Genome Sequences of Eight Shiga Toxin-Producing Escherichia coli Strains Isolated from a Produce-Growing Region in California.

Produce contaminated with Shiga toxin-producing Escherichia coli (STEC) is a continuing source of foodborne illness in the United States. This report documents the complete genome sequences of eight STEC strains isolated from livestock and water samples taken from a major agricultural region for leafy greens in California.

ANNOUNCEMENT

Cases of foodborne illness due to Shiga toxin-producing Escherichia coli (STEC) are a global problem, with approximately 2.8 million cases of acute illness annually (1). Although STEC O157:H7 strains are the cause of the majority of these illnesses, non-O157 STEC infections have emerged as a public health problem in the United States and internationally (2–4). The presence of STEC adjacent to a raw produce production environment must be considered a risk factor for contamination, with several transmission routes possible, including water, air, and animals (5–9). Indeed, raw produce has been the source of multiple outbreaks involving a variety of identified serotypes, including O157, O145, and O104 STEC (10–14). This announcement documents the complete genome sequences of eight non-O157 STEC strains previously isolated from livestock and water samples collected in a major agricultural region for leafy greens production on California’s Central Coast (15).

The STEC cultures were grown overnight on LB agar plates at 37°C, and genomic DNA was extracted using sucrose-Tris with phenol-chloroform cleanup extractions as described previously (16). Sequencing was performed using a Pacific Biosciences (PacBio, Menlo Park, CA) RS II platform with 20-kb SMRTbell libraries as described previously (17). Briefly, SMRTbell libraries were prepared from 10 μg of bacterial genomic DNA with G-tube (Covaris, Woburn, MA) fragmentation using the described PacBio procedure (18) but with 1× AMPure cleanup and DNA repair after 10-kb size selection using BluePippin. Single-molecule real-time (SMRT) cells were run with the 0.1 nM on-plate concentration, P6/C4 sequencing chemistry, MagBead One Cell Per Well v1 collection protocol, and 360-min data collection mode. The PacBio reads were assembled using the hierarchical genome assembly process (HGAP) v3.0 in SMRT Analysis v2.3.0 (PacBio). The chromosomes and plasmids for each STEC strain were circularized from one contig with overlapping ends. All strains were sequenced using an Illumina MiSeq platform and the KAPA LTP library preparation kit (KAPA Biosystems, Wilmington, MA). The pooled libraries were loaded into a MiSeq system and sequenced using a MiSeq reagent kit v2 with 2 × 250 cycles (Illumina, Inc.). A final base call validation of the PacBio contigs was performed using MiSeq reads trimmed using a quality score threshold of 20 or higher (≥Q20) and the reference assembler within Geneious software v11.1 (Biomatters, Ltd., Auckland, New Zealand). The final coverage for each strain was >200×. Protein-, rRNA-, and tRNA-coding genes were annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) (https://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).

The accession numbers, serotypes, and assembly metrics for each complete genome are listed in Table 1. Seven serotypes are represented among these agriculturally relevant STEC genomes, including the first complete STEC O43 genome (strain RM10042) based on BLAST analysis. Features from these STEC genomes include one to three stx-containing prophages per chromosome. It should be noted that not all genomes possess the locus for enterocyte effacement (LEE) pathogenicity island. The sequencing data of these STEC strains will add to our understanding of STEC genome organization, phylogeny, and references for outbreak traceback analyses.

TABLE 1

Accession numbers and genome metrics of 8 Shiga toxin-producing Escherichia coli strains from a produce-growing region in California

Strain name	Serotype	Chromosomal GenBank accession no.	No. of stx phages	LEE region	Chromosome size (bp)	Plasmid size (bp) (GenBank accession no.)
RM8426	O26:H11	CP028116	1	Yes	5,648,177	90,123 (CP028115)
RM10386	O26:H11	CP028126	2	Yes	5,785,882	83,012 (CP028124)
						98,899 (CP028125)
RM10042	O43:H2	CP028122	2	No	5,057,506	8,120 (CP028123)
						86,617 (CP028120)
						109,466 (CP028121)
RM8385	O103:H11	CP028112	1	Yes	5,631,913	6,673 (CP028114)
						94,220 (CP028113)
RM9322	O111:H8	CP028117	1	Yes	5,198,840	7,072 (CP028119)
						78,469 (CP028118)
RM10466	O113:H21	CP028381	3	No	5,044,460	67,750 (CP028383)
						160,675 (CP028382)
RM8352	O121:H19	CP028110	1	Yes	5,391,064	83,211 (CP028111)
RM9872	O145:H28	CP028379	1	Yes	5,385,924	89,518 (CP028380)

Data availability.

The whole-genome sequences have been deposited in GenBank under the accession numbers listed in Table 1.