Draft Genome Sequences of Shiga Toxin-Producing Escherichia coli O157:H7 Strains Recovered from a Major Production Region for Leafy Greens in California

Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen and is responsible for outbreaks of human gastroenteritis. This report documents the draft genome sequences of nine O157:H7 cattle strains, which were identified to be PCR positive for a Shiga toxin gene but displayed different levels of functional toxin activity.

ABSTRACT

Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen and is responsible for outbreaks of human gastroenteritis. This report documents the draft genome sequences of nine O157:H7 cattle strains, which were identified to be PCR positive for a Shiga toxin gene but displayed different levels of functional toxin activity.

ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) strains are enteric pathogens responsible for human gastroenteritis (1, 2). In some cases, human infections progress to hemolytic-uremic syndrome, a life-threatening disease resulting from the production of Shiga toxins (Stx). Serotype O157:H7 has been commonly associated with the development of severe disease symptoms. Cattle are a main reservoir for O157:H7 (3), and meat products are recognized as main sources of human infections (2). Recently, the consumption of leafy vegetables has been significantly linked to foodborne illness due to O157:H7 infections (4). Using a robust isolation method, STEC O157:H7 strains were recovered from cattle in a major agricultural region for leafy greens and were identified based on a typical STEC colony morphology on selective chromogenic medium and positive PCR tests for a stx gene (5). Further proteomic analyses indicated that all O157:H7 cattle strains were found to express the toxin receptor binding B-subunit (6, 7), but some were found not to have a functional toxin (7). The genome sequencing of nine O157:H7 cattle strains with various levels of toxin activity is reported here.

All STEC O157:H7 strains (Table 1) were streaked for isolation from a frozen stock culture on Luria-Bertani (LB) agar (Difco, Detroit, MI) at 37°C for 24 h. A single colony from each strain was further grown in LB broth (Difco) for 18 h with constant shaking (200 rpm) at 37°C. Genomic DNA of the O157:H7 strains was extracted with the Wizard genomic DNA purification kit (Promega Corp., Madison, WI) (8), sheared using a g-TUBE (Covaris, Inc., Woburn, MA), and quantified by fluorometric measurement using a Qubit 4.0 fluorometer (Invitrogen, Carlsbad, CA). Fifteen micrograms of the sample was used to prepare 20-kb SMRTbell libraries using the proprietary P6-C4 sequencing chemistry (Pacific Biosciences, Menlo Park, CA), according to the SMRTbell template prep kit 1.0 protocol (9). Single-molecule real-time (SMRT) sequencing was performed with the PacBio RS II platform (Pacific Biosciences) using the MagBead OneCellPerWell v1 collection protocol and 360-minute data collection mode (10). The sequencing reads were assembled using the PacBio Hierarchical Genome Assembly Process (HGAP; v3.0) and polished using Quiver in the SMRT Analysis v2.3.0 software, with default parameters (11), except for strain RM10645 and RM10646 assemblies (minimum polymerase read quality, 0.70; minimum seed read length, 5,000 bp). Methylation patterns were analyzed with RS_Modification_and_Motif_Analysis.1 using the SMRT Analysis software, with default settings. Annotations were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; version 4.8) (12). Genome sequence comparisons showed that all O157:H7 cattle strains were highly similar and had an average genome size of 5,441,700 bp, with either 50.5% or 50.6% G+C content (Table 1). Analysis of methylation patterns revealed the DNA motifs GATC, GAAABCC, CACNNNNNNNCTGG, and CCAGNNNNNNNGTG to be 98 to 99% methylated (an m6A modification occurred at the nucleotide in bold). The cattle strains harbored only one copy of the stx_2c-carrying prophage, which was inserted in the chromosomal sbcB gene, an insertion site commonly used by stx_2c prophages in virulent O157:H7 strains (13). Only strain RM10024 tested positive for cytotoxic activity (Table 1), but all other O157:H7 cattle strains tested negative due to the presence of the insertion sequence variant IS1203v (14) in the coding sequence of the catalytic A-subunit of Stx. The whole-genome sequencing information of O157:H7 cattle strains, recovered during a short time period and discrete sampling location in a major agricultural region, has provided an explanation for the variability in Stx activity and has set the foundation for future studies on the persistence of strains with an attenuated pathogenic potential.

TABLE 1.

Genome characteristics and accession numbers of nine Escherichia coli O157:H7 strains from cattle from a leafy greens production region on the Central California Coast^a

Characteristic	Data for strain:
	RM10024	RM10641	RM10645	RM10646	RM10649	RM10716	RM10718	RM10719	RM10720
GenBank accession no.	QSMX00000000	QSMW00000000	QSMV00000000	QSMU00000000	QSMT00000000	QSMS00000000	QSMY00000000	QSMR00000000	QSMQ00000000
SRA accession no.	SRX5679060	SRX5679061	SRX5679058	SRX5679059	SRX5679056	SRX5679057	SRX5679054	SRX5679055	SRX5679062
Avg genome coverage (×)b	176	136	130	177	83	123	143	123	171
N50 (bp)b ,c	33,062	28,334	22,969	34,372	21,352	29,210	31,088	26,248	33,302
Mean read length (bp)b	18,917	15,903	15,136	21,691	13,196	15,758	16,804	14,861	18,300
Mapped read length of insert (bp)b	5,651	4,084	4,925	5,041	5,381	4,486	4,414	4,129	4,110
No. of mapped readsb	66,346	60,966	73,290	81,452	42,756	52,256	58,706	57,840	64,823
Genome size (bp)d	5,498,458	5,402,896	5,470,951	5,253,756	5,470,673	5,464,836	5,483,285	5,470,842	5,459,610
G+C content (%)d	50.60	50.50	50.50	50.60	50.50	50.50	50.50	50.50	50.50
Total no. of genesd	5,831	5,793	5,712	5,793	5,784	5,772	5,794	5,767	5,714
No. of pseudogenesd	382	359	412	897	454	353	354	366	328
stx gene subtypee	stx2c	stx2c	stx2c	stx2c	stx2c	stx2c	stx2c	stx2c	stx2c
Cytotoxicity assay resultf	+	−	−	−	−	−	−	−	−
stx screening test resultg	+	+	+	+	+	+	+	+	+

^aSee reference 5.

^bSequencing metrics were obtained using the PacBio RS II platform.

^cN₅₀, minimum contig length required to cover 50% of the total genome size.

^dGenome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline.

^eIn silico serotype and stx subtype were confirmed by PCR (15, 16).

^fCytotoxicity was determined with a fluorescent Vero cell-based assay (17, 18).

^gSample enrichments were screened for stx genes by real-time PCR, and positive samples were further analyzed using selective chromogenic medium (5).

Data availability.

The assembled sequences and sequencing reads have been deposited at DDBJ/ENA/GenBank and the NCBI Sequence Read Archive, respectively, under the accession numbers listed in Table 1.