Genomic diversity, antibiotic resistance, and maturation‑dependent adhesion of F18 enterotoxigenic Escherichia coli strains in porcine intestinal cells

22 Enterotoxigenic Escherichia coli (ETEC) strains expressing F4 and F18 fimbriae are major 23 causes of neonatal and post‑weaning diarrhea in swine. Although epithelial maturation 24 influences susceptibility in vivo, its impact on ETEC-host interactions remains poorly defined. 25 This study characterizes emerging F18 ETEC isolates using a differentiated porcine intestinal 26 cell model. Three F18 strains (3EC1, 27EC1, 3247EC), a porcine F4 strain, and human ETEC 27 H10407 were analyzed by comparative genomics for virulence factors, toxin genes, and 28 antimicrobial resistance determinants. Adhesion assays were performed using IPEC‑1, 29 IPEC‑J2, and Caco‑2 cells conditioned to Early (6 days post‑confluence, DPC), Mid (9 DPC), 30 and Late (16 DPC) maturation states. Transcription of F18‑binding receptors (FUT1, FUT2) was 31 quantified by RT‑qPCR. IPEC‑1 cells exhibited significantly higher FUT1 and FUT2 expression 32 than IPEC‑J2, corresponding to approximately two‑fold stronger adhesion by most F18 isolates. 33 Strain 3EC1 showed a distinct adhesion peak at 9 DPC, approaching F4 levels, while F4 and 34 H10407 consistently displayed the highest adhesion across all models. Genomic analyses 35 revealed substantial heterogeneity among F18 strains in fimbrial loci, flagellin, 36 lipopolysaccharide biosynthesis, and antimicrobial resistance. Strain 3EC1 uniquely carried 37 stx2e, and non‑classical EAST1 variants were detected in 3EC1 and 3247EC. All F18 isolates 38 encoded hlyE and were β‑hemolytic; 3247EC harbored 28 antimicrobial resistance genes. The 39 IPEC‑1/IPEC‑J2 maturation stages recapitulate age‑dependent susceptibility to ETEC, likely 40 driven by FUT1/2 expression levels. The combination of strong adhesion, stx2e, and extensive 41 antimicrobial resistance in F18 strains underscores their evolving virulence and supports this 42 model as a refined platform for studying porcine ETEC pathogenesis. 43 Importance 44 ETEC remains a leading cause of neonatal and post-weaning diarrhea in swine, yet the 45 biological basis for age-dependent susceptibility is not fully understood. This study 46 demonstrates that maturation of porcine intestinal epithelial cells strongly influences F18 ETEC 47 adhesion, driven in part by developmental regulation of the F18-binding receptors FUT1 and 48 FUT2. By integrating comparative genomics with a physiologically relevant in vitro maturation 49 model, we reveal substantial diversity in virulence and resistance among F18 strains, including 50 strong adhesion capacity, stx2e, and extensive antimicrobial resistance in strain 3EC1. These 51 findings highlight the evolution of ETEC toward increased persistence and pathogenic potential 52 in swine populations. The interaction between the maturation-dependent IPEC-1 and IPEC-J2 53 cell lines and ETEC offers a valuable tool for evaluating intervention strategies to reduce 54 weaning piglet susceptibility to ETEC infection. 55

Enterotoxigenic Escherichia coli ETEC, F4, F18, comparative genomics, 56 adhesion, swine epithelium, IPEC-1, IPEC-J2, antibiotic resistance genes. 57 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 3 of 23

58 Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial pathogen responsible for piglet 59 diarrhea and edema disease (ED) (1,2) and contributes to significant weight loss, high 60 morbidity, and mortality. Post-weaning diarrhea (PWD), a hallmark of ETEC infection, imposes 61 substantial financial burdens on the swine industry due to reduced growth rates and increased 62 veterinary costs (3). 63 Current control strategies, including zinc supplementation, antibiotics, probiotics, and prebiotics, 64 are increasingly challenged by the emergence of antimicrobial resistance (AMR), driven by the 65 overuse of antibiotics in livestock feed (4-6), particularly in the United States (7,8). These 66 challenges underscore the urgent need to elucidate the ETEC virulence mechanism to develop 67 sustainable alternatives to antibiotics. 68 Fimbriae are a critical virulence factor that mediates ETEC adhesion and colonization in the 69 porcine small intestine (9). ETEC expressing F4 (K88) or F18 (F107, 2134P, 8813) fimbriae 70 exhibits distinct age-specific pathogenicity: F4 predominantly affects neonate piglets, while F18 71 is associated with PWD in weaned piglets (7,10). F4 fimbriae are categorized into three 72 subtypes (K88ab, K88ac, K88ad), while F18 fimbriae are divided into F18ab (associated with 73 ED) and F18ac (associated with PWD) (11). These differences in tropism are attributed to the 74 dynamic expression of host intestinal receptors. F4 receptors (e.g., MUC4) are highly expressed 75 after birth and start to decline after weaning (12,13), whereas F18 receptors (e.g., FUT1) 76 increase in expression at three weeks of age and persist into adulthood (14). However, 77 conflicting evidence suggests comparable FUT1 mRNA levels in newborn and weaned piglets 78 (15), highlighting unresolved questions about F18 receptor dynamics. The F18 operon 79 comprises five genes (fedA–fedF), with FedA forming the structural backbone. FedE and FedF 80 mediate receptor binding alongside FedA (16). FedA and FedB also facilitate pilus assembly 81 (17). 82 This study characterized three disease-causing E. coli strains isolated from swine farms through 83 biochemical, genetic, and functional assays. We utilized porcine (IPEC-1 and IPEC-J2) (18-21) 84 and human intestinal epithelial (Caco-2) cell lines with differential susceptibility to F4- and F18-85 pathotype ETEC to compare and evaluate their virulence. Two porcine cell lines were assessed 86 at varying stages of maturity to investigate how intestinal development influences bacterial 87 adhesion and pathogenicity. Whole-genome sequencing was applied to interrogate the genetic 88 profiles of these isolates, shedding light on the novel acquisition of virulent features and 89 antibiotic resistance in the current epidemiology. The antibiotic resistance profile of each strain 90 was also examined. Our findings aim to advance understanding of ETEC evolution, host-91 pathogen interactions, and antibiotic resistance to inform the development of targeted 92 interventions. 93 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 4 of 23

94 Virulence factors characterization in ETEC F4 and ETEC-F18 95 ETEC cultures (3EC1, 27EC1, and 3247EC) used in the study were further verified to be lactose 96 fermenting, showing growth on MacConkey agar (Fig. S1) and hemolytic (Fig. 1A). PCR 97 confirmed the presence of the fedA (506 bp) in all F18 E. coli isolates (3EC1, 27EC1, and 98 3247EC) since fedA is a distinctive marker correlated with PWD or edema disease (22) and 99 encodes the core structure of F107 fimbriae used to classify the ETEC-F18 strains (11,23). Non-100 ETEC F18 strains, such as ETEC F4 (K88), ETEC F5 (K99), and Listeria monocytogenes 101 F4244 (Lm), used as controls, tested negative for fedA (Fig. 1B). 102 FIG 1 Characterization of Enterotoxigenic Escherichia coli (ETEC) F18 strains. (A) Hemolytic activity 103 assay on sheep-red blood agar plates showing clear hemolytic zones surrounding the colonies in all 104 strains tested. (B) PCR confirmation of enterotoxigenic Escherichia coli (ETEC) F18 strains showing 105 amplification of fedA gene (506 bp), while ETEC F4 and F5 and Listeria monocytogenes (Lm) F4244 106 showed no amplification as negative controls. 107 108 Age-differentiated adhesion comparison of ETEC F4 and F18 on IPEC-J2 109 The maturation of intestinal epithelial cells influences the relative expression of ETEC fimbriae-110 specific receptors (13,14,18). To investigate this, IPEC-1 and IPEC-J2 cells were categorized 111 into three distinct maturity phases: "Early" (6 days post-confluence, DPC), "Mid" (9 DPC), and 112 "Late" (16 DPC) (Fig. 2). In the "Early" phase, the cell monolayer displayed a clear epithelioid 113 morphology with well-defined cellular boundaries. As differentiation progressed, cellular 114 morphology became increasingly complex. By the "Mid" phase, intercellular overlap became 115 apparent, and cellular boundaries appeared less distinct. At the "Late" phase, notable 116 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 5 of 23 cytoplasmic changes were observed, including granule accumulation and vacuolation (Fig. 2A 117 and C). 118 Adhesion analysis of four swine ETEC strains revealed distinct maturity-dependent 119 adhesion patterns. In the IPEC-1 model, strain 3EC1 showed a marked increase in adhesion at 120 9 DPC, reaching levels nearly comparable to those of F4. However, this increase was not 121 observed at 6 or 16 DPC. In contrast, strains 27EC1 and 3247EC demonstrated consistent 122 adhesion capacity across all maturity phases. Strain F4 and H10407 exhibited the highest 123 adhesion level among all tested strains, surpassing the ETEC-F18 strains by an order of 124 magnitude across all maturity phases (Fig. 2A). Interestingly, a similar trend of adhesion pattern 125 was observed in the IPEC-J2 model (Fig. 2B and D). 126 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 6 of 23 FIG 2 Age-differentiated adhesion comparison of ETEC strains F18 and F4 on porcine intestinal 127 IPEC-1 (A,B) and IPEC-J2 (C,D) cells. (A,C) Adhesion characteristics of ETEC F4 and F18 strains to 128 IPEC-1 (A) or IPEC-J2 (C) cells after 6, 9 and 16 days of post-confluence (DPC), 30 min post-infection at 129 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 7 of 23 MOI 10. Data represent mean ± SEM. ***, p<0.001; ****, p<0.0001. (B,D) Photomicrograph of IPEC-1 (B) 130 and IPEC-J2 (D) after 6,9, and 16 DPC. Scale bar: 50 µm. 131 Since F18 binding to IPEC cells is directly proportional to receptor (FUT1 and FUT2) expression 132 levels (14,15), we analyzed mRNA levels for FUT1 and FUT2 in IPEC cells at 6, 9, and 16 DPC. 133 Compared to IPEC-J2, IPEC-1 showed significantly higher expression of FUT1 at 6 and 16 DPC 134 and FUT2 at 16 DPC (Fig. 3A). When comparing the two models in parallel, most strains exhibit 135 ~2-fold stronger adhesion profiles on IPEC-1 than on IPEC-J2, except for 3EC1 at 6 and 9 DPC. 136 Notably, this enhanced adhesion characteristic in IPEC-1 diminished by 16 DPC to a level like 137 that in IPEC-J2. However, at 16 DPC, two of the three F18 strains (3EC1 and 27EC1) show an 138 increasing trend in adhesion capacity (Fig. 3B). 139 140 FIG 3 F18-specific host receptor analysis. (A) Relative mRNA expression level of FUT1 and FUT2 of 141 IPEC-1 to IPEC-J2 at day 6, 9 and 16 post cell confluence. (B) Fold changes of ETEC adhesion on IPEC-142 1 to IPEC-J2 at day 6, 9 and 16 post cell confluence. Data represent mean ± SEM. *, p<0.05; **, p<0.001. 143 Adhesion comparison of pathogenic ETEC strains on porcine intestinal cell lines 144 Two porcine intestinal epithelium cell lines (IPEC-1 and IPEC-J2) with distinct susceptibility to 145 ETEC fimbriae types were tested for differential ETEC adhesion (21). The IPEC-J2 line is 146 previously documented for its heightened susceptibility to ETEC F4 adhesion, whereas ETEC 147 F18 adheres strongly to IPEC-1 with higher expression of F18 receptors (19). Identical 148 experimental procedures and conditions were applied to both cell lines to ensure compatibility of 149 results. In general, all F18 isolates showed a 1-2 logs reduction in adhesion to both porcine 150 intestinal epithelial cell lines compared to F4 (Fig. 4A). Strains 27EC1 and 3247EC adhered 151 markedly less to IPEC-J2 but only slightly reduced to IPEC-1 than the F4 strain. This aligns with 152 the adhesion characteristic of ETEC F18. By contrast, the ETEC F4 and human ETEC H10407 153 display comparable adhesion to IPEC-1, with H10407 showing slightly higher binding to IPEC-2 154 (Fig. 4A). 155 156 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 8 of 23 FIG 4 Adhesion comparison of enterotoxigenic Escherichia coli (ETEC) strains on porcine 157 intestinal cell lines. (A) Comparative analysis of adhesion of ETEC strains to swine intestinal IPEC-1, 158 IPEC-J2, and Caco-2 cell lines. (B) Giemsa staining showing differential adhesion of ETEC strains to 159 Caco-2 cell monolayers. (C) Cytotoxicity assay using lactate dehydrogenase (LDH) release from cell 160 monolayers after 30 min of ETEC exposure during the adhesion experiment. Triton-X was used as a 161 positive control. Data are mean ± standard error of the mean (SEM). 162 163 These swine-derived ETEC strains were also tested on the human enterocyte-like intestinal 164 Caco-2 cell line as a control. In the Caco-2 cell line, human-origin ETEC H10407 exhibited the 165 highest adhesion as demonstrated by plate counting and Giemsa staining (Fig. 4A and B). Both 166 3EC1 and F4 demonstrated relatively high adhesion levels at 5 logs. In contrast, adhesion by 167 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 9 of 23 27EC1 and 3247EC was 1-2 logs lower than F4, with 3247EC demonstrating the most 168 significant reduction relative to F4 and H10407. 169 170 Post-adhesion lactate dehydrogenase (LDH) levels (% cytotoxicity) in the culture supernatant 171 remained below 10% of the positive control, with no significant alterations observed across all 172 tested groups. These findings indicate that 30 minutes of incubation is sufficient for ETEC to 173 initiate host adhesion but not enough to cause cell damage due to subsequent invasion or toxin 174 release (Fig. 4C). 175 Genomic analysis of three clinical ETEC F18 isolates 176 The Whole Genome Sequencing was performed to systematically characterize the virulence 177 gene content of the three clinical F18 isolates with accession numbers: 3EC1 (CP199153), 178 27EC1 (CP199154), and 3247EC (CP199155) (Fig. 5, Fig. S2). The genomes ranged in size 179 from 5 to 5.5 Mbp, with coding sequences (CDS) varying from 5009 to 5701, and an average 180 GC content of 50.7% (Table 1). Notably, plasmids were detected only in 3247EC, as 181 summarized in Table S1. 182 FIG 5 Genome sequence analysis. Genome-wide comparative analysis to examine the genetic 183 differences and similarities between enterotoxigenic Escherichia coli (ETEC) F18 isolates and 184 F4 (K88). 185 186 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 10 of 23 187 TABLE 1. General information of Escherichia coli strains 3EC1, 27EC1, and 3247EC compared 188 with ETEC F4, H10407, and E. coli Nysø 189 Isolate F4 H10407 3EC1 27EC1 3247EC E. coli Nysø Serogroup O141:H4 O78: H11 O3: H45 O35: H7 O119:H23 O0:H19 MLST 5786 48 4214 1642 224 90 FimCHType 11-560 11-398 4-31 4-31 4-0 11-41 Genome size (Mbp) 5.2 5.3 5.2 5.0 5.6 5.2 GC content (%) 50.4 50.7 50.8 50.6 50.8 50.9 Plasmids IncFIB IncFIC IncFII IncI1-I p0111 IncFII ND ND Col156 IncB/O/K/Z IncFIA IncFIB IncFII(pCo o) IncHI2 IncHI2A IncQ1 IncY Col156 IncFIB IncFIC IncFII IncI1-I IncX1 Total coding sequence 5182 5322 5314 5009 5701 5419 Accession Number CP002729.1 FN64941 4.1 This study (CP1991 53) This study (CP1991 54) This study (CP19915 5) DADUQP00000 0000 190 Phylogenetic analysis showed that 27EC and 3247EC are more closely related to ETEC F4. At 191 the same time, 3EC1 is genetically distant, clustering closer to the same clade of human ETEC 192 isolate H10407 and ancestral ETEC strain E. coli Nysø. For comparison, one historical F18 193 isolate (Nysø) and one human ETEC (H10407) isolate were included in the analysis (Fig. 6). 194 To assess the virulence potential of these newly isolated F18 strains, ETEC F4 was used as the 195

genome for comparative genomics. The analysis identified the top four categories of 196 differential genes as those associated with fimbriae, flagella, LPS synthesis proteins, and 197 phage-associated proteins (Fig. 5, Fig. S2, Table S2). The most notable contrasts were 198 observed in virulent genes contributing to pathogen adhesion and toxin synthesis, such as those 199 encoding fimbriae and LPS-associated synthesis genes. Intriguingly, while fimbrial and LPS 200 synthesis genes from 3EC1 showed limited similarity to those from 27EC1 and 3247EC, they 201 closely aligned with F4 and H10407 (Fig. S2). For instance, YadU and YadC, key adhesins 202 involved in colonization and biofilm formation (24), exhibited less than 50% homology in 27EC1 203 and 3247EC compared to 3EC1 and F4. In contrast, flagellar-associated genes were found to 204 be relatively conserved, showing over 80% amino acid similarity among all swine- and human-205 derived ETEC strains (Fig. 6A, Fig. S2). 206 Classic ETEC toxin genes encoding heat-labile (eltAB) or heat-stable (esta, estb) toxins were 207 absent in all three ETEC F18 strains. However, non-classic ETEC toxins, such as EAST-1, were 208 identified with minimal variation between 3EC1 and 3247EC. Interestingly, stx2e, a Shiga toxin 209 gene, was identified in 3EC1, but not in 27EC1 and 3247EC, indicating potential hybrid virulent 210 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 11 of 23 traits. The avian hemolysin (hlyE) (25) was presented in all ETEC F18 and H10407, which 211 aligned with hemolytic activity (Fig. 1). While both hemolysin A and avian hemolysin were 212 detected in F4 and E. coli Nysø. Outer membrane fimbrial cluster Yeh family proteins (YehA-D) 213 were identified in both human and porcine ETECs, except E. coli Nysø. Additionally, glutamate 214 decarboxylase (GAD), an enzyme that aids in acid resistance (e.g., stomach fluid), was 215 detected in H10407, 3EC1, 27EC, and 3247EC. All ETEC strains harbored the fimH fimbrial 216 genes, except E. coli Nysø. The adhesin iha, prevalent in pathogenic E. coli (26), was identified 217 in 3EC1, 27EC1, and E. coli Nysø (Fig. 6B). A comprehensive list of virulence factors is 218 summarized in Table S2. 219 FIG 6 Comparative genetics of human and swine pathogenic Escherichia coli strains. (A) 220 Phylogenetic tree and similarity of core virulent genes of enterotoxigenic Escherichia coli (ETEC) clinical 221 isolates from swine and human. The phylogenetic tree was constructed based on core conserved genes, 222 root on midpoint. Bootstrap value is 1000. For comparative virulent gene analysis, a scale from 0 to 100 223 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 12 of 23 reflects the similarity of each gene using ETEC F4 as a reference. (B) Comparative genomics showing 224 the presence and absence of virulent genes. (C) Prediction of antibiotic resistance phenotype. Value 225 code: 0, No match found; 1: Match < 100% ID and match length < ref length; 2: Match = 100% ID and 226 match length < ref length; 3: Match = 100% ID and match length = ref length. 227 Antibiotic resistance phenotype 228 Genomic analysis revealed that all three clinical ETEC F18 isolates carry several antibiotic 229 resistance-associated genes (Fig. 6C, Table S3), which are absent in the ETEC F4 and 230 H10407. Interestingly, the isolates harbor genes conferring resistance to aminoglycosides, 231 aminocyclitols, quinolones, beta-lactams, folate pathway antagonists, and tetracyclines. 232 Antibiotic genes conferring resistance to streptomycin, ciprofloxacin, nalidixic acid, and 233 sulfamethoxazole are identified in all three ETEC isolates. 234 We phenotypically verified the antibiotic resistance genotype using microdilution (Table 2) and 235 disc diffusion (Table 3) assays against a panel of antibiotics. Phenotypic results confirmed the 236 resistance in the three clinical F18 isolates. Genomic prediction of strains 3EC1 and 27EC1 237 were resistant to penicillin (amoxicillin, ampicillin, ticarcillin, and piperacillin), aminoglycosides 238 (gentamicin, kanamycin, neomycin, netilmicin, paromomycin, apramycin, sisomicin, 239 streptomycin, tobramycin), aminocyclitol (closely related to aminoglycosides, spectinomycin), 240 cephalothin, sulfamethoxazole, and florfenicol. However, 3247EC was found to be sensitive to 241 β-lactam antibiotics, including penicillin derivatives (amoxicillin, ampicillin, ticarcillin, and 242 piperacillin), as well as aminoglycosides (gentamicin, kanamycin, neomycin, netilmicin, 243 paromomycin, apramycin, sisomicin, streptomycin, and tobramycin), which contrasts with the 244 antimicrobial resistance (AMR) prediction. Interestingly, we observed a morphological change in 245 3247EC upon ampicillin treatment, characterized by bacterial elongation. This atypical 246 morphology reverted to the typical rod shape upon removal of ampicillin. In contrast, no such 247 morphological changes were observed in 27EC or 3EC1 under the same treatment conditions 248 (Fig. S3). 249 TABLE 2. Minimum inhibitory concentrations (MIC, µg/mL) of select antibiotics against 250 Escherichia coli strains 3EC1, 27EC1, 3247EC, H10407, and F4(K88). 251 Escherichia coli strain 3EC1 3247EC 27EC1 H10407 F4(K88) Amoxicillin >128 4 >128 2 8 Ampicillin >128 4 >128 2 16 Apramycin >128 64 >128 32 16 Aztreonam 0.25 0.12 0.12 0.03 0.12 Bleomycin 2 1 1 2 1 Cefepime 0.12 0.06 0.06 0.03 0.03 Cefotaxime 0.06 0.06 0.06 0.03 0.06 Ceftazidime 0.25 0.25 0.5 0.12 0.25 Ceftriaxone 0.12 0.12 0.12 0.06 0.12 Cephalothin 32 32 32 8 32 Chloramphenicol 2 2 2 0.5 2 Colistin 0.25 0.25 0.25 0.25 0.25 Doxycycline 16 8 32 0.5 2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 13 of 23 Florfenicol 8 8 8 2 4 Gentamicin >128 1 32 1 0.5 Kanamycin >128 4 >128 8 2 Neomycin >128 2 128 2 2 Netilmicin >128 2 32 1 0.5 Paromomycin >128 4 >128 4 2 Piperacillin >128 2 >128 1 2 Sisomicin >128 1 16 2 0.5 Spectinomycin >128 32 >128 32 16 Streptomycin >128 >128 >128 8 2 Sulfamethoxazole >512 >512 >512 >512 16 Tetracycline 32 128 >128 2 2 Ticarcillin >128 16 >128 8 16 Tobramycin >128 1 32 1 0.5 Trimethoprim >128 >128 0.12 0.12 0.25 252 TABLE 3: Inhibition zones (mm) of the antibiotics against Escherichia coli strains after agar disc diffusion 253 assay. 254 Escherichia coli strain Antibiotics 3EC1 3247EC 27EC1 H10407 F4(K88) 3247EC+Amp Ampicillin 0 21 0 23 22 25 Sulfamethoxazole 0 0 28 24 27 NT Clindamycin 0 0 8 0 0 NT Penicillin G 0 0 0 11 9 NT Gentamicin 0 19 10 18.5 18 18.2 Tetracycline 23 8 0 30 25 7 Vancomycin 0 9 0 9 10 8 Cephalothin 17 17 17 24 18 22 Ciprofloxacin 0 25 27 35 33 21.6 Chloramphenicol 24 23 29 33 25 24 NT= Not tested 255

256 The swine industry suffers from significant economic losses due to piglet mortality from ETEC 257 strains (8,27). Yet molecular information, antibiotic resistance, and virulence phenotype data are 258 limited (28,29). In this study, we conducted a comprehensive analysis of three clinical E. coli 259 isolates from U.S. pig farms, elucidating their respective pathobiology through molecular & 260 cellular assays and whole-genome sequencing. Notably, we identified the E. coli isolates 261 adhesion efficiency to swine intestinal cells peaks at 9 days post-confluence, coinciding with the 262 progressive upregulation of the F18 receptor (F18R/FUT1) in the neonatal porcine intestine 263 during the suckling phase (30). This temporal correlation underscores the feature of ETEC F18 264 infection. The lack of significant differences in F18 ETEC adhesion between the IPEC-1 and 265 IPEC-J2 cell lines may be a threshold effect in gene expression. The observed differences in 266 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 14 of 23 FUT1/2 expression levels are too subtle to manifest as a functionally distinct adhesion profile. 267 Specifically, ETEC F4 still outcompetes F18 strains in the IPEC-1 model, suggesting that the 268 cell lines express a substantial level of F4-specific receptors as well. Ultimately, these results 269 underscore that ETEC adhesion is a complex multifactorial process determined by factors 270 beyond FUT1/2 expression levels alone. 271 Among the three F18 isolates, 3EC1 exhibited the most pronounced virulent phenotype. 272 Phylogenetic analysis shows a closer evolutionary trajectory between porcine ETEC 3EC1 and 273 human ETEC H10407, suggesting potential cross-species transmission or shared evolutionary 274 pathways. Comparative genomic and phylogenetic analysis revealed that 3EC1 harbors genetic 275 features resembling both porcine F4-like and human ETEC pathovars, particularly in fimbrial 276 biosynthesis and LPS synthesis pathways. These traits were functionally validated across 277 porcine and human intestinal cell line models. Furthermore, WGS also detected stx2e, a 278 defining toxin of Shiga toxin-producing E. coli (STEC), in F4 and 3EC1, consistent with the 279 prevalence of swine ETEC isolates in the U.S. (31) and China (32). This finding suggests the 280 emergence of novel E. coli hybrid pathovars with combined virulence mechanisms, potentially 281 complicating clinical management and zoonotic risk. 282 Another critical concern arising from this study is the pervasive antibiotic resistance observed in 283 these isolates, which contrasts with the resistance profiles of classical ETEC F4 and F18 284 serovars. Both 3EC1 and 3247EC exhibit extended-spectrum β-lactamase (ESBL) phenotypes 285 by carrying blaCTX-M-15 and blaTEM, respectively. Resistance to ampicillin is the most commonly 286 observed trait in E. coli isolates from animal farms in Europe and America in recent years (33). 287 A clinical survey of E. coli isolates from Bangladesh reported blaCTX-M-15 as the most prevalent β-288 lactamase antibiotic-resistant gene, detected in 52% of cases, while blaTEM was identified in 289 20% of cases (34). Surprisingly, no antibiotic resistance phenotypes such as ampicillin and 290 gentamicin were observed during phenotypic testing, despite the presence of resistance genes. 291 Notably, 3247EC is the only isolate found to carry plasmids. The loss of resistance in 3247EC 292 may be due to plasmid loss during sub-culturing (Table 4), which may be associated with 293 changes in morphology (Fig. S3). The dissemination of ESBL is driven mainly via horizontal 294 gene transfer (HGT) from plasmid-borne elements (29). 295 Additionally, 3EC1 and 27EC1 exhibited elevated resistance to aminoglycosides, consistent with 296 previously reported patterns in ETEC F18 (29). This may be attributed to the widespread use of 297 gentamicin in the treatment of neonatal colibacillosis in pig farms (35). The ETEC F18 isolates 298 from animal farms have shown significantly increased resistance to gentamicin and kanamycin; 299 a trend is not observed in ETEC F4 (33). The increasing prevalence of multidrug-resistant ETEC 300 isolates poses a significant public health threat, as HGT facilitates the rapid spread of resistance 301 determinants across microbial communities. Given the diversity and adaptability of these 302 communities, continuous genetic monitoring and surveillance are essential to track emerging 303 resistance patterns and inform mitigation strategies. Collectively, these findings emphasize the 304 dynamic evolution of ETEC pathovars, driven by both virulence gene diversification and 305 antibiotic selection pressures. To mitigate the risks of emergent hybrid strains and multidrug 306 resistance, sustained genomic surveillance and stringent antibiotic stewardship in livestock 307 systems are imperative. Such measures will safeguard animal and human health against these 308 adaptable pathogens. 309

310 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 15 of 23 Bacterial strains and growth conditions 311 Porcine ETEC clinical strains (F4, 3EC1, 27EC1, 3247EC) were initially isolated from swine 312 rectal swabs, while the human reference strain H10407 (ATCC35401), was isolated from human 313 feces (Table 4). Bacterial cultures were inoculated from single colonies and grown in tryptic soy 314 broth (30 g/L) supplemented with yeast extract (6 g/L) (TSBYE) (BD BBL, Franklin Lakes, NJ, 315 USA) in an orbital shaker at 150 rpm, 37°C for 12–16 h. Freshly prepared stationary-phase 316 cultures were used in all experiments, with an OD600 of approximately 1.5. L. monocytogenes 317 F4244 was grown in TSBYE at 37°C for 12–16 h. 318 TABLE 4. Enterotoxigenic Escherichia coli (ETEC) strains (a) and primers (b) used in the study. 319 (a) ETEC strains 320 ETEC strains Isolate Type Source of Culture Provided by F4 (K88) Escherichia coli Swine rectal swab Dr. Paul Ebner, Purdue University F18-3EC1 Escherichia coli Swine rectal swab Dr. Donald Bade, Ft. Collins, CO F18-27EC1 Escherichia coli Swine rectal swab Dr. Donald Bade, Ft. Collins, CO F18-3247EC Escherichia coli Swine rectal swab Dr. Donald Bade, Ft. Collins, CO H10407 (O78:H11) Escherichia coli Human feces ATCC 35401 (Manasas, VA, USA) 321 (b) PCR Primers 322 Genes Sequences Size (bp) Ref fedA (F18ac) F: 5’-GTGAAAAGACTAGTGTTTATTTCTTTT-3’ R: 5’-CTTGTAAGTAACCGCGTAAGC-3’ 506 (9) FUT1 (U70883) F: 5′-TTTTAAGCCCCCAAACTGCC-3′ R: 5′-TAAATCGACCCCATCAGCCTC-3 126 (18) FUT2 (U70881.2) F: 5′-AATCCCTGACCTCACTCCGTG-3′ R: 5′-CGGAACTACAACTGCTGGCC-3 123 (18) GAPDH (NM_001206359.1) F: 5′-ACATCATCCCTGCTTCTACCGG-3′ R: 5′-CTCGGACGCCTGCTTCAC-3 188 (18) 323 DNA isolation and PCR determination of virulence factor in ETEC strains 324 DNA was extracted using the Zymo Research Quick-DNA Fungal/Bacterial Kits according to the 325 manufacturer's instructions. The quality (A260/280 ~1.8) and purity (A260/230 > 2.0) of the 326 extracted DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, 327 NJ, USA) and agarose gel electrophoresis. For PCR analysis (9), approx. 10 ng of genomic 328 DNA (gDNA) served as the template for amplification of the fedA gene (Table 4b). 329 Thermocycling conditions include initial denaturation at 95°C (30s); 30 cycles of 95°C (15s), 330 50°C (15s), 68°C (30s) with a final extension at 68°C (120s). The amplified products were 331 analyzed by agarose gel electrophoresis and visualized under UV light at 320 nm. 332 Reverse-transcriptase quantitative PCR for the expression F18 receptors 333 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 16 of 23 Swine intestinal epithelial cell lines (IPEC-1 and IPEC-J2) were maintained at 37°C under 5% 334 CO2 in RPMI-1640 medium supplemented with 10% FBS. Upon reaching confluence, cells were 335 cultured for an additional 6, 9 and 16 days prior to total RNA extraction. RNA quality is ensured 336 (A280/260 > 2.0) and normalized to 2000 ng before reverse transcription (NEB). Transcribed 337 cDNA (NEB) was 10-fold diluted (the total reverse transcription mixture was 20 µL, diluted to 338 200 µL by adding 180 µL DEPC water, and 3 µL of cDNA from each sample was used for PCR) 339 in ultra-pure water (ThermoFisher). The diluted cDNA was used as a template for qPCR 340 amplification for FUT1 and FUT2, as before (18). GAPDH was used as an internal reference 341 gene. The RT-qPCR primers used are listed in Table 4b. 2-ΔΔCT method was used to calculate 342 the relative expression of the FUT1/2 in IPEC-1 and IPEC-J2 cell lines. 343 Whole genome sequence analysis and genotyping 344 Bacterial genome sequencing was outsourced to GenScript (Piscataway, NJ, USA) for library 345 preparation and sequencing on the Illumina NextSeq platform using 150 bp paired end reads. 346 Raw reads were assembled using Shovill (Galaxy v1.0.4), and the assembly quality was 347 evaluated using QUAST (36). The assembled genomes of ETEC isolates were annotated and 348 comparatively analyzed using the RAST server (37). Plasmids, virulence factors, antibiotic 349 resistance genes, fimbrial type, serotype (O and H serogroups), and multilocus sequence types 350 (MLST) are identified by phenotyping services from the Center for Genomic Epidemiology 351 (https://www.genomicepidemiology.org/services/). 352 Genome annotation, phylogenetic analysis, and comparative genetics 353 In this study, 11 bacterial genomes were annotated using GTDB-Tk (38). A core conserved 354 gene approach was utilized to identify and extract the gene sequences of 120 conserved 355 proteins from the genome data. The resulting protein sequences were aligned through multiple 356 sequence alignments. Phylogenetic analysis was subsequently conducted on the aligned 357 sequences of 120 core genes using MEGA 11 software (39), employing the Neighbor-Joining 358 (NJ) method with 1000 bootstrap replicates. 359 Bacterial adhesion to porcine and human intestinal cell lines 360 To further validate the differential ETEC adhesion, two porcine intestinal epithelium cell lines 361 (IPEC-1 and IPEC-J2) with distinct susceptibility to ETEC fimbriae types were used. The IPEC-362 J2 cell line is a morphologically and metabolically more active cell model with higher expression 363 of microvilli than the IPEC1 cell line (21). IPEC-1 and IPEC-J2 purchased from Leibniz Institute 364 DSMZ (Braunschweig, Germany) were cultured in complete RPMI-1640 medium (Thermo 365 Fisher Scientific) supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal 366 bovine serum (FBS, Atlanta Biologicals), and 0.0001% epidermal growth factor (EGF, Corning 367 Life Sciences). The human colon-derived Caco-2 cells were cultured in complete Dulbecco's 368 Modified Eagle's Medium (DMEM) containing 10% FBS. To investigate the adhesion capacity of 369 ETEC, IPEC-1, IPEC-J2 cells at three maturation stages (6, 9, and 16 days post-confluence), 370 designated as "Early," "Mid," and "Late," were used as a model to mimic the natural physiology 371 of the intestine and assess corresponding adhesion dynamics (Fig. S2A, C). 372 For adhesion assays, stationary-phase bacterial cultures were diluted to a multiplicity of 373 infection (MOI) of 10 (5x106 to 1x107 CFU/well according to cell density) in serum-free RPMI-374 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 17 of 23 1640, unless otherwise stated. Bacterial cultures were centrifuged at 6000× g for 5 min, washed 375 with sterile phosphate-buffered saline (PBS), and resuspended in 500 µL of serum-free RPMI-376 1640. To facilitate bacterial contact with the cell layers, 0.5 mL of the bacterial suspension was 377 added to each well of the 12-well plates, ensuring minimal liquid volume. The cells were 378 incubated at 37°C for 30 min to allow bacterial adherence. After incubation, non-adherent 379 bacteria were removed by washing the cells with serum-free RPMI-1640. To quantify adhered 380 bacteria, the cells were lysed with 0.1% Triton X-100 in serum-free RPMI-1640. This 381 concentration effectively lysed mammalian cells without affecting bacterial viability, releasing 382 any intracellular bacteria. The number of adherent bacteria was determined by serial dilution 383 and plating on MacConkey agar (BD BBL, Franklin Lakes, NJ, USA). Each experiment was 384 conducted in duplicate. 385 ETEC-induced cytotoxicity 386 To evaluate the cytotoxic effects of ETEC, if any, on three different cell models during the 387 adhesion assay, the supernatants were collected after bacterial infection and analyzed for 388 lactate dehydrogenase (LDH) activity using the LDH Cytotoxicity Assay Kit (Cayman Chemical 389 Company, Ann Arbor, MI). The LDH assay, an indicator of cell damage, quantifies the release of 390 LDH from compromised cells into the culture media, providing a measure of the percentage of 391 cytotoxicity (40). Supernatants from untreated cells served as a negative control, while a 392 solution of 1% Triton X-100 in RPMI-1640, which fully lyses cells to release maximal LDH, was 393 used as a positive control. Each experimental condition was performed in triplicate. Absorbance 394 was measured spectrophotometrically at 490 nm using a microplate reader. The percentage of 395 cytotoxicity was then calculated (40) 396 Wright-Giemsa staining 397 Wright-Giemsa staining was employed to visualize infected cellular structures and the binding of 398 bacterial pathogens to cells. Cell lines were grown in chambered Lab-Tek™ II slide flaskets 399 (Thermo Scientific) to about 80% confluence. Following bacterial infection (MOI 10) for 1 h at 400 37ºC, the cells were rinsed twice with PBS and fixed with methanol for 5 min. The cells were 401 then flooded with Giemsa stain solution (10% Giemsa stain, 10% methanol, and 80% PBS) for 402 45 min, rinsed with PBS for 1 min, and then rinsed with deionized (DI) water. The slides were 403 air-dried and examined under a Leica light microscope (Wetzlar, Germany). 404 Microdilution assay to test antibiotic resistance 405 Antibiotics were dissolved in water or dimethyl sulfoxide (DMSO) at a concentration of 10 - 40 406 mg/mL and were visually checked to ensure complete dissolution. E. coli strains were streaked 407 on tryptic soy agar (TSA) plates and incubated aerobically at 37ºC overnight. As recommended 408 by the Clinical and Laboratory Standards Institute (41), colonies were suspended in PBS and 409 diluted in Mueller-Hinton broth to achieve a concentration of 5 x105 CFU/mL. The bacterial 410 suspensions were then mixed with 2-fold serial dilutions of the antibiotics in 96-well plates (in 411 duplicates). Colistin was used as a positive control, while DMSO was used as a negative 412 control. Plates were incubated aerobically at 37ºC for 16 h before inspected for bacterial growth. 413 The minimum inhibitory concentration (MIC) of each antibiotic was the lowest concentration at 414 which the wells were clear, indicating no bacterial growth. 415 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 18 of 23 Agar disk diffusion assay 416 Bacterial isolates were grown overnight on TSA plates at 37ºC. A bacterial suspension of 1 x 417 108 CFU/mL was prepared in PBS and was spread evenly on the surface of Mueller-Hinton agar 418 plates. Antimicrobial susceptibility discs (Oxoid, Lenexa, KS) were aseptically placed on the 419 surface of each inoculated plate and incubated at 37ºC for 16 h. The diameter of the inhibition 420 zone around each disc was measured three times, and the average diameter was reported 421 (Table 3). The test was repeated for strain 3247EC on an ampicillin-containing plate (100 422 µg/mL) to evaluate the effect of including ampicillin on the bacterial resistance pattern. 423 Statistical analysis 424 Experimental data were analyzed using GraphPad Prism 9 (La Jolla, CA). Statistical 425 comparisons between treatments were conducted using one-way or two-way analysis of 426 variance (ANOVA) followed by Tukey’s multiple-comparison test for more than two treatments 427 or Student’s t-test for comparisons involving only two treatments. Unless otherwise specified, 428 data from all experiments are presented as the mean ± standard error of the mean (SEM) from 429 two independent experiments. 430 ACKNOWLEDGMENTS 431 This research was partly supported by United Animal Health, Inc. (Sheridan, IN, USA) and the 432 USDA National Institute of Food and Agriculture (Hatch accession no. 1016249). Any opinions, 433 findings, conclusions, or recommendations expressed in this publication are those of the 434 author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. We 435 thank Dr. Donald Bade and Dr. Paul Ebner for their generosity in providing the ETEC strains. 436 DATA AVAILABILITY 437 Genome sequence information has been deposited at NCBI (PRJNA1311150), and all other 438 data are presented in the manuscript. 439 CONFLICT OF INTEREST 440 The research was partially funded by United Animal Health (UAH), Inc (Sheridan, IN). N.H. is 441 employed by UAH. 442 SUPPLEMENTAL MATERIALS 443 Supplemental Figures S1 to S3 and supplemental Tables S1 to S3 444 Figure S1. (A) Growth of F18 clinical isolates on MacConkey agar plates. 445 Figure S2. Genome sequence of enterotoxigenic E. coli (ETEC) F18 strains. Genomic map of 446 ETEC (A) 3EC1, (B) 27EC1, and (C) 3247EC displaying the differential presence of genes 447 encoding fimbriae or fimbriae-like proteins (box). Panel D shows a comparative analysis of 448 major virulence factors among ETEC F18 and ETEC F4 strains. Differences in genes encoding 449 flagella, fimbrae, and LPS were seen among F18 strains, while no difference in toxin genes 450 were observed. 451 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 19 of 23 Figure S3. Morphological characteristics of enterotoxigenic Escherichia coli (ETEC) cells after 452 growth in ampicillin-containing or ampicillin-deficient tryptic soy broth (TSB) or Mueller-Hinton 453 agar plates. Magnification 1000x. 454 455 Table S1. List of plasmids identified in Escherichia coli strain 3247EC via PlasmidFinder. 456 457 Table S2. List of virulent genes identified in Escherichia coli strains 3EC1, 27EC1, and 3247EC 458 via VirulenceFinder. 459 460 Table S3. List of acquired antimicrobial resistance genes in Escherichia coli strains 3EC1, 461 27EC1, and 3247EC. 462 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 20 of 23

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Swine enteric colibacillosis: diagnosis, therapy and antimicrobial 566 resistance. Porcine health management 3:16. 567 36. Gurevich, A., V. Saveliev, N. Vyahhi, and G. Tesler. 2013. QUAST: quality 568 assessment tool for genome assemblies. Bioinformatics 29:1072-1075. 569 37. Brettin, T., J. J. Davis, T. Disz, R. A. Edwards, S. Gerdes, G. J. Olsen, R. Olson, R. 570 Overbeek, B. Parrello, G. D. Pusch, M. Shukla, J. A. Thomason, 3rd, R. Stevens, V. 571 Vonstein, A. R. Wattam, and F. Xia. 2015. RASTtk: a modular and extensible 572 implementation of the RAST algorithm for building custom annotation pipelines and 573 annotating batches of genomes. Sci Rep 5:8365. 574 38. Chaumeil, P. A., A. J. Mussig, P. Hugenholtz, and D. H. Parks. 2019. GTDB-Tk: a 575 toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics 576 36:1925-1927. 577 39. Tamura, K., G. Stecher, and S. Kumar. 2021. MEGA11: molecular evolutionary 578 genetics analysis version 11. Mol. Biol. Evol. 38:3022-3027. 579 40. Roberts, P. H., K. C. Davis, W. R. Garstka, and A. K. Bhunia. 2001. Lactate 580 dehydrogenase release assay from Vero cells to distinguish verotoxin producing 581 Escherichia coli from non-verotoxin producing strains. J. Microbiol. Methods 43:171-181. 582 41. CLSI. 2025. Performance Standards for Antimicrobial Susceptibility Testing, 35th 583 Edition. CLSI supplement M100. , 35th ed. 584 585 586 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint Page 23 of 23 FIGURE LEGENDS 587 FIG 1. Characterization of Enterotoxigenic Escherichia coli (ETEC) F18 strains. (A) 588 Hemolytic activity assay on sheep-red blood agar plates showing clear hemolytic zones 589 surrounding the colonies in all strains tested. (B) PCR confirmation of enterotoxigenic 590 Escherichia coli (ETEC) F18 strains showing amplification of fedA gene (506 bp), while ETEC 591 F4 and F5 and Listeria monocytogenes (Lm) F4244 showed no amplification as negative 592 controls. 593 594 FIG 2. Age-differentiated adhesion comparison of ETEC strains F4 and F18 on porcine 595 intestinal IPEC-1 (A,B) and IPEC-J2 (C,D) cells. (A,C) Adhesion characteristics of ETEC F4 596 and F18 strains to IPEC-1 (A) or IPEC-J2 (C) cells after 6, 9 and 16 days of post-confluence 597 (DPC), 30 min post-infection at MOI 10. Data represent mean ± SEM. ***, p<0.001; ****, 598 p<0.0001. (B,D) Photomicrograph of IPEC-1 (B) and IPEC-J2 (D) after 6,9, and 16 DPC. Scale 599 bar: 50 µm. 600 FIG 3. F18-specific host receptor analysis. (A) Relative mRNA expression level of FUT1 and 601 FUT2 of IPEC-1 to IPEC-J2 at day 6, 9 and 16 post cell confluence. (B) Fold changes of ETEC 602 adhesion on IPEC-1 to IPEC-J2 at day 6, 9 and 16 post cell confluence. Data represent mean ± 603 SEM. *, p<0.05; **, p<0.001. 604 FIG 4. Adhesion comparison of pathogenic ETEC strains on porcine intestinal cell lines. 605 (A) Comparative analysis of adhesion of ETEC strains to swine intestinal IPEC-1, IPEC-J2, and 606 Caco-2 cell lines. (B) Giemsa staining showing differential adhesion of ETEC strains to Caco-2 607 cell monolayers. (C) Cytotoxicity assay using lactate dehydrogenase (LDH) release from cell 608 monolayers after 30 min of ETEC exposure during the adhesion experiment. Data are mean ± 609 standard error of mean (SEM). 610 FIG 5. Genome sequence analysis. Genome-wide comparative analysis to examine the 611 genetic differences and similarities between enterotoxigenic Escherichia coli (ETEC) F18 612 isolates and ETEC F4. 613 FIG 6. Comparative genetics of human and swine pathogenic Escherichia coli strains. (A) 614 Phylogenetic tree and similarity of core virulent genes of enterotoxigenic Escherichia coli 615 (ETEC) clinical isolates from swine and human. The phylogenetic tree was constructed based 616 on core conserved genes, root on midpoint. Bootstrap value is 1000. For comparative virulent 617 gene analysis, a scale from 0 to 100 reflects the similarity of each gene using ETEC F4 as a 618 reference. (B) Comparative genomics showing the presence and absence of virulent genes. (C) 619 Prediction of antibiotic resistance phenotype. Value code: 0, No match found; 1: Match < 100% 620 ID and match length < ref length; 2: Match = 100% ID and match length < ref length; 3: Match = 621 100% ID and match length = ref length. 622 623 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 25, 2026. ; https://doi.org/10.64898/2026.01.23.701439doi: bioRxiv preprint