethoxazole (69.7 %), 363 tetracycline (47.2 %) and trimethoprim (69.7 %), while AMR to ciprofloxacin, ceftiofur and colistin 364 were not detected (43). MDR in the pig industry has been linked with the wide use of 365 aminoglycosides and beta-lactams in veterinary medicine (44). Here, MDR was detected in 60 366 isolates (66.7 %), far from the 94 % detected among ETEC isolates carrying mcr-1 and causing 367 PWD in Spain (36). It is to highlight that in silico prediction of AMR genes showed an almost 368 perfect agreement with the phenotypic analysis according to Kappa statistical analysis. In addition, 369 genes encoding for macrolides (specifically for erythromycin via MdfA) or lincosamides resistance, 370 which were not phenotypically tested, were also predicted. Both drug classes are commonly 371 employed in Denmark and other countries for treatment against Lawsonia intracellularis, an 372 intracellular pathogen causing enteric disease in pigs (45-47). The increase in the administration of 373 macrolides during last years and the steady use of lincosamides in the pig industry in Denmark (41) 374 could have selected for resistance to the drugs in E. coli. 375 Studies of ETEC across several countries worldwide, including old studies from Denmark, describe 376 ETEC F4 as the most common type associated to PWD, followed by F18 (48-52). However, in 377 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 18 other countries such as Poland, Cuba, Japan and Spain (36, 53-55), the highest prevalence was 378 found for ETEC-F18. In a recent study in Denmark, the number of F4 and F18 strains detected were 379 similar (annual report from 2018; https://diagnostik.dtu.dk/raadgivning/aarsrapporter-for-380 diagnostik_overvaagning/aarsrapporter-svin). As demonstrated in previous studies, the 90 strains 381 under study were all F4ac or F18ac (6, 7). Denmark has further had a breeding strategy to reduce 382 susceptibility of pigs against ETEC F4ac. The strategy consisted on the inactivation (based on one 383 SNP change) of the candidate gene of the F4ac receptor MUC4 (5, 56), however, according to our 384 results, F4 strains type ac are still recovered. Since information on the farms is confidential, we 385 acknowledge if the herds under study have this strategy implemented and/or piglets were 386 vaccinated. Thus the reason why F4ac is still been detected could be that some of the isolates tested 387 were recovered from herds where the strategy and/or vaccination has not been applied. Besides, 388 some studies suggest that MUC13 and not MUC4 is the most likely gene governing susceptibility to 389 ETEC F4ac, and this might explain also why ETEC F4ac is still the predominant causative agent of 390 PWD right after weaning (57). 391 In two F4 isolates, F4 and F6 fimbriae genes were detected concurrently. ETEC isolates encoding 392 for more than one fimbriae have been previously described (28, 48, 49, 58, 59) and such strains 393 have been suggested to have a pathogenetic advantage (27). The most prevalent enterotoxin 394 detected in our study was STb (85 isolates) consistent with previous studies performed in other 395 countries (28, 48, 52). All F4-positive isolates carried astA, ltcA and stb genes, in line with results 396 of previous studies, where the F4 gene was strongly associated with lt and stb (28, 48, 60). 397 However, in our study, only a proportion of F18 positive isolates (n=8) carried astA, ltcA and stb 398 genes, while the remaining strains harbored ltcA and/or stb and sta genes. 399 Additionally, 16 other virulence genes were predicted among strains, and their distribution was 400 associated with the type of fimbria, suggesting that F4 and F18 positive isolates may use different 401 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 19 virulent strategies to cause disease. As suggested, ETEC strains harboring additional fimbriae 402 adhesins, could potentially exploit other alternative pathways for colonization of the host (17). 403 Overall, the analysis showed that ETEC from Danish pigs harbor other virulence factors besides 404 their characteristic adhesins and toxins, but currently the role for such factors, if any, in intestinal 405 disease is not known. 406 Plasmids play an important role in the spread and dissemination of both AMR and virulence genes 407 (61). IncF, IncI and IncX replicon types were the most prevalent and at least one IncF replicon was 408 detected in all the strains, as previously reported in porcine ETEC isolates from Australia and Spain 409 (32, 62). In general, F4 isolates showed more plasmid replicon diversity (21 different plasmid 410 replicons) than the F18 isolates (eight different plasmid replicons). IncF plasmid type is the most 411 commonly described in bacteria from humans and animals, particularly in E. coli, and is known to 412 carry virulence and AMR genes (63, 64). IncI1 plasmids, the most dominant among F18 strains, 413 were also detected in all O141-F18 isolates and in 87.1% of O149:F4 isolates from Australia (32). 414 IncI1 type plasmids are often associated with AMR and known to be ESBL carriers (65), however, 415 in the current study, ESBL were not detected, and the link to AMR was not investigated. IncX 416 plasmids are narrow host range plasmids of Enterobactericeae, which are known to provide 417 additional advantages, commonly associated with AMR and biofilm formation (64, 66). Since 418 ETEC fimbriae and toxin genes have been reported often to be plasmid located (67), we 419 investigated the putative plasmid localization of these genes and potential co-occurrence with AMR 420 determinants. F4 and F18 fimbria encoding genes were found to be plasmid located in all the 421 strains), toxins were plasmid located in more than 70% of the isolates, and in addition, the majority 422 of the AMR genes were predicted to be plasmid encoded. Interestingly, AMR and virulence genes 423 were often predicted to be part of the same plasmid component, however, further detailed studies 424 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 20 are needed to confirm this, since the program used for predictions (plasmidSPAdes) do not separate 425 plasmids of the same type present in a single strain. 426 Recent studies have reported that AMR might be associated with tolerance to heavy metals existing 427 naturally or used in food animal production, such as zinc oxide and copper (41, 68-70). Here, we 428 predicted that aminoglycoside, tetracycline and ampicillin resistance genes and zinc or copper 429 resistance genes were located on the same plasmid component, however, as mentioned above for 430 AMR and virulence genes, more detailed studies are needed to confirm this. Co-localization would 431 imply that use of ZnO and Cu may co-select for AMR. In addition to heavy metals, biocides, 432 including disinfectants and antiseptics, widely used in farms, could also promote the spread of 433 AMR (68). All isolates analyzed here were predicted to carry genes responsible for biocidal 434 resistance, but co-occurrence with AMR was not investigated. 435 Each of the 90 isolates under study contained at least seven prophages, mostly similar to the 436 coliphages P88 (from 3.9% to 93.3%) and Phi27 (from 4.8% to 60.9%). Despite Phi27 is known to 437 encode Stx2e, none of the ETEC isolates encoded for Shiga toxins, thus indicating that, despite the 438 similarity, it is not the same phage. 439 In conclusion, the current study showed a high clonal diversity among F18 isolates, while, in 440 contrast, similar F4 clonal groups might be circulating in Danish herds. Besides, high rates of AMR 441 against aminoglycosides, sulfamethoxazole, tetracycline, trimethoprim and ampicillin as well as 442 high diversity of virulence genes were detected -including toxin genes (ltcA, astA, sta, stb), and 443 fimbriae encoding genes typed as F4ac and F18ac. 444 MATERIALS AND METHODS 445 Bacterial strains and PCR detection of fimbriae types F4 and F18 446 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 21 ETEC F4 isolates were collected from pigs with diarrhea in 2018 (n= 34, from 30 different farms), 447 2019 (n=29, from 29 different farms), and 1989-1992 (n=6, from six different farms). Presumptive 448 ETEC F18 isolates (n=20) were recovered from five different farms (collected at the same time 449 point in each farm) in 2019. In addition, E. coli Nysø, a well characterized ETEC strain recovered 450 in the 70 ´s (71), was included as F18 historical control. Strains were obtained during routine 451 diagnostic procedures and use for research purposes did not require ethical clearance, as long as 452 farm identity was not disclosed. 453 Strains were confirmed positive for F4 or F18 fimbriae using PCR with primers and conditions as 454 previously reported (52). 455 Antimicrobial resistance phenotype 456 MIC values for E. coli isolates were determined for amoxicillin–clavulanic acid (2/1-32/16 µg/ml), 457 ampicillin (1-32 µg/ml), apramycin (4-32 µg/ml), cefotaxime (0.125-4 µg/ml), ceftiofur (0.5-8 458 µg/ml), chloramphenicol (2-64 µg/ml), ciprofloxacin (0.015-4 µg/ml), colistin (1-16 µg/ml), 459 florfenicol (2-64 µg/ml), gentamicin (0.5-16 µg/ml), nalidixic acid (4-64 µg/ml), neomycin (2-32 460 µg/ml), spectinomycin (16-256 µg/ml), streptomycin (8-128 µg/ml), sulphamethoxazole (64-1024 461 µg/ml), tetracycline (2-32 µg/ml) and trimethoprim (1-32 µg/ml) by the broth microdilution method 462 using Sensititre microtiter trays (DKMVN4, Sensititre system; Thermo Fisher Scientific, West 463 Sussex, United Kingdom). E. coli ATCC 25922 was used for quality control. Results were 464 interpreted according to EUCAST epidemiological cut-off values, EUCAST clinical breakpoints for 465 amoxicillin-clavulanic acid (www.EUCAST.org) and DANMAP for apramycin (72). Isolates were 466 defined as “susceptible” when classified as “wild type” and “resistant” when classified as “no wild 467 type”. Multidrug-resistant (MDR) strains were those resistant to one agent from three or more 468 different antimicrobial classes (73). 469 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 22 PCR for detection of sta and stb toxin genes 470 E. coli DNA was extracted from a single overnight-grown colony by the boiling lysis method as 471 reported (52), and PCR amplification of sta and stb toxin genes was performed using the primers 472 and PCR conditions previously described (52). 473 DNA extraction and whole genome sequencing (WGS) 474 DNA was extracted using the Maxwell® system (Promega) following the instructions provided by 475 the Maxwell® RSC cultured cells DNA kit (Promega). Quality of the DNA was determined by 476 NanoDrop-1000 (Thermo Fisher Scientific) and DNA quantification was performed using dsDNA 477 BR Assay kit with the Qubit 2.0 fluorometer (Invitrogen, USA). 478 The libraries for sequencing were prepared using the Nextera DNA Flex Library Prep Kit (Illumina, 479 Inc., San Diego, CA, USA) according to the manufacturer´s protocol and sequenced using Illumina 480 NextSeq (Illumina). The paired-end raw reads were assembled using SPAdes Genome Assembler 481 v.3.13.0 (74) and the quality of assembly was evaluated with QUAST v.5.0.2. (75). The raw 482 sequences were submitted to the European Nucleotide Archive (ENA) under the study accession 483 number PRJEB38608. 484 Whole genome characterization 485 The assembled contigs, with genomic size ranging between 5.1 and 5.7 Mbp (mean size 5.4 Mbp) 486 (Supplementary Table S1), were analyzed using the bioinformatics tools of the Center for Genomic 487 Epidemiology (CGE) for the presence of antibiotic resistance (ResFinder v.3.2) (76), virulence 488 genes (VirulenceFinder v.2.0) (77), plasmid replicon types (PlasmidFinder v.2.1) (78), and 489 identification of clonotypes (CHTyper v.1.0), sequence types (ST) (MLST v.2.0) (79) and serotypes 490 (SerotypeFinder v.2.0) (75). All the CGE predictions were called using default settings. 491 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 23 Identification of antibacterial biocide- and metaltolerance genes was assessed using BacMet-Scan 492 v.2.0 (80).The ClermonTyping tool (http://clermontyping.iame-research.center/) and PHASTER 493 webserver (http://phaster.ca/) were used to predict the phylogroups and putative prophage 494 sequences in the bacterial genomes, respectively (81, 82). 495 In silico prediction of localization of antimicrobial resistance, virulence and metal tolerance genes 496 and potential co-occurrence on plasmids 497 The putative localization of AMR and virulence genes (F4, F18, ltcA, astA, sta and stb) was 498 predicted using a combination of plasmidSPAdes v.3.13.0 (83), ResFinder and VirulenceFinder 499 tools. Briefly, plasmidSPAdes was used to identify contigs most likely belonging to plasmid DNA 500 and to assign them to components. Each component is considered as a putative plasmid consisting 501 of one or more contigs. This tool is not able to separate similar plasmids (for example similar 502 plasmids of the same type present in a single strain), and thus their contigs may be assigned to the 503 same id (same component). ResFinder and VirulenceFinder were used to analyze the presence of 504 AMR and virulence genes in all contigs identified as putative DNA regions of plasmids. The output 505 from both tools provides the contig ID and component on which the specific genes were located. 506 Genes contained in the same contig and/or component were predicted to be plasmid located, while 507 those antimicrobial and/or virulence genes not detected in plasmid DNA contigs were assumed to 508 be chromosome encoded. 509 The BacMet database (BacMet-Scan v.2.0.), which includes metal tolerance and biocide resistance 510 genes (metal tolerance genes include also those genes that are indirectly related to metals) was used 511 to investigate the presence of these genes in all the genomes. The genome of the E. coli K12 512 substrain MG1655 (GenBank accession number NC_000913.3) was also included in the analysis. 513 Plasmid contigs identified and assigned to components with plasmidSPAdes v.3.13.0 as described 514 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 24 above were analyzed with BacMet-Scan v.2.0. Next, the plasmid contigs were manually inspected 515 for the presence of metal tolerance genes (zinc, copper, silver, mercury and tellurium) previously 516 identified on the genome assembly by using the BacMet database (Supplementary Table S4). Co-517 occurrence of AMR genes (detected as indicated above) and the metal tolerance genes on the same 518 plasmid was predicted when they were found to belong to the same contig. 519 Phylogenetic analysis 520 Phylogenetic relationships between the isolates were analyzed based on SNP trees constructed using 521 the bioinformatics tool CSI Phylogeny v.1.4 (84) available at CGE. The genome of the E. coli K12 522 substrain MG1655 was included as a reference strain, and CGE default parameters were used 523 during SNP analysis. The phylogenetic tree was visualized and edited by using the bioinformatics 524 tool iTOL v5 (85). 525 Besides, the ETEC isolates under study were compared with 20 swine ETEC isolates from three 526 different countries (China, USA and Spain) as mentioned above. The accession numbers and 527 MLST-types of the 20 ETEC strains used in this phylogenetic analysis are indicated in the 528 Supplementary Table S10. 529 Statistical analysis 530 Differences between F4 and F18 positive strains regarding serogroups, antimicrobial resistance, 531 virulence gene content and plasmid replicons were analyzed using two-tailed Fisher's exact test with 532 the GraphPad Prism version 8.3 software (GraphPad Inc). P values <0.05 were considered 533 statistically significant. 534 Cohen's Kappa statistical analysis was used to analyze the correlation between phenotypic resistance 535 and in silico gene predictions using SPSS version 26 (IBM, USA). Kappa values ≤ 0 indicate no 536 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 25 agreement; 0.01–0.20 none to slight; 0.21–0.40 fair; 0.41– 0.60 moderate; 0.61–0.80 substantial, and 537 0.81–1.00 indicate values with an almost perfect agreement (86). 538 Acknowledgments 539 The authors are grateful to Joakim Larsson and Johan Bengtsson-Palme for their assistance with the 540 use of the Bac-Met database as well as K. Aagard and M. Carlsen for their technical assistance. 541 Funding 542 V. García acknowledges the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta 543 de Galicia for her post-doctoral grant (grant number ED481B-2018/018). This work was supported 544 by Innovationsfonden (grant number 8088-00032B) and the Danish Ministry of Food of 545 Environment (VetforligIII). 546 Conflicts of interest 547 All authors declare that they have no competing interests. 548 Data availability 549 The draft genome sequences of E. coli isolates from pigs in Denmark in this study are available in 550 the European Nucleotide Archive (ENA) under the study accession number PRJEB38608. 551 References 552 1. Fairbrother JM, Nadeau E, Gyles CL. Escherichia coli in postweaning diarrhea in 553 pigs: an update on bacterial types, pathogenesis, and prevention strategies. 2005. Anim Health Res 554 Rev 6(1):17-39. 555 2. Luppi A. 2017. Swine enteric colibacillosis: diagnosis, therapy and antimicrobial 556 resistance. Porcine Health Manag 3:16. 557 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 32 52. Zhang W, Zhao M, Ruesch L, Omot A, Francis D. 2007. 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SNP-based phylogeny of the 90 ETEC isolates from pigs in Denmark and the 20 ETEC 805 isolates from different countries. Colours in the ring correspond to Sequence Type. Isolates 806 highlighted in red, orange and blue correspond to isolates from China, USA and Spain, 807 respectively. 808 Figure 3. Distribution of metal tolerance and biocides resistance among the 90 ETEC isolates 809 from pigs (A), the F4-positive isolates (B) and the F18-positive isolates (C). 810 811 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 37 Table 1. Serotype, phylogroup, Sequence Type and clonotypes association with fimbrial antigens in the 90 812 ETEC isolates from pigs. 813 Serotype-PG-ST-CH Nº (%) Fimbrial antigen O149:H10-A-ST100-CH27-0 49 (54.4) F4 ONT:H10-A-ST100-CH27-0 6 (6.7) F4 O149:HNT-A-ST100-CH27-0 1 (1.1) F4 O6:H16-A-ST48-CH11-34 11 (12.2) F4 O8:H19-C-ST90-CH4-54 2 (2.2) F4 O8:H17-C-ST23-CH4-54 6 (6.7) F18 ONT:H4-A-ST10-CH11-24 5 (5.6) F18 O141:H4-A-ST10-CH11-24 2 (2.2) F18 O147:H14-D-ST42-CH28-65 3 (3.3) F18 ONT:H14-D-ST42-CH28-65 2 (2.2) F18 O29:H12-B1-ST155-CH4-121 1 (1.1) F18 ONT:H19-C-ST90-CH4-0 1 (1.1) F18 O8:H31-E-ST3524-CH23-31 1 (1.1) F18 PG: phylogroup, ST: MLST-type, CH: clonotype. 814 815 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 38 Table 2. Prevalence of antimicrobial resistance among the 90 ETEC isolates from pigs. 816 Antimicrobiala Number of resistant isolates (%) F4 (%)b F18 (%)b p-value OR at 95% CI Ampicillin 47 (48.3) 35 (50.7) 12 (57.1) 0.6282 Amoxicillin/Clavulanic acid 1 (1.1) 1 (1.5) 0 >0.9999 Chloramphenicol/florfenicol 15 (16.7) 9 (13.4) 6 (28.5) 0.1064 Apramycin 8 (8.9) 7 (10.14) 1 (4.7) 0.6752 Gentamicin 6 (6.7) 8 (11.6) 1 (4.7) 0.6792 Neomycin 23 (25.6) 16 (23.2) 7 (33.3) 0.3964 Spectinomycin 50 (55.6) 35 (50.5) 15 (71.42) 0.1327 Streptomycin 62 (68.9) 53 (76.8) 9 (42.9) 0.0061 4.417 (1.491-12.48) Sulfamethoxazole 61 (67.8) 46 (66.7) 15 (71.4) 0.7932 Tetracycline 51 (56.7) 42 (60.9) 9 (42.8) 0.2084 Trimethoprim 48 (53.3) 35 (50.7) 13 (62) 0.4568 Nalidixic acid 8 (8.9) 8 (11.6) 0 0.1901 aThe breakpoints used correspond to EUCAST Epidemiological cut-off values (ECOFFs) for ampicillin (8 µg/ml), 817 chloramphenicol/florfenicol (8 µg/ml), gentamicin (2 µg/ml), nalidixic acid (8 µg/ml), neomycin (8 µg/ml), 818 spectinomycin (64 µg/ml), streptomycin (16 µg/ml), sulphamethoxazole (64 µg/ml), tetracycline (8 µg/ml) and 819 trimethoprim (2 µg/ml), EUCAST clinical breakpoints for amoxicillin/clavulanic acid (R > 8 µg/ml ) and DANMAP 820 2015 for apramycin (R >32 µg/ml ) 821 bPercentage is estimated based on the total number of strains associated to each fimbria type. 822 OR: Odds ratio is indicated when p-value <0.05; CI: confidence interval; ∞: infinity. Significant differences (p-value 823 <0.05) are indicated in bold 824 825 826 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 39 Table 3. Antimicrobial resistance genes detected among the 90 ETEC isolates from pigs. 827 Antimicrobial group Genes Total (%) F4 (%)a F18 (%)a kappa pvalue Beta-lactams bla TEM-1A 4 (4.4) 4 (5.8) 0 0.933 0.0001 bla TEM-1B 42 (46.7) 30 (43.5) 12 (57.1) bla TEM-30 1 (1.1) 1 (1.4) 0 Aminoglycosidesb aph (phosphotransferases) 58 (64.4) 50 (72.5) 8 (38.1) 0.927 0.0001 aadA (nucleotidyltransferases) 57 (63.3) 42 (60.9) 15 (71.4) aac (acetyltranspherases) 9 (10) 8 (11.6) 1 (4.8) Phenicols catA1 3 (3.3) 3 (4.3) 0 1.000 0.0001 cmlA1 8 (8.9) 2 (2.9) 6 (28.6) floR 5 (5.6) 5 (7.2) 0 Macrolides mdf(A) 90 (100) 69 (100) 21 (100) ND ND mph(A) 8 (8.9) 8 (11.6) 0 mph(B) 7 (7.8) 7 (10.1) 0 erm(B) 9 (10) 9 (13) 0 Lincosamides lnu(F) 5 (5.6) 5 (7.2) 0 ND ND lnu(G) 5 (5.6) 5 (7.2) 0 Sulphonamides sul1 30 (33.3) 28 (40.6) 2 (9.5) 0.898 0.0001 sul2 42 (46.7) 35 (50.7) 7 (33.3) sul3 9 (10) 3 (4.3) 6 (28.6) Tetracycline tet(A) 40 (44.4) 32 (46.4) 8 (38.1) 1.000 0.0001 tet(B) 13 (14.4) 12 (17.4) 1 (4.8) tet(X) 1 (1.1) 1 (1.4) 0 Trimethoprim dfrA1 34 (37.8) 27 (39.1) 7 (33.3) 0.978 0.0001 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 40 dfrA5 2 (2.2) 2 (2.9) 0 dfrA12 8 (8.9) 2 (2.9) 6 (28.6) dfrA14 5 (5.6) 5 (7.2) 0 dfrA17 2 (2.2) 2 (2.9) 0 aPercentage is estimated based on the total number of strains associated to each fimbria type. A p-value <0.05 is 828 considered significant. 829 830 831 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from 41 Table 4. Distribution of virulence genes (other than F4 and F18-encoding fimbria) among the 90 ETEC 832 isolates. 833 Function Genes Total (%) F4 (%)a F18 (%)a p-value OD 95% CI Toxins astA 77 (85.6) 69 (100) 8 (38.1) <0.0001 ∞ (25.71-∞) ltcA 82 (91.1) 69 (100) 13 (62) <0.0001 ∞ (9.402-∞ Sta 21 (23.3) 13 (18.8) 8 (38.1) 0.0820 Stb 85 (94.4) 69 (100) 16 (76.2) 0.0005 ∞ (5.465-∞) Fimbriae lpfA 15 (16.7) 2 (2.9) 13 (61.9) <0.0001 0.01837 (0.003635- 0.08) fasA 2 (2.2) 2 (2.9) 0 >0.9999 Adhesion iha 77 (85.6) 57 (82.6) 20 (95.2) 0.2854 air 4 (4.4) 0 4 (19) 0.0023 0 (0.0-0.02885) Colicin cba 33 (36.7) 33 (47.8) 0 <0.0001 ∞ (4.748-∞) cma 47 (52.2) 42 (60.9) 5 (23.8) 0.0053 4.978 (1.631-13.29) celb 10 (11.1) 8 (11.6) 2 (9.5) >0.9999 Microcin mchB 9 (10) 9 (13) 0 0.1096 mchC 9 (10) 9 (13) 0 0.1096 mchF 9 (10) 9 (13) 0 0.1096 mcmA 9 (10) 9 (13) 0 0.1096 Others capU 55 (61.1) 54 (78.3) 1 (4.8) <0.0001 72 (11.75-759.7) gad 40 (44.4) 35 (50.7) 5 (23.8) 0.0439 3.294 (1.09-8.811) sepA 10 (11.1) 10 (14.5) 0 0.1088 iss 20 (22.2) 1 (1.4) 19 (90.5) <0.0001 0.001548 (0.0001506- 0.0) eilA 5 (5.6) 0 5 (23.8) 0.0005 0 (0.0-0.1830) aPercentage is estimated based on the total number of strains associated to each fimbria type. 834 on September 30, 2020 at THE INST OF MOLECULAR BIOLOGYhttp://aem.asm.org/Downloaded from