www.sciencetranslationalmedicine.org/cgi/content/full/5/184/184ra60/DC1

Supplementary Materials for

Genomic Diversity and Fitness of E. coli Strains Recovered from the Intestinal and Urinary Tracts of Women with Recurrent Urinary Tract

Infection

Swaine L. Chen, Meng Wu, Jeffrey P. Henderson, Thomas M. Hooton, Michael E. Hibbing, Scott J. Hultgren, Jeffrey I. Gordon*

*Corresponding author. E-mail: jgordon@wustl.edu

Published 8 May 2013, Sci. Transl. Med. 5, 184ra60 (2013)

DOI: 10.1126/scitranslmed.3005497

This PDF file includes:

Fig. S1. Maximum likelihood fimH gene tree incorporating the UPEC strains characterized in the present study. Fig. S2. Hierarchical clustering of 404 E. coli strains including the 26 characterized in the present study. Fig. S3. The episode 3 UTI strain Ec72_E3U1 forms IBCs. Fig. S4. Assays of urinary tract colonization with episode 1 and 3 strains from patient 72. Fig. S5. Parameter testing and validation of FitSeq. Fig. S6. In vitro characterization of Ec72_U1E1 and Ec72_U3E1. Fig. S7. Genes encoding flagellar proteins that are present/absent in the genomes of UTI episode 1 (Ec72_E1U1) and episode 3 (Ec72_E3U1) strains. Fig. S8. UPEC virulence–associated elements present/absent in the genomes of UTI episode 1 (Ec72_E1U1) and episode 3 (Ec72_E3U1) strains recovered from patient 72 compared to UPEC strain UTI89. Fig. S9. PTS pathway components involved in L-sorbose utilization. Fig. S10. Comparison of growth phenotypes of Ec72_E1U1 and Ec72_E3U1 strains from UTI episodes 1 and 3. Legends for Tables S1 to S9

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/5/184/184ra60/DC1)

Table S1. Clinical characteristics and treatment of the four patients with three episodes of recurrent UTI. Table S2. Summary of 45 isolates analyzed for this study, including those subjected to whole-genome sequencing.

Table S3. Genes targeted for MLST and primers used for generating PCR amplicons for DNA sequencing. Table S4. Genome sequencing and assembly metrics for urine and fecal isolates obtained from patients 13 and 72 during their three episodes of UTI. Table S5. Reference genomes used for OGU- and SNP-based analyses. Table S6. Representation of known virulence factors in the genomes of the 26 isolates from the present study and in 54 other E. coli genomes classified as complete in the NCBI RefSeq database. Table S7. Comparison of YESCA pellicle, Congo Red staining, and swarming phenotypes of strains EC72_E1U1, EC72_E3U1, and UTI89. Table S8. Representation of genes involved in flagellar assembly and PTS-sorbose systems in the 26 clinical isolates from the present study and in the 54 reference complete E. coli genomes. Table S9. Representation of genes (OGUs) assigned to KEGG pathways in the shared and variable components of the EC72_E1U1 and EC72_E3U1 genomes.

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Genomic diversity and fitness of E. coli strains recovered from the intestinal and

urinary tracts of women with recurrent urinary tract infection

SUPPLEMENTAL MATERIALS

Supplemental Figures

Fig. S1. Maximum likelihood fimH gene tree incorporating the UPEC strains characterized in the present study. fimH sequences analyzed in (33) are included to

876

1 2 3

Patient 72 Fecal

Patient 72 Urine

Patient 13 Fecal

Patient 13 Urine

1 1 2 32 32 3321

3 3 3 33

1 1 11 22

2

provide background fimH variability within E. coli. Squares indicate fecal isolates while circles are urine isolates. The number inside each symbol indicates the UTI episode from which the strain was isolated.

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Fig. S2. Hierarchical clustering of 404 E. coli strains including the 26 characterized in the present study. (A) SNP-based unsupervised hierarchical clustering of the clinical isolates from the present study with 378 strains with ‘finished’, ‘complete’, or draft genomes available in the NCBI RefSeq and PATRIC databases.

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Fig. S2. Hierarchical clustering of 404 E. coli strains including the 26 characterized in the present study. (B) OGU-based unsupervised clustering of 26 clinical isolates with 378 strains with finished or draft genomes available in public database. All isolates from Patient 13 are colored red while all isolates from Patient 72 are colored green.

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Fig. S3. The episode 3 UTI strain Ec72_E3U1 forms IBCs. Confocal images of IBCs found in infections of the mouse bladder using the reference UPEC strain, UTI89 (A,B) and the episode 3 isolate from patient 72, Ec72_E3U1 (C,D). Bacterial strains express GFP and appear green. Epithelial cells are stained with wheat germ agglutinin (red) to show uroplakin expression. Images are taken 6 h post-infection (A,C) or 12 h post-

infection (B,D). Scale bar in (A), 10µm (same in all panels).

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Fig. S4. Assays of urinary tract colonization with episode 1 and 3 strains from patient 72. C3H/HeN mice were inoculated transurethrally with 1-5x108 CFUs of (A) UTI89, (B) Ec72_E1U1, or (C) Ec72_E3U1. 24 h after infection, urine samples were collected and mice were sacrificed. Bladders and kidney pairs were homogenized in PBS to a final volume of 1 ml and bacterial titers defined in urine and tissue homogenates (n=10 mice

Urine

Bladde

r

Kidney

UTI-89

CFU

s/m

lC

FUs/

ml

Ec72_E3U1

100

101

102

103

104

105

106

107

108

109

Urine

Bladde

r

Kidney

Ec72_E1U1

CFU

s/m

l

100

101

102

103

104

105

106

107

108

109

Urine

Bladde

r

Kidney

100

101

102

103

104

105

106

107

108

109A

B

C

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for each strain from patient 72, n=5 animals for the reference UTI89 strain; lines connect data points generated from a given animal). Fig. S5. Parameter testing and validation of FitSeq. (A) In silico simulation of the accuracy of FitSeq at different levels of sequencing coverage. Three different defined fractional representations of Ec72_E1U1 in a mixture of Ec72_E1U1 and Ec72_E3U1 were used to test the coverage requirement. The x-axis is the total number of reads while the

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y-axis plots the detected representation as calculated by FitSeq. (B) Validation of FitSeq by in silico simulation sequencing of defined mixtures. The expected representation and the detected representation are highly correlated in both the in silico simulation and the defined mixtures at a coverage of 500,000 reads/sample (R2=0.999).

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Fig. S6. In vitro characterization of Ec72_U1E1 and Ec72_U3E1. (A) Biofilm formation assayed after a 48 h static incubation, at room temperature in LB media, in 96-well polyvinyl chloride assay plates. Adherent biomass was defined by staining the material in each well with crystal violet, solubilizing the crystal violet with acetic acid, and recording A595 values for each well (data normalized to UTI89). (B) Guinea pig red blood cell hemagglutination titers, with and without mannose (determined with cells normalized to OD600 = 1.0, as described previously (76); (C) Swimming motility. Cultures of each strain were incubated statically at 37oC for 24 h, subcultured and incubated again under the same conditions and inoculated into 0.25% LB agar. Swim diameter was assessed 6.5 h after inoculation. Mean values ± SD are plotted

A

B

C

0.0

2.0

4.0

6.0

8.0

10.0

UTI89 Ec72E1U1 Ec72E3U1

HA

Tite

r

Mannose -

Mannose +

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

UTI89 Ec72E1U1 Ec72E3U1

A59

5 R

elat

ive

to U

TI8

9 LB

0.0

0.5

1.0

1.5

2.0

UTI89 Ec72E1U1 Ec72E3U1

Sw

im D

iam

eter

(cm

)

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Fig. S7. Genes encoding flagellar proteins that are present/absent in the genomes of UTI episode 1 (Ec72_E1U1) and episode 3 (Ec72_E3U1) strains. Figure adapted from the KEGG database (version 58). A red ‘X’ indicates genes that are absent in the Ec72_E1U1 but present in Ec72_E3U1.

Hook-filamentjunction

Filament cap

FliK

L ring

P ring

Distal rod

FlgH

FlgI

Proximal rod

MS ring

C ring

Proximal rod

Outer membrane

Peptidoglycan layer

Cytoplasmicmembrane

BasalBody

Motor switch

FliD

FliC

FlgL

FlgK

FlgDFlgE

FlgG

FliE FlgB FlgC

FliF

FliGFliMFliN

FlgF

MotB

MotA

FlhA FlhB

FliO

FliHFliI

FliPFliQFliR

Filament

Hook

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Fig. S8. UPEC virulence–associated elements present/absent in the genomes of UTI episode 1 (Ec72_E1U1) and episode 3 (Ec72_E3U1) strains recovered from patient 72 compared to UPEC strain UTI89. Each gene is represented by a square and is colored red if present, and black if absent, as determined by the OGU-based pan-genome analysis.

yad sfa fml yeh yfc yqi auf pap F17 fim ECP

HeminIron/

Manganese Salmochelin Yersiniabactin

Prophage 43016 Prophage 43020 Prophage 43023

Prophage 43025 Prophage 43027

UTI89 plasmid

PAI-II/leuX

PAI-III/serX PAI-IV/asnT

PAI-V PAI-VI

IAHP/aspV IAHP/metV ibeA

UTI89

Ec72_E1U1

Ec72_E3U1

Pat

hoge

nici

ty is

land

sP

roph

ages

Pili

Iron

Toxi

ns

Pla

smid

foc pixType IV(c2395)

Type IV(hof/ppd)

Aerobactin

cytotoxicnecrotizing

factor 1alpha

hemolysincytolethaldistending Shiga-like

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Fig. S9. PTS pathway components involved in L-sorbose utilization. The components of L-sorbose-specific enzymes II (EII-A, EII-B, EII-C and EII-D) are absent in the Ec72_E1U1 strain but present in the Ec72_E3U1 strain.

sorbose

periplasmic space

inner membrane

cytosol

sorbose 1-phosphatemetabolism

phosphpenolpyruvate (PEP)

pyruvateEII-AEII-B

EII-CEII-D

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Fig. S10. Comparison of growth phenotypes of Ec72_E1U1 and Ec72_E3U1 strains from UTI episodes 1 and 3. The color key indicates the level of growth during a 48 h incubation relative to the reference UPEC strain UTI89.

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Supplemental Tables

Table S1. Clinical characteristics and treatment of the four patients with three

episodes of recurrent UTI.

Table S2. Summary of 45 isolates analyzed for this study, including those subjected to

whole-genome sequencing.

Table S3. Genes targeted for MLST and primers employed for generating PCR

amplicons for DNA sequencing.

Table S4. Genome sequencing and assembly metrics for urine and fecal isolates

obtained from patients 13 and 72 during their three episodes of UTI.

Table S5. Reference genomes used for OGU- and SNP-based analyses.

Table S6. Representation of known virulence factors in the genomes of the 26 isolates

from the present study and in 54 other E. coli genomes classified as complete in the

NCBI RefSeq database. (A) Distribution of virulence factors in each strain. “Reference

genome” refers to the sequenced genome whose annotation was used to assign “Gene

name”. (B) The number of virulence factors per virulence system (e.g. adherence, iron

update) represented in each of the 80 genomes.

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Table S7. Comparison of YESCA pellicle, Congo Red staining, and swarming

phenotypes of strains EC72_E1U1, EC72_E3U1, and UTI89.

Table S8. Representation of genes involved in flagellar assembly and PTS-sorbose

systems in the 26 clinical isolates from the present study and in the 54 reference

complete E. coli genomes. (A) Flagellar assembly system. (B) Phosphotransferase

system (PTS) for L-sorbose.

Table S9. Representation of genes (OGUs) assigned to KEGG pathways in the shared

and variable components of the EC72_E1U1 and EC72_E3U1 genomes.