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Comparative genomic analysis of enterotoxigenic Escherichia coli O159 strains isolated from diarrheal patients in Korea

Background Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection that leads to diarrhea. Although some studies have proposed a potential association between the toxic profile and genetic background, association between toxin of ETEC and phylo-group has not been reported yet. The objective of this study was to examine genomic and phylogenetic characteristics of ETEC strain NCCP15731 and NCCP15733 by whole genome sequencing and comparative genomic analysis of two phylo-groups of O159 reference strains. Results Whole genome sequencing showed that genome size of NCCP15731 strain was 4,663,459 bp, containing 4435 CDS and 19 RNAs. The genome size of NCCP15733 was 4,645,336 bp, containing 4369 CDS and 23 RNAs. Both NCCP15731 and NCCP15733 were classified in the phylo-group A, which is one of major E. coli phylogenetic groups. Their serotype was O159:H34. They possessed the virulence factor such as adherence systems, auto transporter systems, and flagella segments of major driving force for ETEC pathogenicity. They also harbored STh enterotoxin. Hierarchical clustering result based on the presence or absence of a total of 108 major virulence factors of 14 O159 ETEC strains showed that seven strains in phylo-group A and seven strains in phylo-group B1 were clustered each other, respectively. Colonization factors (CFs) of NCCP15731 or NCCP15733 were not detected. Conclusions Serotype of NCCP15731 and NCCP15733, representing major types of ETEC in Korea, was O159:H34 and their MLST type was ST218. Comparison with other O159 strains revealed that NCCP15731 was specialized for transporter system and secretion system whereas NCCP15733 had unique genes related to capsular polysaccharide. Compared with E159, the most recent common ancestor, these two strains had different toxin type and virulence factors. These results will improve our understanding of ETEC O159 strains to prevent ETEC disease. Electronic supplementary material The online version of this article (10.1186/s13099-019-0289-6) contains supplementary material, which is available to authorized users.


Background
Escherichia coli is a Gram-negative bacterium belonging to family Enterobacteriaceae. Most of E. coli are typical members of the normal microflora of the humans and animals [1]. However, they could be classified as pathogenic strains that cause serious disease like diarrhea. Pathogenic E. coli can be divided to intestinal pathogenic E. coli (IPEC) and extraintestinal pathogenic E. coli (ExPEC) [2]. Intestinal pathogenic E. coli include enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli (STEC), while extraintestinal pathogenic E. coli include uropathogenic E. coli (UPEC) depending on the various virulence genes and phenotypes that play important roles in the pathogenesis [3][4][5][6][7][8]. ETEC is a common cause of bacterial infection that leads to diarrhea in infants, young children, and travelers in developing countries [9,10]. Because ETEC outbreaks are gained by consuming contaminated food or water, food contamination is an important concern for public health [11,12]. ETEC can be defined by the ability to produce a heat-labile toxin (LT) and/or heat-stable toxin (ST, including STh and STp) that can disturb the intestinal secretory state, thereby causing watery diarrhea of infected patients [13][14][15]. While STh is produced by ETEC isolated from humans, STp originally found in pig ETEC is also associated with disease in humans [13,16]. ETEC harbors one or more cell adhesion factors called colonization factors (CFs) to colonize epithelial cells of intestinal surfaces of hosts. ETEC CFs are named as coli surface antigens (CS) with a number except CFA/I. Currently, more than 30 colonization factors (CFs) have been identified in human ETEC. They are co-expressed with one, two, or three CFs and/or toxic factors, such as CS1 + CS3 (± CS21) with LT + STh, CS2 + CS3 (± CS21) with LT + STh, CS5 + CS6 with LT + STh, CS6 with STp, CFA/I (± CS21) with STh and CS7 with LT [17][18][19]. However, between 30 and 50% of ETEC have undetectable CFs, suggesting that there are still unknown CFs [20,21]. Phylogenetic analysis is important for investigating the evolution and diversity of E. coli and evaluating bacterial toxicity [22]. Most commensal and pathogenic E. coli strains belong to phylo-groups A and B1 [23,24]. About 90% of foodborne E. coli isolates in Korea belong to phylo-groups A and B1 [25].
In a previous study [26], 258 isolates from patients with diarrhea in Korea were analyzed for CFs and subjected to multi-locus sequence typing (MLST). ST171 (24%) was identified as the most prevalent ETEC type in Korea, followed by ST955, ST964, and ST656. NCCP15740 [27], representing the major MLST type ST171 of ETEC in Korea, has been investigated about its genomic features, CF genes, and virulence factors. However, other MLST types have not been investigated. In this study, we selected one of ST964 strains and one of ST656 strains identified in the previous work namely NCCP15731 and NCCP15733 [26], because these strains are the most representative strains of ST964 and ST656, and performed whole-genome sequencing. We compared whole-genome sequences of these two strains with NCCP15732 of O6 isolate and those of other ETEC strains reported as serotype O159 isolates.

Bacteria and strain isolation
Escherichia Coli were isolated from patients with diarrhea outbreak and identified as third and fourth highest prevalent MLST type (ST964 and ST656) of ETEC in Korea based on 7 isolates obtained from 2003 to 2011, respectively [26]. Candidate colonies of E. coli were identified based on phenotypes and biochemical properties using the API20E system (Biomerieux, Marcy l'Etoile, France). These isolated strains were deposited at National Culture Collection for Pathogens (NCCP) under the registration numbers NCCP15731 and NCCP15733. We selected 12 E. coli O159 strains as reference strains with the same O serotype as NCCP15731 and NCCP15733 listed in Table 2. These 12 E. coli O159 strains had been isolated globally from 1980 to 2011 [18]. Illumina short reads of reference strains were obtained at NCBI SRA (Sequence Read Archive) under the accession numbers listed in Table 2. De novo assemblies were performed with Spades (version 3.5.0) [28]. E. coli NCCP15732 was used as the reference strain because this strain represents the second highest prevalent ST955 in Korea. In comparison with 12 strains of E. coli O159 and NCCP15732 in Korea, we anticipated that we could identify genomic and pathogenic characteristics of NCCP15731 and NCCP15733.

Whole genome sequencing, assembly and annotation
Genomic DNAs of a single bacterial isolate of NCCP15731 or NCCP15733 were extracted from a pure culture. Potential contamination of other microorganisms was checked using a BLAST search against non-redundant database. A sequencing library was created using TruSeq sample preparation kit (Illumina, San Diego, CA, USA). Whole genome sequencing of NCCP15731 and NCCP15733 was performed using the Illumina HiSeq 2000 platform (Theragen Etex Bio Institute, Suwon, Republic of Korea). High-quality reads were assembled by discarding low-reads, quality scores < Q20, and duplicated reads using SOAP de novo (version 1.05) [29]. Assembled contigs of NCCP15731 and NCCP15733 were annotated-using the Rapid Annotation using Subsystem Technology (RAST version 4.0) [30] server pipeline.

Genomic analysis
The same method as described by Clermont et al. [22] was performed in silico to identify E. coli phylogenetic groups (A, B1, B2, D, E, and F) using Primersearch program from the European Molecular Biology Open Software Suite(EMBOSS) [31]. In silico SerotypeFinder (version 1.1) [32] was used to identify serotype of 14 O159 strains including NCCP15731 and NCCP15733 and NCCP15732. Colonization factors and MLST type were identified using CF primers [33] and E. coli MLST database [34]. To identify the genes encoding virulence factors, the total CDSs of NCCP15731, NCCP15732, NCCP15733 and 12 O159 strains were analyzed using BLASTP [35] against the virulence factor genes of E. coli listed in VFDB with an e-value of 1e−5 [36]. We selected genes with coverage of at least 60%. Resistance genes in Whole-genome sequence of all isolates were identified by ResFinder [37] Default thresholds of coverage of at least 60% and identity of at least 90% were employed.

Phylogenetic analysis
To identify evolutionary relationship of 14 O159 strains and NCCP15732, phylogenetic analysis was performed. Multiple sequence alignments obtained from concatenated whole genome sequences of each of all strains were performed with Mugsy (version 1.2.3) [38]. The generalized time reversible + CAT model [39] was used for maximum-likelihood phylogenetic tree construction using FastTree (version 2.1.7) [40]. Resulting trees were visualized with FigTree (ver 1.3.1) (http://tree.bio.ed.ac. uk/softw are/figtr ee). In order to exclude the effect of HGT (Horizontal gene transfer) in phylogenetic analysis, multi-locus sequence analysis method was used [41,42]. Seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) from each of all ETEC sequences were obtained according to the protocol described in E. coli MLST database (http://mlst.warwi ck.ac.uk/mlst/ dbs/Ecoli /docum ents/prime rsCol i_html). A phylogenetic tree based on MLST genes was created using the method employed for whole genome phylogenetic analysis.

Genomic analysis
The genome size of NCCP15731 was 4,663,459 bp with G+C content of 50.7%. The genome size of NCCP15733 was 4,645,336 bp with G+C content of 50.6%. Based on RAST analysis, 4435 coding sequences and 19 tRNA genes were detected in the genome of NCCP15731, of which 3855 (87%) were functional. A total number of 4369 coding sequences and 23 tRNA genes were detected in the genome of NCCP15733, of which 3852 (88%) were functional (Fig. 1). In silico analysis revealed that serotype of both NCCP15731 and NCCP15733 was O159:H34 belonging to phylo-group A, one of major E. coli phylogenetic groups [23]. Genomic and phylogenetic  Table 1. We investigated toxin types and colonization factors of NCCP15731 and NCCP15733. Both NCCP15731 and NCCP15733 possessed STh enterotoxin, but not LT enterotoxin. As shown in Table 2, two strains, E133 and E1679sc, contained both STp and LT. Four strains contained STh or STp, while seven strains contained LT. CFs were not found in eight strains of all isolates, including NCCP15731 and NCCP15733. NCCP15732 had STh and LT enterotoxin and harbored CS1 and CS3 of CFs.

Phylogenetic analysis
Phylogenetic comparison of candidate genes implemented in SEED [43] showed that NCCP15731 and NCCP15733 were most close to E. coli O157:H7 str. 88.1467 (score: 531 and 530, respectively). Whole genome phylogenetic analysis and MLST phylogenetic analysis were performed based on multiple sequence alignments of whole genomes, and seven MLST genes (adk, fumC, gyrB, icd, mdh, purA, and recA) of 15 E. coli isolates including NCCP15731, NCCP15732 and NCCP15733, respectively (Fig. 2). Whole genome phylogenetic tree showed that NCCP15732 and 14 E. coli O159 strains were clustered into two phylo-groups (A and B1) like previous study [18]. NCCP15731 and NCCP15733 in this study belonged to phylo-group A and were clustered with E. coli O159:H34 str. E159 with sequence type of ST218 (Fig. 2a). MLST phylogenetic analysis also showed that NCCP15731 and NCCP15733 belonged to phylogroup A (Fig. 2b). E. coli E159 strain was also placed with NCCP15731 and NCCP15733 in MLST phylogenetic tree.

Virulence factors
The acquisition of virulence factors has been suggested to be a major driving force for ETEC pathogenicity [44,45]. ETEC causes disease by colonizing the small bowel through attachment to the host epithelial lining by surface proteins called CFs and possibly other surface structures. Subsequently, adherent ETEC elaborates enterotoxins that cause typical clinical manifestations of ETECinduced diarrhea. Thus, virulence factors can be used as important guide for understanding their pathogenicity. Virulence factors of NCCP15731 and NCCP15733 were investigated and these factors were compared with those of 14 O159 strains and NCCP15732. We identified 222 virulence factors grouped into 28 categories and 74 subcategories (Additional file 1: Table S1).  In silico analysis revealed that major virulence factors belonged to the following six categories: adherence, auto transporter, iron uptake, non-LEE encoded TTSS effectors, toxin, and secretion system. Hierarchical clustering based on the presence or absence of 108 major virulence genes of 14 O159 strains was constructed using R (version 3.4.3) (Fig. 3) [46]. 14 O159 strains of phylo-group A and B1 shown in Fig. 2a again completely clustered each other, respectively. These results suggested that the virulence factors were related with the phylo-group [23,44,[47][48][49][50].
ECP (E. coli Common Pilus), EaeH, Type I fimbriae, Curli fimbriae, Stg fimbriae, AIDA-I type and UpaG, which belong to 13 potentially functional systems in adherence and auto transporter categories, are known to be produced by pathogenic E. coli [51]. These functional systems suggest that the ability of ETEC to attach to the host surface is the most important step in successful colonization [52]. Compared with NCCP15732 strain, all of O159 strains had EaeH system in adherence category. A virulence factor, espC, was only identified in NCCP5731 and NCCP15733. Conversely, ehaA auto transporter gene and stg fimbriae (stgABCD) systems were only found in phylo-group B1. It is known that stg fimbriae (stgABCD) systems contribute to the attachment of human epithelial cells. They are associated with phylogenetic group B1 [53].

Resistance factors
All isolates in the study were analyzed for antimicrobial susceptibility. We identified 11 resistance factors of six phenotypes (Table 3). One resistance factor, mdf(A) of MLS (Macrolide Lincosamide and Streptogramin) phenotype was found in all of isolates. We identified 11 resistance factors of six phenotypes (Table 3). NCCP15731 had six resistance factors of six phenotypes, but NCCP15733 had only one resistance factor. In phylo-group A, except NCCP15731 and E1573, five strains had only a mdf(A) resistance factor

Comparison with other E. coli strains in phylo-group A
To calculate Average Nucleotide identity (ANI), five genomes of O159 of phylo-group A including NCCP15731 and NCCP15733 were compared with each other using ANIu calculator [56]. E. coli E1573, and E1679sc were excluded due to the presence of strains with the same MLST type and toxin type. Results indicated that NCCP15731 was most similar to NCCP15733, with OrthoANIu value of 99.97% (Table 4). Both NCCP15731 and NCCP15733 were most similar to E. coli strain E159 among reference Fig. 3 Hierarchical clustering of 14 ETEC strains according to virulence factors. The dendrogram and associated heatmap based on the presence or absence of 108 major virulence genes were constructed using R version 3.4.3 [43]. Grey and white colors indicate gene presence and gene absence, respectively

aadA1 aph(6)-Id aph(3′')-Ib blaTEM-1B mdf(A) mph(A) sul1 sul2 tet(A) dfrA1 dfrA8
NCCP15731 strains in phylo-group A, with OrthoANIu values of 99.90% and 99.91%, respectively. Venn diagram of the virulence factors of the five strains including NCCP15731 and NCCP15733 in phylo-group A was obtained using InteractiVenn [57]. They shared 137 genes encoding E. coli common pilus (ECP) proteins, flagella-biosynthetic proteins, enterobactin transport proteins, and proteins related to Type II secretion system (Fig. 4). Comparison of NCCP15731, NCCP15733, and E159 (the most recent common ancestor of NCCP15731 and NCCP15733) showed that these three strains shared 142 genes. Plus, NCCP15731 and NCCP15733 shared In enterobactin synthesis of virulence systems, entF gene was found in NCCP15731, but not in NCCP15733 whereas entD gene was found in NCCP15733, but not in NCCP15731.

Conclusions
Escherichia coli NCCP15731 and NCCP15733, previously identified as MLST types ST964 and ST656, respectively, were isolated from diarrheal patients. However, multi-locus sequence typing (MLST) profiles showed that the MLST type of each of two strains was ST218. NCCP15731 and NCCP15733 belonged to phylogroup A and their serotype was O159:H34. In both whole genome and MLST phylogenetic analyses, NCCP15731 and NCCP15733 also belonged to phylo-group A. Hierarchical clustering based on the presence or absence of major virulence factors suggest that the virulence factors are associated with the phylogenetic group. In comparison of virulence genes of 14 strains, NCCP15733 has unique genes related to capsular polysaccharide. NCCP15731 has no unique virulence gene. However, in comparison with NCCP15731 and E159, it showed differences in the auto transporter, ABC transporter, and secretion system. Genomic analysis of NCCP15731 and NCCP15733 will be useful for further study on the development of ETEC vaccines.

Future directions
In summary, serotype and MLST type of NCCP15731 and NCCP15733 were O159:H34 and ST218, respectively. Unlike other O159 strains, CF gene of NCCP15731 and NCCP15733 was not detected. These strains have unique genes related to capsular polysaccharide, auto transporter, and secretion system. Moreover, both strains do not contain LT genes. These results will improve our understanding of ETEC O159 strains to prevent ETEC disease. However, because the results were obtained from in silico analysis, experimental confirmation of these results is required.