Complete Genome Sequences of Two Shiga Toxin-Producing Escherichia coli Strains Isolated from Crows.

Escherichia coli strains RM9088 and RM10410 were isolated from crows near a leafy greens-growing region in California in April and July 2009, respectively. Both strains carry genes encoding Shiga toxins and other virulence factors in enteric pathogens. Here, we report the complete genome sequences of RM9088 and RM10410.

ANNOUNCEMENT

Shiga toxin-producing Escherichia coli (STEC) is one of the main bacterial causal agents of foodborne illness outbreaks associated with fresh produce (1). Although STEC O157:H7 has been considered the most frequent cause of STEC-associated outbreaks, recent studies suggest that non-O157 STEC strains are causing a large number of human infections worldwide (2–4). STEC naturally resides in ruminant animals, primarily cattle; however, diverse STEC strains have been isolated from birds (5–7), implying that birds might be an environmental source of STEC transmission. To better understand the pathogenicity of avian STEC, we sequenced the genomes of the two isolates obtained from crows.

Strains RM9088 and RM10410 were isolated by cloacal swab, as described previously (8). Genomic DNA was extracted from the mid-exponential-phase cultures grown in LB broth, as described previously (9). Genomic libraries were prepared according to the PacBio 20-kb library standard protocol (10) using the SMRTbell DNA template prep kit 3.0, followed by size selection with the BluePippin size selection system (Sage Science, Inc.) and then template binding with the P6v2 kit. DNA sequencing was performed on an RS II instrument (Pacific Biosciences) with P6-C4 sequencing chemistry and a 360-min data collection protocol. The sequence reads were filtered with PreAssembler filter prior to de novo assembly with RS_HGAP_Assembly v.3. Detailed sequencing metrics and filter parameters for each strain are listed in Table 1. The closed genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (11).

TABLE 1

Genome attributes of two avian STEC strains

Feature	Value for strain:
	RM9088	RM10410
Serotypea	O109:H48	O113:H4
PacBio sequencing metrics
No. of reads	81,165	67,867
Avg read length (bp)	6,931	10,205
No. of bases	1,351,682,302	1,354,076,155
Sequence coverage (×)	152	200
PreAssembler Filter v.1
Minimum subread length	1,000	500
Minimum polymerase read quality	0.80	0.80
Minimum polymerase read length	100	100
Chromosome
Size (bp)	5,270,611	5,227,472
G+C content (%)	50.8	50.5
No. of CDSs	5,188	5,114
No. of tRNAs	94	91
No. of rRNAs	22	22
No. of prophages	14	11
stx gene(s)	stx1a	stx1a, stx2d
Plasmids
pEHEC	p1RM9088	None
Size (bp)	167,256	None
G+C content (%)	45.9	None
No. of CDSs	198	None
Second plasmid	p2RM9088	None
Size (bp)	86,529	None
G+C content (%)	49.2	None
No. of CDSs	114	None

The serotype of each strain was determined in silico.

The RM9088 genome is composed of a 5,270,611-bp chromosome and two plasmids, encoding a total of 5,500 coding DNA sequences (CDSs). The RM10410 genome is composed of only a 5,227,472-bp chromosome, encoding 5,114 CDSs (Table 1). The serotypes of RM9088 and RM10410 were determined to be O109:H48 and O113:H4, respectively, using SerotypeFinder 2.0 (12), with the default settings (thresholds for percent identity [%ID], 85%, and minimum length, 60%). The sequence types (STs) of RM9088 and RM10410 are ST339 and ST10, respectively, using the Warwick scheme (13). In silico phylo-typing using the Clermont method (14) placed both strains in phylogroup A.

The stx genes in strain RM9088 encode Stx1a, located on a 43,766-bp prophage (chromosome positions, base pairs 4689207 to 4732972) that was identified using PHASTER (15, 16). The p1RM9088 plasmid (167,256 bp) is a typical pEHEC (the large virulence plasmid of enterohemorrhagic E. coli [EHEC]) (17) containing genes (hlyCABD) encoding enterohemolysin. Interestingly, this plasmid also carries genes such as EAST1 (annotated as astA in RM9088) (GenBank accession number AB042002) and sta1 (GenBank accession number AJ555214) encoding heat-stable toxins, a common virulence factor of enterotoxigenic E. coli. No known STEC virulence genes were identified on the second plasmid, p2RM9088 (86,529 bp). The two sets of stx genes in strain RM10410 encode Stx1a and Stx2d, respectively. The stx1a gene is located on a 94,727-bp prophage (chromosome positions, base pairs 1786478 to 1881204); the stx2d gene is located on a 59,845-bp prophage (chromosome positions, base pairs 2420694 to 2480538). A search of additional virulence factors using VirulenceFinder 2.0 (18), with the default settings (threshold for %ID, 90%; minimum length, 60%), failed to identify the pathogenicity island locus of enterocyte effacement (LEE) or genes encoding any type III secretion effectors in either of the two genomes. Since strain RM10410 has no plasmids, it lacks genes encoding enterohemolysin.

Data availability.

The sequences described in this study are available under BioProject accession number PRJNA557687. The GenBank accession numbers are CP042298, CP042296, CP042297, and CP042350 for the RM9088 chromosome, plasmid p1RM9088, plasmid p2RM9088, and RM10410 chromosome, respectively. The raw reads are available under Sequence Read Archive (SRA) accession numbers SRR9953605 and SRR9953217 for RM9088 and RM10410, respectively.