Investigating the Whole-Genome Sequence of a New Locus of Enterocyte Effacement-Positive Shiga Toxin-Producing Escherichia coli O157:H7 Strain Isolated from River Water

Diverse Shiga toxin-producing Escherichia coli (STEC) strains have been isolated from several environmental samples. Rivers are associated with the distribution of STEC pathogens in the environment. Thus, we report the complete genome sequence of a locus of enterocyte effacement (LEE)-positive STEC O157:H7 strain isolated from the Mississippi River.

ABSTRACT

Diverse Shiga toxin-producing Escherichia coli (STEC) strains have been isolated from several environmental samples. Rivers are associated with the distribution of STEC pathogens in the environment. Thus, we report the complete genome sequence of a locus of enterocyte effacement (LEE)-positive STEC O157:H7 strain isolated from the Mississippi River.

ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) is one of the major bacterial pathogens associated with numerous foodborne outbreaks around the world (1, 2). E. coli O157:H7 has been the most persistent serotype in STEC-associated outbreaks causing severe human illnesses, such as hemolytic uremic syndrome (HUS), and high mortality among immunocompromised patients (3). Previous studies showed that diverse STEC strains have been isolated from several environmental samples, such as animal feces, leafy greens, and soil (4; https://www.cdc.gov/nationalsurveillance/ecoli-surveillance.html). Moreover, rivers were considered a distributing source of STEC due to its association with the aforementioned environmental factors (5–8). Thus, whole-genome sequencing was conducted on a STEC O157:H7 strain isolated from river water to unveil the pathogenicity of the strain.

Escherichia coli O157:H7 strain RM19259 was previously isolated from a sample collected from the Mississippi River watershed in 2016 using Moore swabs. For strain isolation, the environmental sample was enriched with tryptic soy broth (TSB) and subsequently incubated at 25°C for 2 hours, followed by 42°C for 8 hours prior to the further isolation process as previously described (9). Genomic DNA was extracted from the cultures grown to mid-exponential phase in 10 ml tryptic soy broth (TSB; Difco, Becton, Dickinson, Sparks, MD) using a Quick-DNA Miniprep Plus kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. The DNA library was constructed using an Express template prep kit 2.0 (Pacific Biosciences) and subsequently sequenced using PacBio Sequel II with V1 reagents (Pacific Biosciences), resulting in 398,925 single-end reads. De novo assembly was performed using Flye 2.4.1 with the default parameters, resulting in 3 contigs with an N₅₀ value of 5,511,015 bp (10). Two contigs were identified as circular contigs represented by a complete chromosome and a plasmid using BUSCO 3 (11) and Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides). The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The predictions of serotype, prophages, and virulence genes were performed using SerotypeFinder 2.0 (12), PHASTER (13), and VirulenceFinder 2.0 (14), respectively. The genomic island was analyzed with IslandViewer 4 using the IslandPath-DIMOB method (15). Default parameters were used for all software unless otherwise specified.

The strain contains a 5,511,015-bp chromosome and a 98,304-bp plasmid. The chromosome has an average GC content of 50.5% and carries a total of 5,440 predicted protein-coding sequences (CDSs), 22 rRNAs, and 106 tRNAs. The strain is an E. coli O157:H7 strain that harbors two sets of stx genes, including one stx_2a located on an 83,266-bp prophage (chromosome position, base pairs 260410 to 343675) and one stx_2c located on a 35,769-bp prophage (chromosome position, base pairs 5403730 to 5439498). Additionally, a 41,255-bp pathogenicity island—locus of enterocyte effacement (LEE)—was identified between base pairs 1729493 and 1770747 of the chromosome. This strain also carried enterohemorrhagic E. coli (EHEC)-associated non-LEE-encoded type III translocated virulence factors (nleA, nleB, and nleC). In addition, the strain contains a virulence gene, EAST-1 heat-stable toxin (astA), commonly related to many E. coli pathovars, such as enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and EHEC (16). The plasmid pRM19259 is a typical F-like plasmid pO157, which contains several virulence-related genes, such as ehxA (enterohemolysin), espP (serine protease), etpD (type II secretion protein), katP (catalase-peroxidase), and toxB (toxin B) (17). The findings of this study provide valuable insights into the epidemiological surveillance of E. coli O157:H7 infections.

Data availability.

The sequence described in this study is available under BioProject accession number PRJNA573729. The GenBank accession numbers of the RM19259 chromosome and plasmid pRM19259 are CP046527 and CP046526, respectively. The raw reads of the strain are available under Sequence Read Archive (SRA) accession number SRR10598574.