Insights into Evolution of Escherichia coli O157:H7 from Complete Genome Sequence of Closely Related O55:H7 PrecursorJennifer L. Kyle1, Craig T. Parker1, Craig A. Cummings, Beatriz Quiñones1, Steven Huynh1, Lovorka Degoricija2, Paolo Vatta2, Elizabeth Newton2,

Olga Petrauskene2, Melissa Barker2, Robert Tebbs2, Rixun Fang2, Manohar Furtado2, Robert E. Mandrell1

1Produce Safety and Microbiology, ARS/USDA, Albany, CA; 2Life Technologies, Foster City, CAB-2055

Background Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant mortality and morbidity in developing countries. In spite of this, only two EPEC genomes have been fully sequenced: the typical, model EPEC strain E2348/69 (O127:H6), and the contemporary, atypical EPEC strain CB9615 (O55:H7, Germany, 2003). This latter serotype has been established as a recent precursor to the virulent, enterohemorrhagic E. coli serotype O157:H7. We chose strain RM12579 (O55:H7, California, 1974) due to its proximity in time and space to the first O157:H7 isolated in the US, a California isolate from 1975. Methods

Multiple second generation sequencing technologies were used to ensure complete and accurate coverage. The long reads from 454 Sequencing™

at 33X coverage yielded an

initial assembly that was further refined by high fidelity SOLiD™ system reads at 730X coverage. The remaining gaps in repetitive regions were filled by PCR and Sanger sequencing.

Results

The RM12579 genome is approximately 5.3 million bases, not including five plasmids. Three plasmids show similarity to those sequenced from other EPEC strains; the remaining two appear to be small, cryptic plasmids. A total of 18 prophage and prophage-like elements were identified, including 16 shared with the previously completed O55:H7 strain CB9615.

Conclusions

The overall pattern of phage insertion elements appears intermediate between the

previously sequenced O157:H7 and O55:H7 strains. Thus, analysis of the complete sequence of this 1975 California isolate permits further insight into the evolution and emergence of the deadly O157:H7 serotype.

1 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000 3,500,000 4,000,000 4,500,000 5,000,000 5,386,352

1 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000 3,500,000 4,000,000 4,500,000 5,000,000 5,498,450

A

Figure 1. (A) Linear representation of RM12579 chromosome (middle) and comparison to E. coli O157:H7 strain Sakai (top) and E. coli O55:H7 strain CB9615 (bottom). Phage are represented as black boxes; phage-like regions are represented as grey boxes. tRNA loci are marked with pink triangles; ribosomal loci with blue triangles. Phage found only in the two O55:H7 strains are marked with a blue rectangle; phage unique to only one strain are marked with a red rectangle. A green line indicates a phage insertion at the same location in two strains, but without homology. Two gene loci are marked: wrbA, the insertion site for the Stx2-containing phage Sp5 and yehV(mlrA), the insertion site for the Stx1-containing phage Sp15. (B) Diagrams of the five plasmids found in strain RM12579, have gene annotations marked in green. Also shown is the homology of the common backbone shared by two of these plasmids (p6KB and p12KB) to p5217 from E. coli O127:H6 strain E2348/96.

Sp1,2 Sp3 Sp4,5 Sp6,7,8 Sp9 Sp10 Sp11.12 Sp13,14 Sp15 Sp16 Sp17 Sp18

Ep1,2 Ep3 Ep4 Ep5 Ep6 Ep7 Ep8,9 Ep10 Ep11 Ep12 Ep13 Ep14 Ep15

B 5 , 0 0 0

1 0 , 0 0 0

1 5 , 0 0 0

2 0 , 0 0 0 2 5 , 0 0 0

3 0 , 0 0 0

3 5 , 0 0 0

4 0 , 0 0 0 4 5 , 0 0 0 5 0 , 0 0 0

5 5 , 0 0 0

6 0 , 0

0 0

6 5 , 0

0 0

7 0 , 0

0 0

7 5 , 0

0 0

8 0 , 0

0 0

8 5 , 0 0 0

9 0 , 0 0 0 9 4 , 0 1 6

P1-like 94,016 bp

1 0 , 0 0 0 2 0 , 0 0 0

3 0 , 0 0 0 4 0 , 0 0 0

5 0 ,0

0 0

6 0 , 0 0 0 6 6 , 0 7 8

pO55-like 66,078 bp

gene

gene

gene

gene

gene

gene gene

strA gene

sulII gene

strB gene

1 , 0 0 0 2 , 0 0 0

3 , 0 0 0

4 , 0 0 0

5 ,

0 0 0

6 , 2 1 1

pE2348-2 like 6,211 bp

homology to p5217 mobC

rop/rom tnpA

tnpR

bla

mobA

2 , 0 0 0 4 , 0 0 0

6 , 0 0 0

8 , 0 0 0

1 0

, 0 0 0

1 2 , 0 6 8

p12KB 12,068bp

homology to p5217

mobD

mobB

mobC

rop/rom

endonuclease

mobA DNA methylase

1 , 0 0 0 2 , 0 0 0

3 , 0 0 0

4 , 0 0

0 5 ,

0 0 0

5 , 9 5 4

p6KB 5,954bp

homology to p12KB

homology to p6KB

gene

mobB gene

mobC gene rop gene

gene

mobA gene

5 0 0

1 , 0 0 0 1 , 5 0 0

2 , 0 0 0 2 , 5 0 0 3 , 0 0 0

3 , 5 0

0 4 ,0

0 0

4 , 5 0 0

5 , 2 1 7

NC_011799 5,217 bp

Cryptic plasmid p5217 from E. coli O127:H6 strain E2348/69

mlrA (yehV) wrbA

1 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000 3,500,000 4,000,000 4,500,000 5,000,000 5,262,299

RM12579 Cp1,2 Cp3 Cp4 Cp5 Cp6 Cp7 Cp8,9 Cp10 Cp11 Cp12 Cp13

Size ( ) bGenome a bp ORFsb Plasmids tRNA lambda (total) prophage

RM12579 (EPEC O55:H7) 5,262,299 5027 94/66/12/6/6 98 10 (13)

CB9615 (EPEC O55:H7)8 5,386,352 5028 66 101 10 (15)

Sakai (EHEC O157:H7)9 5,498,450 5361 93/3 105 13 (18)

E2348/69 (EPEC O127:H6)10 4,965,553 4703 97/6/5 92 4 (13)

A

Figure 2. Phylogenetic analysis was performed using sequence information from genomes available at NCBI (both completed genomes as well as contigs available for unfinished genomes). SNPs identified in areas common to all genomes analyzed were used to construct maximum likelihoodtrees . (A) Unrooted tree demonstrating a close relationship between E. coli O157:H7 and O55:H7 lineages in comparison to strains of enterohemorrhagic E. coli (O26:H11, O111:H-, O103:H2) and Shigella dysenteriae. (B) Cladogram of O55:H7, O157:H7 and H- strains. Numbers on internal nodes indicate bootstrap support. Clades are labeled according to Feng et al (1998); Feng et al (2003) and Eppinger et al (2011).

B

RM2012 DEC 5A 1950 USA (NY)RM2027 DEC 5B 1979 USA (FL)RM2042 DEC 5C 1966 USA (NJ)RM2057 DEC 5D 1965 Sri LankaRM2072 DEC 5E 1963 Iran

RM2163CDC-5644/ TW01967 1962 USA (IL)

RM3654 CPK100 CanadaRM12506 BB2RM12579* 10591-12-74 1974 USA (CA)RM13616 99107 (90) BrazilRM13617 21443 (24) BrazilRM14503* 10591-12-74 1974 USA (CA)RM14504 4694-9-75 1975 USA (CA)RM14505 2022-7-76 1976 USA (CA)RM14506 5150-10-74 1974 USA (CA)RM14507 7305-10-74 1974 USA (CA)

Table 2. E. coli O55:H7 strains in this study.

USDA # Other name Year of Place ofIsolationIsolation

*Duplicate isolates.

DEC 5

A (N

Y)DE

C 5B

(FL)

DEC 5

C (N

J)DE

C 5D

(Sri

Lank

a)DE

C 5E (

Iran)

CDC-

5644

(IL)

CPK1

00 (C

anad

a)99

107 (

Braz

il)21

443 (

Braz

il)RM

1450

3 (CA

)*RM

1450

4 (CA

)RM

1450

5 (CA

)RM

1450

6 (CA

)RM

1450

7 (CA

)

H2O

RM12

579 (

CA)*

BB2

(stx1

+)Or

igO1

57:H

7Sa

kai

EDL9

33Sp

inac

h-En

vSp

inac

h-Cl

inSp

inac

h-Ba

gO2

6:H2

2

A

B

C

DEC 5

A (N

Y)DE

C 5B

(FL)

DEC 5

C (N

J)DE

C 5D

(Sri

Lank

a)DE

C 5E (

Iran)

CDC-

5644

(IL)

CPK1

00 (C

anad

a)99

107 (

Braz

il)21

443 (

Braz

il)RM

1450

3 (CA

)*RM

1450

4 (CA

)RM

1450

5 (CA

)RM

1450

6 (CA

)RM

1450

7 (CA

)

H2O

RM12

579 (

CA)*

BB2

(stx1

+)Or

igO1

57:H

7Sa

kai

EDL9

33Sp

inac

h-En

vSp

inac

h-Cl

inSp

inac

h-Ba

gO2

6:H2

2

Pattern 1: O157:H7 Sakai, EDL933

wrbA mlrA (yehV)

sbcB argWyecE

Pattern 2: O157:H7 Spinach outbreak strains (2006)

Stx2 Stx1

D

Pattern 3: O55:H7 (CPK100, RM12579*, BB2 )

Pattern 4: O55:H7 (DEC 5B, CDC-5644, 99107, 21443, RM14507)

Figure 4. Analysis of phage insertion sites for E. coli O157:H7 andO55:H7 strains (see Table 2). Red arrows indicate target product at an intact locus. (A) Diagram highlighting know insertion sites forStx-containing lambda phage in O157 & non-O157 EHEC. (B) PCR analysis of yehV/mlrA locus. (C) PCR analysis of wrbA locus. (D) Insert at wrbA in O55:H7 is a simple insertion element.

= non-Stx lambda phage

= other insertion element

Stx2 Stx2

Figure 3. PFGE analysis of 15 unique E. coli O55:H7 isolates (see Table 2) performed using (A) XbaI or (B) BlnI enzymes. Dendrograms were created using BioNumerics.

Stra

in eae

ehxA

ent/

espL

2

espP

etpD

katP

nleA per

saa

stx1

(gen

eral

)

stx2

(gen

eral

)

subA

chuA

yjaA

TSPE

4.C2

papC

hlyA

ehly

A

ehx

sheA

nleB

nleB

2

nleD

nleE

nleF

nleG

nleG

2-1

nleG

2-3

nleG

9

espX

2

espX

7

espK

espV paa

2012 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - - - +2027 + - + - - - + - - - - - + - - + - - - + + + + + + + + - + - - - +2042 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +2057 + - + - - - - - - - - - + - - + - - - + + + + + + + + + - + - + - +2072 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + + + + +2163 + - + - - - + - - - - - + - - + - - - + + + + + + + + - + - + - +3654 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +

12506 + - + - + - - - - + - - + - - + - - - + + + + + + + + + + - - + + +12579 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +13616 + - + - - - + - - - - - + - - + - - - + + + + + + + + - - + - + - +13617 + - + - - - + - - - - - + - - + - - - + + + + + + + + - - + - + - +14503 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +14504 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +14505 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +14506 + - + - + - - - - - - - + - - + - - - + + + + + + + + + + + - + - +14507 + - + - - - + - - - - - + - - + - - - + + + + + + + + - - + - + - +

O157:H7 + + + + + + + + - + - - + - - + - - + + + + + + + + + + + + + + + +

Hemolysin Nle-e ector STEC Virulence Factors AIEC

Table 3. PCR detection of select virulence genes O55:H7 strains (see Table 2) and 1st O157:H7 isolate.

Introduction

Enterohemorrhagic E. coli (EHEC) of the O157:H7 serotype isestimated to result in over 73,000 infections a year in the US,causing in its most severe manifestation the potentially deadlyhemolytic uremic syndrome (1). Multiple analyses have shown that this serotype appears to have evolved from enteropathogenic E. coli O55:H7, possibly as recently as 500 years ago (2-7). This hypothesis is best evaluated using multiple strains of bothserotypes. Although several O157:H7 genomes have been finished, and sequence information is available from manyother O157:H7 strains, only one O55:H7 genome has been completed to date (8). A complete understanding of the emergence of this deadly pathogen will benefit from the avaiability of several O55:H7 finished genomes, specificallyones that are separated in time and geographic space.

Methods

> Shotgun and 8KB paired-end sequencing using 454 pyrosequencing technology (33X coverage)> Shotgun and mate-pair sequencing by the SOLiD system (730X coverage)> Gap closure and verification of repeat regions by PCR and Sanger sequencing

Summary

AcknowledgementsWe thank Sharon Abbott (Calif. Dept. of Health) and Lee Riley (Univ. of Calif., Berkeley) for providing strains; Samar Fontanar, Michelle S. Swimley and Ronak N. Patel for excellent technical assistance.

™

™

References(1)(2)(3)(4)(5)

a enteropathogenic Escherichia coli (EPEC); enterohemorrhagic E. coli (EHEC)b chromosome only

Table 1. Genome details for strain RM12579 and comparison to selected published strains.

BA

> Due to multiple, repetitive phage regions, having a second finished O55:H7 allows broader analysis of the diversity in these areas, often sites of laterally acquired virulence factors.>