Fecal Metagenome Sequences from Lactating Dairy Cows Shedding Escherichia coli O157:H7.

Cattle are primary reservoirs of Escherichia coli O157:H7, a causative agent of severe human infections. To facilitate analyses of the communities in which this pathogen is found, we sequenced the fecal metagenomes of 10 dairy cows shedding E. coli O157:H7 and added them to the public domain.

ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) species are major zoonotic bacterial pathogens responsible for foodborne infections worldwide, and Escherichia coli O157:H7 is the most common etiological agent of hemolytic uremic syndrome in the United States. (1). Cattle can harbor this pathogen in their intestines without symptoms of infection and intermittently shed it in their feces. Human exposure may occur through consumption of contaminated products (milk and meat) and through cross-contamination of produce, water, wildlife, and the farm environment (2–5). In addition to several contributing factors, such as seasonal variation, nutritional condition, and host physiology, cattle gastrointestinal microbiota may have direct or indirect effects on the carriage and shedding (and super shedding) of E. coli O157:H7 (3, 5–8). These effects may be exerted by competitive exclusion (from mucosal adhesion sites), release of secondary metabolites, bioavailability of nutrients, or functional synergy. Understanding the ecological dynamics of E. coli O157:H7 in the ruminant intestine in relation to the common gut microflora is important to develop strategies for reducing shedding and mitigating human outbreaks.

During a longitudinal study of an individual commercial dairy farm and its associated offsite heifer raising operation in Pennsylvania (USA), E. coli O157:H7 isolates were sporadically recovered from fecal grab, manure composite, milk filter, and environmental (fly, trough water) samples (9, 10). Ten fecal samples collected from the lactating cows that were shedding E. coli O157:H7 were stored at −80°C (Table 1), and the samples were later thawed for metagenome sequencing after DNA extraction using a QIAamp fast DNA stool minikit (Qiagen, Waltham, MA). Nextera DNA library preparation kits (Illumina, San Diego, CA) were used to prepare sequencing libraries of the 10 samples. Paired-end sequencing (2 × 151 bp) was conducted on an Illumina NextSeq 500 sequencing platform with a NextSeq 500/550 v2 high-output flow cell. Raw reads were demultiplexed using the BCL2FastQ program and cleaned using DeconSeq (11) and Trimmomatic v0.36 (12).

TABLE 1

Metadata and accession numbers of deposited metagenome sequences

Sample identifiera	Sampling date (mo/day/yr)	Total no. of reads	MeTaxa2 analysisb			NCBI accession no.
			Bacteria	Archaea	Eukaryotes
B-27-100	6/14/2010	20,884,502	20,113	474	205	SRX4547096
B-S104-6	6/30/2010	11,094,746	10,998	73	49	SRX4547097
B-27-102	6/14/2010	43,291,458	51,593	1,238	72	SRX4547098
B-S104-5	6/30/2010	17,717,456	22,118	335	18	SRX4547099
B-27-111	6/14/2010	8,684,666	10,070	261	13	SRX4547086
B-S104-4	6/30/2010	73,242,474	52,393	2,075	694	SRX4547087
B-27-118	6/14/2010	105,817,744	75,028	1,618	249	SRX4547094
B-S104-3	6/30/2010	32,199,304	19,687	853	46	SRX4547095
B-27-142	6/14/2010	93,199,932	72,797	2,297	108	SRX4547091
B-S104-7	6/30/2010	23,886,090	15,971	455	37	SRX4547088

Fecal samples from individual animals.

Data are numbers of reads assigned to specific taxa.

Cleaned and curated reads were used to identify bacterial, archaeal, eukaryotic, and mitochondrial small subunits (12S, 16S, and 18S) rRNA sequences with MeTaxa v2 (13). Assigned taxonomic profiles of the fecal metagenomes included Archaea (Crenarchaeota and Euryarchaeota), Bacteria (mainly Firmicutes, Bacteroidetes, and Actinobacteria), and eukaryotes (Fungi, Excavata, Metazoa, Viridiplantae, and others). The metagenomic data will be used to investigate the phylogenetic and functional diversity of the fecal microflora, metabolic pathways, microbial stress and virulence factor-related gene profiles, and other potential biomarkers that may be associated with E. coli O157:H7 carriage and shedding in ruminants. A detailed report of the results of comparative analyses of these metagenomes will be published elsewhere.

Data availability.

The metagenomes of these 10 fecal samples have been deposited in GenBank under the accession numbers listed in Table 1.