Is Shiga Toxin-Producing Escherichia coli O45 No Longer a Food Safety Threat? The Danger is Still Out There

Many Shiga toxin-producing Escherichia coli (STEC) strains, including the serogroups of O157 and most of the top six non-O157 serotypes, are frequently associated with foodborne outbreaks. Therefore, they have been extensively studied using next-generation sequencing technology. However, related information regarding STEC O45 strains is scarce. In this study, three environmental E. coli O45:H16 strains (RM11911, RM13745, and RM13752) and one clinical E. coli O45:H2 strain (SJ7) were sequenced and used to characterize virulence factors using two reference E. coli O45:H2 strains of clinical origin. Subsequently, whole-genome-based phylogenetic analysis was conducted for the six STEC O45 strains and nine other reference STEC genomes, in order to evaluate their evolutionary relationship. The results show that one locus of enterocyte effacement pathogenicity island was found in all three STEC O45:H2 strains, but not in the STEC O45:H16 strains. Additionally, E. coli O45:H2 strains were evolutionarily close to E. coli O103:H2 strains, sharing high homology in terms of virulence factors, such as Stx prophages, but were distinct from E. coli O45:H16 strains. The findings show that E. coli O45:H2 may be as virulent as E. coli O103:H2, which is frequently associated with severe illness and can provide genomic evidence to facilitate STEC surveillance.


Introduction
Shiga toxin-producing Escherichia coli (STEC), as one of the major foodborne pathogens, can produce Shiga toxins and cause severe human disease, such as diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) [1][2][3]. STEC strains have been characterized by serotyping based on the O-antigen, determined by the polysaccharide portion of the cell wall lipopolysaccharide (LPS) and the H-antigen related to the flagella protein [4]. E. coli O157 and the "top six" non-O157 serogroups, including O26, O45, O103, O111, O121, and O145, are the primary STEC pathotypes frequently associated with foodborne outbreaks around the world [5,6]. Among the non-O157 STEC serotypes, STEC O45 has been identified as a cause of sporadic cases of bloody diarrhea [1]. In 2005, the first STEC O45 outbreak occurred in New York City, in which a total of 52 inmates were sick with diarrhea or bloody diarrhea, likely resulting from exposure to an ill food worker [7,8]. Subsequently, two E. coli O45:H2-associated outbreaks were reported to have caused 18 illnesses in the United States, and contaminated smoked game meat and goats were implicated as sources of contamination in these outbreaks [9]. , which were sequenced in this study with the given accession numbers. Additionally, "reference" indicates the chromosome and plasmid of two clinical strains (FWSEC0003 and 2011C-4251) obtained from the National Center for Biotechnology Information (NCBI) with the accession numbers indicated in Table S1. α The strains contained two different plasmids.

Determination of Virulence and Antibiotic Resistance Genes
Two reference E. coli O45:H2 strains (FWSEC0003 and 2011C-4251) were obtained from the NCBI database to facilitate the genomic analyses of virulence and antibiotic resistance genes of STEC O45 strains. E. coli clinical O45:H2 (FWSEC0003) was previously isolated from human feces in Canada [35], and the E. coli O45:H2 strain (2011C-4251) was isolated from samples of human feces obtained in the United States [36]. The virulence gene profiles of six strains (four experimental strains and two reference strains) were obtained using VirulenceFinder 2.0 [37]. The prediction of antibiotic resistance genes in each bacterial chromosome was conducted using ResFinder 3.2 [38] and Abricate Microorganisms 2020, 8,782 4 of 16 0.5 (https://github.com/tseemann/abricate). The setting with a minimum of both 95% nucleotide sequence identity and coverage was used to screen for these genes.

Identification of the Mobile Genetic Elements: Prophages and Genomic Islands
The identification of prophage and prophage-like sequences from each bacterial genome was conducted using the PHASTER web server [39]. Genomic islands were analyzed using the IslandViewer4 web server [40]. The default parameters were used for all the software.

Whole-Genome-Based Phylogenetic Analysis
Six STEC O45 strains, including four strains sequenced in this study and two reference strains from the NCBI database, as well as nine different STEC strains of clinical and outbreak origin (Table S1) were subject to phylogenetic analysis by whole-genome multilocus typing (wg-MLST). Specifically, a total of 14,837 loci and 2513 core loci located on the entire genomes of 15 E. coli strains were analyzed and phylogenetic networks were constructed via a complete linkage method using BioNumerics 7.6 (Applied Maths, Kortrijk, Belgium). The GenBank accession numbers of 11 reference bacterial genomes obtained from the public database are indicated in Table S1.
2.6. Comparative Genomics of E. coli O45:H2 and E. coli O103:H2 Based on the results of wg-MLST, two reference E. coli O103:H2 strains (12009 and 2015C-3163) of clinical origin were subject to comparative genomics analysis with STEC O45:H2 on the virulence genes and mobile genetic elements to evaluate their genetic relatedness using the BLAST Ring Image Generator (BRIG) with a minimum nucleotide sequence identity of 50% [41]. The crucial virulence factors of STEC pathogens, including Stx prophages and LEE pathogenicity islands, were extracted from each genome and subsequently aligned using the MAFFT algorithm in Geneious (Version 11.1.5, Biomatters, New Zealand). The phylogenetic trees of Stx prophages were constructed using Mega-X (Version 10.0.5) with the maximum likelihood algorithm and visualized using the Interactive Tree Of Life (ITOL) webserver [42]. The aligned sequences and the consensus identity of the LEE pathogenicity islands were visualized using Geneious (Version 11.1.5, Biomatters, New Zealand) [43,44].

Genomic Features of Three E. coli O45:H16 Environmental Strains
The chromosomes of the three E. coli O45:H16 strains RM11911, RM13745, and RM13752 had genome sizes of 5,310,338, 5,264,698, and 5,264,517 bp, containing a total of 5194, 5264, and 5264 regions of coding DNA sequence (CDS), respectively; 91% of which were annotated with known functions (Table 1). RM11911, RM13745, and RM13752 strains also contained 22 rRNA and 90-91 tRNAs in each chromosome. Regarding the prediction of numerous mobile elements, RM11911 harbored 14 prophages, including a 65,626 bp Stx prophage carrying an stx 1a gene, whereas both RM13745 and RM13752 contained 12 prophages, including a 65,626 bp Stx1a prophage located at the same insertion site of each bacterial genome. Three E. coli O45:H16 strains also contained two additional virulence factors, iroN and lpfA genes, which were associated with the utilization of the siderophore enterobactin and bacterial adhesion and colonization in the intestine, respectively. Additionally, the antibiotic resistance gene mdfA was detected in each chromosome of E. coli O45:H16, which was attributed to a broad spectrum of drug-resistant features of these strains.
The numerical value indicates the number of genes present, and "-" means that no virulence gene was detected.

Genomic Features of a Clinical E. coli O45:H2 Strain
The E. coli O45:H2 strain SJ7, isolated from a patient stool, contained a 5,444,105 bp chromosome which was approximately 130-180 kb smaller than the genomes of the three E. coli O45:H16 strains ( Table 1). The chromosome of SJ7 consisted of 5278 CDSs, 22 rRNA, and 96 tRNA (Figure 1a). Ninety-one percent of CDSs were annotated with known functions. A total of 17 prophages, including one Stx1a prophage which was 65,813 bp in length, were predicted in the bacterial chromosome. Most of all, strain SJ7 contained a LEE pathogenicity island integrated at the pheV tRNA locus. Several non-LEE-encoded type III translocated virulence genes, including nelA, nelB, nelC, espI, and cif, were also present in the chromosome. No antibiotic resistance genes were found in the genome of SJ7. , and plasmid pSJ7-2 (c) for the clinical STEC O45:H2 strain SJ7 generated using CGview server. For the map of chromosome (a), the rings from the inside out represent the GC skew (green and pink), GC content (brown), coding DNA sequences (CDSs) (blue), tRNA (red), and rRNA (yellow). For the maps of two plasmids (pSJ7-1 and pSJ7-2), the same color codes are used, and no tRNA and rRNA were detected. The annotated functions of the CDSs are not indicated.
The two other clinical E. coli O45:H2 strains (FWSEC0003 and 2011C-4251) were obtained from the NCBI database to facilitate the genomic characterization of STEC O45:H2 strains (Table 1). Similar to the strain SJ7, FWSEC0003 and 2011C-4251 had a genome size of 5,532,455 and 5,440,026 bp, respectively, with an average GC content of 50.7% and a similar number of CDSs (>5700). FWSEC0003 contained an Stx1a prophage (56,560 bp), whereas 2011C-4251 had two Stx prophages consisting of a 64,500 bp Stx1a prophage and a 62,499 bp Stx2a prophage. Furthermore, both FWSEC0003 and 2011C-4251 strains encoded the LEE pathogenicity island, and several non-LEE-encoded type III translocated virulence factors, including nelA, nelB, espI, and cif (Table 2).
2011C-4251 had a plasmid (68,062 bp) which contained the virulence genes ehxA, etpD, and stcE, similar to SJ7. FWSEC0003 had two plasmids (95,228 and 52,940 bp), and the virulence genes etpD and stcE were found in the smaller plasmid. Furthermore, no antibiotic resistance genes were found in the plasmids of these two strains ( Table 2).

Phylogenetic Analysis of 15 STEC Strains Using Whole-Genome Multilocus Typing
The evolutionary relatedness of 15 STEC strains, including four E. coli O45 strains (RM11911, RM13745, RM13752, and SJ7) sequenced in this study, two reference E. coli O45 strains (FWSEC0003 and 2011C-4251), and nine reference STEC strains with different serotypes, was investigated through whole-genome multilocus typing analysis. The results show that these STEC strains grouped into five The phylogenetic tree indicates five distinct clusters, with the strain names shown in black color for cluster 1, green color for cluster 2, purple color for cluster 3, blue cluster for cluster 4, and orange color for cluster 5. The strains highlighted in yellow were sequenced this study. The sources of isolation (outbreak, clinical, and environmental) for these 15 strains are indicated right next to the strain names.

Comparative Genomics of E. coli O45:H2 and E. coli O103:H2 Strains
Due to the close genetic relatedness, the comparative genomics analysis was further performed on the backbones and mobile genetic elements of the three E. coli O45:H2 and two E. coli O103:H2 strains to investigate their evolutionary relationship. The whole-genome sequences of two E. coli O45:H2 (FWSEC0003 and 2011C-4251) reference strains and two E. coli O103:H2 (12009 and 2015C-3163) reference strains were compared against the E. coli O45:H2 strain (SJ7) using BLASTn, and the results show that the genomic backbones of these strains shared more than 95% identity covering over 95% of the SJ7 chromosome ( Figure 3). Furthermore, non-homologous regions were identified among the genomes of E. coli O45:H2 and E. coli O103:H2 strains, particularly in the sequences of their mobile genetic elements. In the three E. coli O45:H2 chromosomes, prophage sequences were the primary non-homologous regions. For example, a 42,590-bp prophage (prophage_17) present in the genome of SJ7 was absent in both genomes of FWSEC0003 and 2011C-4251. Additionally, both FWSEC0003 and 2011C-4251 contained a prophage sequence sharing a low nucleotide sequence similarity to prophage_7 located in similar regions in the chromosome of SJ7. Another prophage sequence (prophage_6) in SJ7 shared low homology to the counterpart prophage sequence in 2011C-4251. On the other hand, genomic heterology of the mobile genetic elements was also observed for the E. coli O45:H2 strain (SJ7) and two E. coli. O103:H2 (12009 and 2015C-3163) strains. The prophage_1 and prophage_17 sequences found in SJ7 were absent in similar regions in the chromosomes of 2015C-3163 and 12009, respectively. Additionally, there was a region of low homology located at the sequence of genomic island_7 in SJ7 compared to the counterpart in both E. coli O103:H2 strains. Minor variations in specific regions, such as genomic island_1, prophage_7, and Due to the close genetic relatedness, the comparative genomics analysis was further performed on the backbones and mobile genetic elements of the three E. coli O45:H2 and two E. coli O103:H2 strains to investigate their evolutionary relationship. The whole-genome sequences of two E. coli O45:H2 (FWSEC0003 and 2011C-4251) reference strains and two E. coli O103:H2 (12009 and 2015C-3163) reference strains were compared against the E. coli O45:H2 strain (SJ7) using BLASTn, and the results show that the genomic backbones of these strains shared more than 95% identity covering over 95% of the SJ7 chromosome ( Figure 3). Furthermore, non-homologous regions were identified among the genomes of E. coli O45:H2 and E. coli O103:H2 strains, particularly in the sequences of their mobile genetic elements. In the three E. coli O45:H2 chromosomes, prophage sequences were the primary non-homologous regions. For example, a 42,590-bp prophage (prophage_17) present in the genome of SJ7 was absent in both genomes of FWSEC0003 and 2011C-4251. Additionally, both FWSEC0003 and 2011C-4251 contained a prophage sequence sharing a low nucleotide sequence similarity to prophage_7 located in similar regions in the chromosome of SJ7. Another prophage sequence (prophage_6) in SJ7 shared low homology to the counterpart prophage sequence in 2011C-4251. On the other hand, genomic heterology of the mobile genetic elements was also observed for the E. coli O45:H2 strain (SJ7) and two E. coli. O103:H2 (12009 and 2015C-3163) strains. The prophage_1 and prophage_17 sequences found in SJ7 were absent in similar regions in the chromosomes of 2015C-3163 and 12009, respectively. Additionally, there was a region of low homology located at the sequence of genomic island_7 in SJ7 compared to the counterpart in both E. coli O103:H2 strains. Minor variations in specific regions, such as genomic island_1, prophage_7, and prophage_10, in E. coli O45:H2 (SJ7) were observed in comparison to both E. coli O103:H2 strains. These results indicate that the presence of different mobile genetic elements, and prophages in particular, was primarily attributed to the heterology of the closely related E. coli O45:H2 and E. coli O103:H2 strains.

Virulence Factors Located on Mobile Genetic Elements
The virulence factors of E. coli O45:H2 and E. coli O103:H2 were analyzed, and the results show that all virulence genes of each strain, except for gad, were located on different prophages and genomic islands (Figure 3). Stx prophages and LEE pathogenicity islands were among the most important virulence factors contributing to the pathogenicity of these pathogenic strains. The Stx prophages in the chromosomes of E. coli O45:H2 and E. coli O103:H2 were predicted and compared to investigate their genetic similarity (Figure 4). The phylogenetic analysis of Stx prophages showed that the Stx2a prophage in E. coli O103:H2 (12009) was classified as being alone in cluster 1. The Stx1a prophages from two E. coli O45:H2 strains (SJ7 and 2011C-4251) and two E. coli O103:H2 strains (12009 and 2015C-3163) were grouped into cluster 2. The Stx1a prophage and Stx2a prophage from E. coli O45:H2 (FWSEC0003) and E. coli O45:H2 (2011C-4251), respectively, were classified into cluster 3. Based on the phylogenetic results, the high sequence homology between the Stx1a prophage of E. coli O45:H2 strain (SJ7) and the two E. coli O103:H2 strains likely indicates that E. coli O45:H2 might exhibit a similar Stx prophage-associated pathogenicity to E. coli O103:H2.
Additionally, one of the essential virulence factors of STEC strains, the LEE pathogenicity island, was also present among the genomes of these E. coli O45:H2 and E. coli O103:H2 strains ( Figure 5).

Virulence Factors Located on Mobile Genetic Elements
The virulence factors of E. coli O45:H2 and E. coli O103:H2 were analyzed, and the results show that all virulence genes of each strain, except for gad, were located on different prophages and genomic islands (Figure 3). Stx prophages and LEE pathogenicity islands were among the most important virulence factors contributing to the pathogenicity of these pathogenic strains. The Stx prophages in the chromosomes of E. coli O45:H2 and E. coli O103:H2 were predicted and compared to investigate their genetic similarity (Figure 4). The phylogenetic analysis of Stx prophages showed that the Stx2a prophage in E. coli O103:H2 (12009) was classified as being alone in cluster 1. The Stx1a prophages from two E. coli O45:H2 strains (SJ7 and 2011C-4251) and two E. coli O103:H2 strains (12009 and 2015C-3163) were grouped into cluster 2. The Stx1a prophage and Stx2a prophage from E. coli O45:H2 (FWSEC0003) and E. coli O45:H2 (2011C-4251), respectively, were classified into cluster 3. Based on the phylogenetic results, the high sequence homology between the Stx1a prophage of E. coli O45:H2 strain (SJ7) and the two E. coli O103:H2 strains likely indicates that E. coli O45:H2 might exhibit a similar Stx prophage-associated pathogenicity to E. coli O103:H2.
island. The comparative genomics of LEE pathogenicity islands from the selected strains also showed that the E. coli O103:H2 strain (12009), associated with a diarrhea patient from a previous foodborne infection in Japan, and the three clinical E. coli O45:H2 strains (SJ7, FWSEC00033, and 2011C-4251) shared a high nucleotide sequence identity, demonstrating that these E. coli O45:H2 strains could harbor similar human pathogenesis, causing diarrhea.  The consensus identity of the alignment of five LEE pathogenicity islands indicates the mean pairwise nucleotide sequence identified for all pairs in the column: green = 100% identity; green-brown = <100% but >30% identity; and red = <30% identity. Virulence genes within the sequence of each LEE pathogenicity island (black) are annotated and colored (yellow).

Discussion
Due to the advancement of next-generation sequencing technology, the high accuracy of complete bacterial genome information has dramatically facilitated the genomic characterization of STEC pathogens and investigation of the evolutionary relationship between different pathogenic E. coli strains. A number of studies have utilized whole-genome sequencing technology to predict the parallel evolution of distinct STEC strains, including the O26, O111, O103, O145, and O157 serogroups [14,45]. Their findings have not only provided genomic evidence related to the clinical impact of these strains, but also facilitated the surveillance of these STEC serotypes. However, similar studies, including on published complete genomes of STEC O45 strains, are lacking. Currently, only two complete genome sequences of STEC O45:H2 strains (FWSEC0003 and 2011C-4251) are available in the public database. Therefore, in this study, four STEC O45 strains, from environmental and clinical samples, were sequenced and subjected to comprehensive genomic characterization, Additionally, one of the essential virulence factors of STEC strains, the LEE pathogenicity island, was also present among the genomes of these E. coli O45:H2 and E. coli O103:H2 strains ( Figure 5). LEE pathogenicity islands had an average sequence size ranging from 61,863 to 88,869 bp and were located at the pheU or pheV tRNA locus. All LEE pathogenicity islands contained the core virulence genes (espF, espB, espA, eae, and tir) in the highly conserved region ( Figure 5). All strains, except for E. coli O103:H2 (2015C-3163), contained the LEE pathogenicity islands harboring extra virulence genes, nleB and efa1, which were located in the non-homologous region of the LEE pathogenicity island. The comparative genomics of LEE pathogenicity islands from the selected strains also showed that the E. coli O103:H2 strain (12009), associated with a diarrhea patient from a previous foodborne infection in Japan, and the three clinical E. coli O45:H2 strains (SJ7, FWSEC00033, and 2011C-4251) shared a high nucleotide sequence identity, demonstrating that these E. coli O45:H2 strains could harbor similar human pathogenesis, causing diarrhea. island. The comparative genomics of LEE pathogenicity islands from the selected strains also showed that the E. coli O103:H2 strain (12009), associated with a diarrhea patient from a previous foodborne infection in Japan, and the three clinical E. coli O45:H2 strains (SJ7, FWSEC00033, and 2011C-4251) shared a high nucleotide sequence identity, demonstrating that these E. coli O45:H2 strains could harbor similar human pathogenesis, causing diarrhea.  identity; green-brown = <100% but >30% identity; and red = <30% identity. Virulence genes within the sequence of each LEE pathogenicity island (black) are annotated and colored (yellow).

Discussion
Due to the advancement of next-generation sequencing technology, the high accuracy of complete bacterial genome information has dramatically facilitated the genomic characterization of STEC pathogens and investigation of the evolutionary relationship between different pathogenic E. coli strains. A number of studies have utilized whole-genome sequencing technology to predict the

Discussion
Due to the advancement of next-generation sequencing technology, the high accuracy of complete bacterial genome information has dramatically facilitated the genomic characterization of STEC pathogens and investigation of the evolutionary relationship between different pathogenic E. coli strains. A number of studies have utilized whole-genome sequencing technology to predict the parallel evolution of distinct STEC strains, including the O26, O111, O103, O145, and O157 serogroups [14,45]. Their findings have not only provided genomic evidence related to the clinical impact of these strains, but also facilitated the surveillance of these STEC serotypes. However, similar studies, including on published complete genomes of STEC O45 strains, are lacking. Currently, only two complete genome sequences of STEC O45:H2 strains (FWSEC0003 and 2011C-4251) are available in the public database. Therefore, in this study, four STEC O45 strains, from environmental and clinical samples, were sequenced and subjected to comprehensive genomic characterization, particularly for crucial virulence factors, and a comparison with other reference bacterial genomes was conducted to understand whether or not STEC O45 poses a similar food safety threat as the other serogroups.

STEC O45:H16 Strains Are Genomically Conserved
In this study, the results showed that the genome features of three E. coli O45:H16 strains (RM11911, RM13745, and RM13752) contained similar virulence factors, most of which were located on similar prophage sequences. All three E. coli O45:H16 strains contained the same Stx1a prophage (65,626 bp) with 100% nucleotide sequence homology (Figure 4). A previous study in our lab also demonstrated that Stx1a prophages from STEC O45 genomes were more conservative than those detected from other serotypes of STEC genomes [46]. Additionally, these three STEC O45:H16 strains contained a virulence factor-the iss gene-contributing to the serum resistance of E. coli strains [47,48]. A previous study revealed that the tested E. coli strains contained three iss alleles, which were likely disseminated among E. coli bacteria through horizontal gene transfer [49]. In this study, each of the three O45:H16 strains contained two different iss alleles, which were located on the Stx1a prophage and a suspected 50,703-bp prophage sequence, respectively. These findings show that the three environmental E. coli O45:H16 strains in this study were genomically conserved, with the virulence factors being carried by certain prophages.

STEC O45:H2 Evolved from a Common Ancestor with STEC O103:H2
The current results of wg-MLST analysis showed that three STEC O45:H2 strains (SJ7, FWSEC0003, 2011C-4251) and two STEC O103:H2 strains (12009 and 2015C-3163) have a close evolutionary relationship ( Figure 2). Moreover, the comparative genomics of E. coli O45:H2 and E. coli O103:H2 revealed that the genomic backbone of these strains share 99% homology. Previous studies have demonstrated that a large percentage of E. coli O103:H2 strains have a high level of virulence in human infection and are commonly associated with diarrhea and HUS [50,51]. In particular, E. coli O45:H2 strains (SJ7, FWSEC0003, 2011C-4251) were shown to contain the crucial virulence factors, eae and tir, that are found in most STEC O103:H2 strains and are involved in the formation of A/E lesions [52]. Additionally, E. coli O45:H2 and E. coli O103:H2 used in this study were found to contain non-LEE-encoded type III translocated virulence genes, which are related to bacterial colonization and the development of HUS [53,54].
Additionally, the wg-MLST result in this study showed that E. coli O145:H28 (RM13514) and E. coli O157:H7 (Sakai) shared a close phylogenetic relationship, which is in agreement with the findings of a previous study demonstrating that STEC O145:H28 and O157:H7 strains evolved from a common evolutionary lineage [55]. The author also found that these two STEC serotypes could be further classified into sublineages based on the presence of different virulence factors, such as Stx prophages and the large virulence plasmids. However, in this study, the most highly heterogeneous regions between E. coli O45:H2 and E. coli O103:H2 strains fell on the sequences corresponding to prophages and genomic islands which were not associated with virulence factors (Figure 3). These findings likely suggest that due to a high homology of the virulence factors, E. coli O45:H2 may possess a similar pathogenicity to E. coli O103:H2 and could potentially cause severe human diseases such as HUS. Thus, the critical findings of the close genetic relatedness between the clinical STEC O103:H2 and STEC O45:H2 strains could facilitate future development of the antimicrobial strategies for the control of STEC O45 strains because some antimicrobial agents, such as weak acids, or lytic bacteriophages, effective in mitigating STEC O103 could also be tested to control STEC O45 strains [56,57]. Other natural antimicrobials that are capable of displaying a wide spectrum of antimicrobial activity and overcoming the emergence of antibiotic resistance in bacterial pathogens could also be considered [58].

Mobile Elements Play a Key Role in Driving the Virulence Evolution of STEC
Mobile genetic elements play a key role in shaping the bacterial genome and virulence evolution of STEC [59][60][61]. The current results also show that most virulence factors were located on mobile genetic elements, including plasmids, prophages, and genomic islands. In this study, the four STEC O45 strains contained different plasmids. Even the three E. coli O45:H16 environmental strains, which shared high nucleotide sequence similarity of the bacterial chromosomes, had plasmids with different genome sizes and contained various virulence genes, including afa, cdt, esp, cnf1, and iha. Several studies have indicated that the plasmids of LEE-negative STEC strains, which were found to contain encoded virulence factors, can also cause severe human diseases such as HUS, an ability which is frequently observed for LEE-positive STEC pathogens [62][63][64][65]. Additionally, a previous study conducted by Michelacci et al. reported that one LEE-negative STEC strain, isolated from an HUS patient, contained a plasmid harboring several virulence factors, including ehxA, sta1, and a novel variant of the faeG, in particular [64]. Notably, the faeG, encoding the production of ETEC F4 fimbriae, is commonly found in the pathogenic E. coli strains related to causing swine diseases, but not in those that could affect human health. Therefore, the presence of faeG in the plasmid of this human clinical strain provided genetic evidence for virulence gene transfer among plasmids of different STEC strains, which contributed to the pathogenicity evolution of the STEC strains.
Prophages represent an essential member of mobile genetic elements and are highly distributed in the genomes of different E. coli strains [66]. Many studies have indicated that stx, encoded in Stx prophage sequence, is the main virulence factor of STEC [46,[67][68][69]. The results of this study showed that three E. coli O45:H16 strains carried a similar Stx prophage, whereas the phylogenetically related E. coli O45:H2 and E. coli O103:H2 strains carried distinct Stx prophages. The current results were supported by the finding of our previous study that the distribution of different Stx prophages is highly associated with specific STEC serotypes. Some serotypes of STEC strains, such as O103, were able to accept a wide range of different Stx prophages, but other serotypes, like O121, were only susceptible to infection with genetically conserved Stx prophages [46]. Therefore, the diversity of Stx prophages from E. coli O45:H2 (SJ7) and E. coli O103:H2 strains used in this study likely implies that these strains are susceptible to accepting exogenous genes, rather than E. coli O45:H16 strains. Additionally, other prophages predicted from all of the STEC O45 and O103 strains in this study also contained several virulence factors, including non-LEE encoded type III translocated effectors (nleA, nleB, nleC, and cif ), serine protease autotransporters (espI), and increased serum survival gene (iss). Similar results were also found in a previous study, in which the genes encoding type III secretion in E. coli were located on a vast prophage region, which acted as a crucible for the evolution of pathogenicity in numerous E. coli species [70]. Interestingly, the results of this study show that the prophages carrying non-LEE encoded type III translocated effectors were only identified in LEE-positive strains, including E. coli O45:H2 and E. coli O103:H2 strains, but were absent in the E. coli O45:H16 LEE-negative strains. This phenomenon will need to be investigated in future studies.

Conclusions
This is the first study to report the genomic characterization of STEC O45 strains of different origins. The whole-genome-based phylogenetic analysis revealed that the environmental E. coli O45:H16 strains were phylogenetically distinct from the clinical E. coli O45:H2 strains, whereas both clinical E. coli O45:H2 and E. coli O103:H2 shared a common evolutionary ancestor. Furthermore, most of the crucial virulence factors from E. coli O45:H2 and E. coli O103:H2 shared high nucleotide sequence similarity and were located on mobile genetic elements, such as prophages, genomic islands, or plasmids, which is strongly associated with the pathogenicity evolution of these STEC strains. The findings of this study provide better insights into the genomic characterization, evolutionary relatedness, and virulence evolution of STEC O45 strains and indicate that E. coli O45:H2 strains still pose potential threats to public health and should, therefore, be included in epidemiological surveillance.
Author Contributions: Y.Z. was responsible for strain preparation, data analysis, and manuscript preparation. Y.-T.L. was responsible for manuscript preparation. X.S. assisted the experiment design. V.C.H.W. conceived and supervised the study, aided in experiment design, and reviewed and edited the manuscript. All authors have read and agreed to the manuscript.