Genome Sequences of 30 Escherichia coli O157:H7 Isolates Recovered from a Single Dairy Farm and Its Associated Off-Site Heifer-Raising Facility.

Cattle are the primary reservoir of Escherichia coli O157:H7, the most frequently isolated serotype of enterohemorrhagic E. coli infections among humans in North America. To evaluate the diversity of E. coli O157:H7 isolates within a single dairy herd, the genomes of 30 isolates collected over a 7-year period were sequenced.

GENOME ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) is a major causative agent of foodborne gastroenteritis in North America, with serotype O157:H7 being responsible for approximately 36% of human clinical cases caused by STEC strains (1). The primary reservoirs of E. coli O157:H7 are known to be cattle and other ruminants, with carriage by other food animals, such as poultry and swine, rarely occurring (2). To evaluate the genomic diversity of E. coli O157:H7 within a single dairy farm and its associated off-site heifer-raising operation, we sequenced the genomes of 30 isolates recovered from these 2 farms and added the sequence data to the public database.

During the semiannual testing of an individual dairy farm and its associated heifer-raising operation for the presence of bacterial foodborne pathogens, at least 30 Shiga toxin-possessing E. coli O157:H7 isolates were recovered (Table 1). To prepare the isolates for genome sequencing, colonies were grown overnight in Luria-Bertani (LB) broth at 37°C, and one milliliter of this overnight growth was concentrated via centrifugation. DNA was extracted using the QIAcube sample preparation system (Qiagen, Hilden, Germany). Sequencing libraries were constructed using the Nextera XT library prep kit (Illumina, La Jolla, CA), which were sequenced using a high-output version 2.0 flow cell on a NextSeq 500 platform (Illumina). Raw reads were cleaned and trimmed using DeconSeq (3) and Trimmomatic (4) and assembled using SPAdes version 3.8.0 (5). After assembly, the genomes were analyzed in silico using the Center for Genomic Epidemiology webserver (http://www.genomicepidemiology.org/).

TABLE 1 

Metadata and accession numbers for the deposited genome sequences

Isolate	Serotype	MLST as determined by methods of:		Isolation date (mo/day/yr)	Source	NCBI accession no.
		Wirth et al. (6)	Jaureguy et al. (7)
ARS-CC2601	O157:H7	ST-5560	ST-628	3/14/2005	Feces	NFRY00000000
ARS-CC2203	O157:H7	ST-5560	ST-628	12/4/2005	Fecal composite (postweaned calves)	NFRX00000000
ARS-CC2204	O157:H7	ST-11	ST-822	12/4/2005	Feces	NFRW00000000
ARS-CC2205	O157:H7	ST-5560	ST-628	6/5/2006	Fecal composite (postweaned calves)	NFRV00000000
ARS-CC2207	O157:H7	ST-11	ST-628	12/4/2006	Fecal composite (heifer grower)	NFRU00000000
ARS-CC2248	O157:H7	ST-5560	ST-628	1/8/2008	Feces	NFRT00000000
ARS-CC5318	O157:H7	ST-11	ST-628	1/26/2010	Feces (entering heifer)	NFRS00000000
ARS-CC5541	O157:H7	ST-11	ST-628	3/22/2010	Fecal composite	NFRR00000000
ARS-CC5513	O157:H7	ST-11	ST-628	4/27/2010	Feces (entering heifer)	NFRQ00000000
ARS-CC5517	O157:H7	ST-11	ST-628	4/27/2010	Feces	NFRP00000000
ARS-CC5525	O157:H7	ST-11	ST-628	6/3/2010	Feces (calf at heifer grower)	NFRO00000000
ARS-CC5526	O157:H7	ST-11	ST-628	6/14/2010	Feces (calf at heifer grower)	NFRN00000000
ARS-CC5530	O157:H7	ST-11	ST-628	6/14/2010	Flies	NFRM00000000
ARS-CC5534	O157:H7	ST-11	ST-628	6/14/2010	Manure collected from manure pit	NFRL00000000
ARS-CC5536	O157:H7	ST-11	ST-628	6/14/2010	Fecal composite	NFRK00000000
ARS-CC5539	O157:H7	ST-11	ST-628	6/14/2010	Trough water	NFRJ00000000
ARS-CC5545	O157:H7	ST-11	ST-628	6/14/2010	Fecal composite	NFRI00000000
ARS-CC5546	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRH00000000
ARS-CC5548	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRG00000000
ARS-CC5549	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRF00000000
ARS-CC5550	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRE00000000
ARS-CC5552	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRD00000000
ARS-CC5587	O157:H7	ST-11	ST-628	6/14/2010	Feces	NFRC00000000
ARS-CC5555	O157:H7	ST-11	ST-628	6/22/2010	Feces	NFRB00000000
ARS-CC5560	O157:H7	ST-11	ST-628	6/30/2010	Feces (calf at heifer grower)	NFRA00000000
ARS-CC6195	O157:H7	ST-11	ST-628	12/6/2010	Fecal composite	NFQZ00000000
ARS-CC6335	O157:H7	ST-11	ST-628	6/13/2011	Fecal composite	NFQY00000000
ARS-CC6338	O157:H7	ST-11	ST-628	6/13/2011	Feces (calf at heifer grower)	NFQX00000000
ARS-CC7094	O157:H7	ST-11	ST-628	9/24/2012	Fecal composite	NFQW00000000
ARS-CC7983	O157:H7	ST-11	ST-628	12/4/2012	Fecal composite	NFQV00000000

Based on the multilocus sequence typing (MLST) scheme developed by Wirth et al. (6), the 30 E. coli O157:H7 isolates consisted of 2 sequence types (STs), ST-11 (n = 26) and ST-5560 (n = 4). These STs have only a single allele difference between them. In a maximum parsimony phylogenetic analysis with 465 other E. coli O157:H7 genomes, the study isolates clustered into 3 divergent groups and one singleton (ARS-CC2204). The four ST-5560 isolates (ARS-CC2601, ARS-CC2248, ARS-CC2205, and ARS-CC2203) clustered closely together on a lineage nested within a larger clade composed mainly of ST-11 isolates, indicating that ST-5560 is a subclade of ST-11. Using the MLST scheme described by Jaureguy et al. (7), 29 isolates were identified as ST-628, but the single divergent isolate (ARS-CC2204) mentioned above was identified as ST-822. Isolates collected from the heifer farm clustered with those from the dairy farm, indicating that strains were likely transferred between these two locations. Using the Harvest package (8), 2,151 single-nucleotide polymorphisms (SNPs) were detected among all strains excluding ARS-CC2204 (ST-628 strains only). When the ARS-CC2204 genome was added, the number of SNPs among the study isolates increased to 2,469 SNPs.

All genomes encoded sequences homologous to eae (intimin), ehxA (plasmid-carried O157 enterohemolysin gene), and stx2 (Shiga-toxin) virulence factors, all of which are integral in the enterohemorrhagic disease process of E. coli O157:H7. Transferable antibiotic resistance genes were not identified in any of the isolates.

Accession number(s).

The genome sequences of these 30 E. coli isolates have been deposited in GenBank under the accession numbers listed in Table 1.