ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) serotype O103 is one of the primary pathogenic contaminants of beef products, contributing to several foodborne outbreaks in recent years. Here, we report the whole-genome sequence of a STEC O103:H2 strain isolated from cattle feces that contains a locus of enterocyte effacement (LEE) pathogenicity island.
ANNOUNCEMENT
Shiga toxin-producing Escherichia coli (STEC) O103 strains have been frequently associated with foodborne outbreaks (1, 2). During 2019 to 2020, three STEC O103-associated outbreaks were reported and related to the contamination of beef products and clover sprouts, causing 272 illnesses in the United States (3). Additionally, previous studies showed that cattle are the primary reservoir of STEC strains, which could be further disseminated in beef production and processing environments through direct and indirect contamination with feces (4–6). Thus, the complete genome sequence and the virulence factors of a STEC O103:H2 strain isolated from cattle feces are reported here to unveil the potential pathogenicity of this pathogen.
Strain RM13322 was previously isolated from cattle feces collected in California and confirmed as E. coli serotype O103 via an E. coli isolation procedure, including the use of selective media, enzyme-linked immunosorbent assay (ELISA), and PCR, as previously described (7). The bacterial culture was grown to the mid-exponential phase in 10 ml tryptic soy broth (TSB; Difco, Sparks, MD) before being subjected to genomic DNA extraction 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 following the manufacturer’s procedure without DNA shearing (Pacific Biosciences, Menlo Park, CA). Subsequently, the pooled library was subjected to size selection using BluePippin with a cutoff of 8 kb (Sage Science, Beverly, MA) and sequenced on a PacBio Sequel II instrument with v1 reagents, resulting in a total of 100,000 single-end reads with an N50 value of 14,710 bp. The raw reads were filtered and demultiplexed using PacBio SMRTLink v7.0 before being assembled into a circular 5,622,341-bp contig and a 76,269-bp linear contig utilizing Flye v2.4.1 (8). The two contigs were identified as a complete chromosome and a plasmid using BUSCO v3 (9), blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides), and PlasmidFinder 2.0 (10). The complete genome was annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (11). The prediction of serotype, prophages, virulence genes, and antibiotic resistance genes was performed via SerotypeFinder 2.0 (12), PHASTER (13), VirulenceFinder 2.0 (14), and ResFinder 3.2 (15), respectively. The genomic island was analyzed via IslandViewer4 using IslandPath-DIMOB methods (16). Default parameters were used for all software unless otherwise specified.
The result indicated that this E. coli O103:H2 strain contains a 5,622,341-bp circular chromosome (201× coverage) and a 76,269-bp linear plasmid (63× coverage). The chromosome has an average GC content of 50.8% and encodes a total of 5,534 coding DNA sequences (CDSs), 22 rRNAs, and 98 tRNAs. The genomic characterization showed that this strain harbors a 117,904-bp Stx1a prophage and a 71,689-bp locus of enterocyte effacement (LEE) pathogenicity island. Additionally, several non-LEE-encoded type III translocated virulence factors (nleA, nleB, espJ, and cif) and adherence-associated genes (iha and efa1) were detected in the bacterial chromosome. Plasmid pRM13322 has a GC content of 45.9% and contains several phage-associated essential CDSs encoding functional proteins, such as structurally related proteins and lysis-related proteins. The findings of this study provide valuable insights into the potential virulence factors of STEC O103:H2 of bovine origin.
Data availability.The sequence described in this study is available under BioProject accession number PRJNA573729. The GenBank accession numbers of the E. coli O103:H2 strain RM13322 chromosome and plasmid pRM13322 are CP050498 and CP050499, respectively. The raw read of the strain is available under Sequence Read Archive (SRA) accession number SRR12005555.
ACKNOWLEDGMENT
This research was funded by the U.S. Department of Agriculture, Agricultural Research Service, CRIS project 2030-42000-050-00D.
FOOTNOTES
- Received 30 July 2020.
- Accepted 17 October 2020.
- Published 5 November 2020.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
