Transcriptomics

and

adaptive

genomics

of

the

asymptomatic

bacteriuria

Escherichia

coli

strain

Escherichia

coli

strains

are

the

major

cause

of

urinary

tract

infections

in

humans

Such

strains

can

be

divided

into

virulent

UPEC

strains

causing

symptomatic

infections

and

asymptomatic

commensal-like

strains

causing

asymptomatic

bacteriuria

ABU

The

best-characterized

ABU

strain

is

strain

Global

gene

expression

profiling

of

strain

has

been

carried

out

under

seven

different

sets

of

environmental

conditions

ranging

from

laboratory

minimal

medium

to

human

bladders

The

data

reveal

highly

specific

gene

expression

responses

to

different

conditions

A

number

of

potential

fitness

factors

for

the

human

urinary

tract

could

be

identified

Also

presence/absence

data

of

the

gene

expression

was

used

as

an

adaptive

genomics

tool

to

model

the

gene

pool

of

using

primarily

UPEC

strain

as

a

scaffold

In

our

analysis

of

the

transcripts

filtered

present

in

strain

can

be

found

in

and

genes

on

six

of

the

seven

pathogenicity

islands

were

expressed

in

Despite

the

very

different

patient

symptom

profiles

the

two

strains

seem

to

be

very

similar

Genes

expressed

in

but

not

in

were

identified

and

can

be

considered

as

virulence

factor

candidates

Strain

is

a

deconstructed

pathogen

rather

than

a

commensal

strain

that

has

acquired

fitness

properties

Introduction

Urinary

tract

infection

-LRB-

UTI

-RRB-

is

one

of

the

most

common

infectious

diseases

in

humans

and

a

major

cause

of

morbidity

It

is

estimated

that

of

adult

healthy

women

have

experienced

at

least

one

UTI

episode

-LRB-

Foxman

-RRB-

UTI

can

be

caused

either

by

pathogenic

strains

leading

to

symptomatic

UTI

or

by

asymptomatic

bacteriuria

-LRB-

ABU

-RRB-

strains

resulting

in

a

symptom-free

carriage

resembling

commensalism

Escherichia

coli

is

responsible

for

more

than

of

all

UTIs

Acute

pyelonephritis

is

a

severe

acute

systemic

infection

caused

by

uropathogenic

E.

coli

-LRB-

UPEC

-RRB-

clones

with

virulence

genes

clustered

on

pathogenicity

islands

-LRB-

PAIs

-RRB-

-LRB-

Eden

et

al.

Funfstuck

et

al.

Stenqvist

et

al.

Orskov

et

al.

Johnson

Welch

et

al.

-RRB-

Paradoxically

a

large

proportion

of

UTIs

are

caused

by

ABU

E.

coli

Individuals

infected

with

ABU-class

E.

coli

may

carry

high

urine

titres

of

a

single

E.

coli

strain

for

months

or

years

without

provoking

a

host

response

Escherichia

coli

is

a

prototype

ABU

strain

and

undoubtedly

the

best-characterised

ABU-class

E.

coli

to

date

Strain

was

originally

isolated

in

the

from

a

young

girl

who

had

carried

it

for

at

least

years

without

symptoms

-LRB-

Lindberg

et

al.

Andersson

et

al.

-RRB-

The

strain

is

well

adapted

for

growth

in

the

human

urinary

tract

-LRB-

UT

-RRB-

where

it

establishes

long-term

bacteriuria

-LRB-

Hull

et

al.

-RRB-

It

has

been

used

for

prophylactic

purposes

in

numerous

studies

as

such

it

has

been

used

as

an

alternative

treatment

in

patients

with

recurrent

UTI

who

are

refractory

to

conventional

therapy

-LRB-

Hull

et

al.

-RRB-

An

ongoing

study

on

patients

infected

with

strain

has

so

far

reported

over

patient

years

with

no

serious

side

effects

-LRB-

Sundén

et

al.

-RRB-

ABU

patients

may

carry

a

single

strain

for

months

or

years

creating

a

condition

that

resembles

commensalism

but

with

a

strain

that

may

have

evolved

from

a

pathogenic

ancestor

Several

lines

of

evidence

support

the

notion

that

the

ancestor

of

strain

was

a

pyelonephritic

UPEC

strain

it

belongs

to

the

clonal

group

a

group

associated

with

pyelonephritis

and

other

extra-intestinal

invasive

clinical

syndromes

such

as

bacteremia

prostatitis

and

meningitis

the

strain

also

contains

gene

clusters

in

various

stages

of

erosion

encoding

the

three

UPEC-class

fimbriae

i.e.

the

fim

pap

and

sfa/foc

clusters

-LRB-

Klemm

et

al.

Roos

et

al.

-RRB-

Several

studies

have

investigated

the

virulence

characteristics

of

uropathogenic

E.

coli

-LRB-

UPEC

-RRB-

isolates

and

ABU

isolates

in

order

to

get

better

understanding

of

how

some

UTI

strains

can

cause

severe

disease

while

others

can

be

used

prophylactically

to

prevent

the

same

-LRB-

Blanco

et

al.

Dobrindt

et

al.

Vranes

et

al.

Johnson

et

al.

Marrs

et

al.

-RRB-

ABU

strains

have

been

shown

to

lack

many

of

the

virulence-associated

phenotypes

many

of

them

are

nonhaemolytic

nonadherent

and

lack

haemagglutination

ability

-LRB-

Vranes

et

al.

-RRB-

Strain

lacks

many

of

the

virulence-associated

phenotypes

but

has

been

shown

to

carry

many

of

the

virulence-associated

genes

such

as

kps

iutA

fyuA

and

malX

-LRB-

Dobrindt

et

al.

-RRB-

However

apart

from

the

fimbrial

clusters

the

strain

has

not

been

sequenced

and

it

is

not

known

which

genes

it

shares

with

other

E.

coli

isolates

which

of

the

genes

in

the

E.

coli

core

genome

it

carries

and

which

genes

it

shares

with

other

UTI

isolates

Thus

far

although

a

number

of

UPEC

isolates

-LRB-

i.e.

and

-RRB-

have

been

completely

sequenced

no

genomic

sequencing

of

any

ABU

strain

has

been

reported

Comparative

genomics

profiling

using

microarray

chips

designed

to

cover

entire

genomes

is

one

strategy

to

obtain

information

about

variability

between

different

strains

of

the

same

species

and

indication

of

horizontal

gene

transfer

-LRB-

Willenbrock

et

al.

-RRB-

DNA

microarray-assisted

functional

genomics

provides

the

global

expression

profile

of

a

strain

revealing

which

genes

are

expressed

under

certain

conditions

Global

gene

expression

profiling

of

ABU

strain

employing

the

GeneChip

E.

coli

Genome

Array

-LRB-

Affymetrix

-RRB-

containing

four

E.

coli

genomes

including

that

of

the

UPEC

isolate

will

not

only

provide

information

regarding

the

up

and

down-regulation

of

genes

comparing

different

conditions

but

will

also

reveal

which

genes

are

actually

present

-LRB-

expressed

-RRB-

in

the

genome

of

Bacterial

pathogens

differ

from

commensals

by

expression

of

specific

virulence

factors

such

as

those

that

mediate

histological

damage

Commensals

in

contrast

have

generally

been

regarded

as

bacteria

lacking

such

virulence

factors

or

other

specific

mechanisms

for

interaction

with

host

tissues

Here

we

compare

the

global

expression

profiles

of

E.

coli

ABU

strain

grown

under

a

number

of

different

in

vitro

conditions

and

in

three

patients

in

order

to

get

a

representative

picture

of

which

genes

are

present/expressed

in

the

genome

of

this

asymptomatic

UTI

strain

Materials

and

methods

Bacterial

strain

Escherichia

coli

is

a

prototype

ABU

strain

and

lacks

defined

O

and

K

surface

antigens

-LRB-

Lindberg

et

al.

-RRB-

It

belongs

to

the

ECOR

group

together

with

many

other

UTI

strains

such

as

the

well-characterized

and

virulent

E.

coli

isolates

and

Growth

conditions

and

stabilisation

of

RNA

for

microarray

experiments

of

E.

coli

grown

on

urine

agar

plates

Human

urine

was

collected

from

four

healthy

men

and

women

volunteers

who

had

no

history

of

UTI

or

antibiotic

use

in

the

prior

months

The

urine

was

pooled

filter

sterilised

stored

at

°C

and

used

within

the

following

day

E.

coli

was

grown

aerated

in

triplicates

in

ml

of

human

urine

for

h.

Thereafter

μl

of

each

culture

was

spread

on

urine

plates

-LRB-

ratio

of

human

urine

and

NaCl

-RRB-

containing

agar

The

plates

were

incubated

at

°C

for

h.

Subsequently

μl

of

a

mixture

of

PBS

and

RNAprotect

Bacteria

Reagent

-LRB-

QIAGEN

AG

-RRB-

was

poured

on

the

plates

mixed

with

the

lawn

of

cells

and

incubated

for

min

at

room

temperature

to

stabilise

RNA

The

stabilised

mixture

was

then

centrifuged

and

pellets

were

stored

at

°C

The

samples

from

grown

exponentially

in

MOPS

and

urine

in

urine

biofilms

and

in

patients

-LRB-

CFU/ml

-RRB-

were

all

treated

identically

with

RNAprotect

Bacteria

Reagent

and

have

been

described

previously

-LRB-

Roos

and

Klemm

Hancock

and

Klemm

-RRB-

RNA

isolation

and

microarray

hybridisation

Total

RNA

was

isolated

using

the

RNeasy

Mini

Kit

-LRB-

QIAGEN

AG

-RRB-

and

on-column

DNase

digestion

was

performed

using

RNase-Free

DNase

Set

-LRB-

QIAGEN

AG

-RRB-

The

quality

of

the

total

RNA

was

examined

by

agarose

gel

electrophoresis

and

by

measuring

the

absorbance

at

and

nm

to

ensure

intact

high-quality

RNA

Purified

RNA

was

precipitated

with

ethanol

and

stored

at

°C

until

further

use

Conversion

of

RNA

-LRB-

μg

per

sample

-RRB-

to

cDNA

labelling

and

microarray

hybridisation

were

performed

according

to

the

GeneChip

Expression

Analysis

Technical

Manual

Rev.

-LRB-

Affymetrix

Inc.

Santa

Clara

CA

-RRB-

GeneChip

E.

coli

Genome

Arrays

-LRB-

Affymetrix

-RRB-

were

used

for

hybridisation

of

the

labelled

cDNA

The

microarrays

were

scanned

using

the

GeneChip

Scanner

Data

analysis

The

raw

intensities

from

the

microarray

experiments

were

background

corrected

and

quantile-normalised

All

microarray

data

in

the

study

were

obtained

from

mRNA

being

converted

to

cDNA

i.e.

no

genomic

DNA

was

used

for

hybridisation

Probe

intensities

were

summarised

to

yield

expression

values

for

each

probe

set

or

gene

These

calculations

were

performed

using

the

implementation

of

GCRMA

-LRB-

Wu

et

al.

-RRB-

in

Bioconductor

-LRB-

Gentleman

et

al.

-RRB-

-LRB-

-RRB-

In

order

to

derive

a

cut-off

expression

value

for

making

presence/absence

calls

we

made

use

of

intensities

due

to

control

probe

sets

with

IDs

beginning

with

AFFY

There

were

such

probes

The

cut-off

value

was

set

so

that

only

the

top

of

these

control

probes

would

be

flagged

as

present

As

a

result

genes

in

the

array

were

marked

as

present

the

remaining

genes

are

referred

to

as

absent

throughout

this

report

i.e.

these

genes

could

be

truly

absent

non-homologous

or

not

expressed

during

any

of

the

seven

different

growth

conditions

Orthologs

of

all

the

genes

in

the

array

across

E.

coli

E.

coli

Sakai

E.

coli

and

E.

coli

were

identified

using

bidirectional

best

hit

BLAST

Microarray

data

accession

number

The

supporting

microarray

data

have

been

deposited

in

ArrayExpress

-LRB-

-RRB-

with

accession

numbers

-LRB-

MOPS

urine

and

patient

arrays

-RRB-

-LRB-

biofilm

arrays

-RRB-

and

-LRB-

urine-agar

plate

arrays

-RRB-

Results

Genes

expressed

in

ABU

E.

coli

The

bacterial

transcriptome

is

a

dynamic

entity

that

reflects

the

organism

's

immediate

ongoing

response

to

its

environment

DNA

microarray-assisted

functional

genomics

provides

the

global

expression

profile

of

the

genome

The

genomic

expression

profiles

of

the

urinary

tract

infectious

E.

coli

isolate

were

analysed

under

several

different

growth

conditions

and

in

different

media

using

the

GeneChip

E.

coli

Genome

Array

-LRB-

Affymetrix

-RRB-

This

array

contains

approximately

probe

sets

for

all

genes

present

in

E.

coli

strains

-LRB-

-RRB-

-LRB-

UPEC

-RRB-

-LRB-

EHEC

-RRB-

and

-LRB-

EHEC

-RRB-

Due

to

the

high

degree

of

similarity

between

the

E.

coli

strains

whenever

possible

a

single

probe

set

is

tiled

to

represent

the

equivalent

ortholog

in

all

the

four

strains

In

total

microarrays

were

included

in

the

study

arrays

in

triplicates

were

hybridised

with

RNA

of

the

ABU

strain

cultured

-LRB-

-RRB-

aerobically

to

exponential

phase

in

MOPS

minimal

medium

-LRB-

-RRB-

aerobically

to

exponential

phase

in

pooled

human

urine

-LRB-

-RRB-

on

urine

agar

plates

-LRB-

-RRB-

statically

in

urine

biofilm

on

Petri

dishes

and

finally

-LRB-

-RRB-

in

three

patients

-LRB-

and

-RRB-

in

vivo

Figure

shows

the

expression

levels

of

all

genes

in

strain

during

growth

in

the

different

environments

many

genes

were

similarly

expressed

during

all

seven

conditions

However

some

genes

were

expressed

only

during

one

or

a

few

of

the

conditions

For

example

the

genes

encoding

yersiniabactin

in

the

high

pathogenicity

island

-LRB-

HPI

-RRB-

i.e.

PAI-asnT

were

highly

expressed

in

-LRB-

and

in

biofilm

-RRB-

but

much

lower

during

the

other

conditions

The

genes

-LRB-

around

M

in

Fig.

-RRB-

involved

in

nucleotide

sugar

and

mannose

metabolism

and

encoding

hypothetical

proteins

were

highly

expressed

in

but

not

under

any

other

condition

Another

example

is

the

genes

-LRB-

around

M

-RRB-

i.e.

ydfI

encoding

a

d-mannonate

oxidoreductase

ydfJ

encoding

a

metabolite

transport

protein

and

rspAB

involved

in

the

starvation

response

which

also

were

highly

expressed

only

in

Fig.

expression

levels

of

genes

in

strain

during

seven

different

growth

conditions

The

outer

blue

circle

shows

the

calculated

absence

-LRB-

-RRB-

and

presence

-LRB-

-RRB-

of

genes

in

ABU

strain

The

seven

PAIs

of

are

indicated

in

red

In

total

there

were

genes

that

were

significantly

changed

in

all

six

urine

environments

compared

with

MOPS

Twenty

of

these

genes

were

up-regulated

in

all

six

urine

conditions

whereof

half

were

related

to

different

iron

systems

i.e.

iroN

fepA

fecI

iucBC

fhuA

and

exbD

as

well

as

and

involved

in

iron

storage

and

encoding

a

putative

haemin

receptor

respectively

The

other

urine

up-regulated

genes

were

marA

a

multiple

antibiotic

resistance

gene

sodA

encoding

superoxide

dismutase

ahpC

encoding

hydroperoxide

reductase

lysA

rrsG

rrsH

and

yrbL

Most

iron

acquisition

systems

were

expressed

in

all

the

six

urine

environments

the

enterobactin

salmochelin

aerobactin

haem

and

sitABCD

systems

were

all

expressed

in

all

the

six

urine

conditions

-LRB-

although

weaker

in

the

urine

plates

-RRB-

Interestingly

the

fec

system

which

is

a

citrate-dependent

iron

uptake

system

found

in

but

missing

in

and

other

UPEC

strains

was

highly

expressed

in

Up-regulation

of

all

these

iron-uptake

systems

revealed

that

the

strain

has

an

impressive

array

of

iron

acquisition

systems

and

all

of

these

are

active

in

the

human

bladder

Nineteen

of

the

top

highest

expressed

genes

overall

were

genes

involved

in

ribosomal

synthesis

The

high

expression

of

ribosomal

genes

in

E.

coli

suggests

a

rapid

growth

rate

the

highest

expression

values

were

obtained

in

followed

by

MOPS

urine

and

indicating

a

growth

rate

just

as

fast

in

the

patients

in

vivo

as

in

exponential

growth

phase

in

a

shake

flask

This

supports

our

hypothesis

that

the

strain

's

optimized

growth

properties

in

human

urine

explain

its

ability

to

successfully

colonize

the

human

urinary

tract

in

the

absence

of

functional

fimbriae

-LRB-

Roos

et

al.

-RRB-

Figure

reveals

that

strain

almost

exclusively

expresses

the

iron

uptake

and

transport

systems

in

the

seven

PAIs

almost

of

the

other

genes

in

these

islands

are

expressed

There

are

only

two

exceptions

located

in

PAI-aspV

encoding

a

hypothetical

protein

and

located

in

PAI-pheV

encoding

YrbH

and

KpsEDC

The

yrbH

gene

belongs

to

the

genes

that

were

recently

identified

as

UPEC

specific

and

it

was

the

second

highest

expressed

UPEC-specific

gene

in

mice

-LRB-

Lloyd

et

al.

-RRB-

in

our

samples

the

highest

expression

was

found

in

the

three

patients

and

in

MOPS

Outside

the

PAIs

there

are

a

few

genes/gene

clusters

that

are

highly

expressed

in

all

urine

samples

or

only

in

the

patients

The

enterobactin

system

was

up-regulated

during

all

urine

conditions

and

the

chu

cluster

-LRB-

involved

in

haem

uptake

and

transport

-RRB-

was

highest

up-regulated

in

the

patients

followed

by

in

vitro

urine

growth

The

ycdO

and

ycdB

genes

were

highly

expressed

in

the

three

patients

these

have

recently

been

identified

to

encode

haemoproteins

probably

involved

in

iron

transport

induced

at

acidic

conditions

-LRB-

Sturm

et

al.

-RRB-

Looking

at

the

significantly

changed

genes

for

all

six

urine

conditions

compared

with

MOPS

-LRB-

in

total

genes

-RRB-

revealed

that

and

shared

the

largest

number

of

similarly

changed

genes

of

all

changed

genes

in

are

regulated

in

the

same

way

-LRB-

i.e.

up

or

down-regulated

-RRB-

in

-LRB-

Fig.

-RRB-

Interestingly

shared

the

largest

number

similarly

regulated

genes

with

the

biofilm

growth

mode

also

for

and

the

biofilm

growth

mode

showed

a

larger

number

of

similarly

regulated

genes

than

or

any

other

condition

This

could

indicate

that

the

expression

profile

of

strain

during

in

vivo

growth

is

closer

related

to

biofilm

growth

than

to

growth

in

shake

flasks

or

plates

Fig.

of

significantly

up

and

down-regulated

genes

in

strain

during

the

different

growth

conditions

-LRB-

i.e.

exponential

growth

in

urine

on

urine-agar

plates

in

urine

biofilm

in

vivo

in

three

patients

-RRB-

compared

with

exponential

growth

in

MOPS

minimal

lab

medium

The

diagonal

boxes

-LRB-

dark

blue

colour

-RRB-

show

the

number

of

significantly

changed

genes

during

cultivation

in

that

specific

condition

compared

with

MOPS

-LRB-

e.g.

genes

were

up

or

down-regulated

in

urine

compared

with

MOPS

and

genes

were

changed

in

plates

compared

with

MOPS

-RRB-

and

the

other

boxes

show

the

number

of

significantly

changed

genes

shared

between

two

conditions

-LRB-

e.g.

of

the

and

significantly

changed

genes

in

urine

and

plates

compared

with

MOPS

were

shared

between

these

two

conditions

i.e.

up

or

down-regulated

in

both

urine

and

plates

compared

with

MOPS

-RRB-

Stronger

blue

colour

indicates

larger

number

of

significantly

changed

genes

shared

between

two

conditions

Closeness

to

Given

the

different

growth

conditions

analysed

it

is

not

unrealistic

to

assume

that

most

genes

present

in

strain

would

be

expressed

to

some

extent

under

at

least

one

of

these

seven

different

conditions/environments

i.e.

growth

in

liquid

and

on

solid

media

during

exponential

phase

in

biofilm

and

during

colony-forming

conditions

in

different

growth

media

-LRB-

human

urine

and

minimal

lab

medium

-RRB-

as

well

as

in

vivo

in

three

different

individuals

Data

analysis

of

the

microarrays

revealed

that

of

the

E.

coli

transcripts

on

the

microarray

-LRB-

not

including

probes

representing

intergenic

regions

and

controls

-RRB-

transcripts

-LRB-

-RRB-

showed

expression

levels

above

detection

limit

during

at

least

one

of

the

growth

conditions

investigated

-LRB-

referred

to

as

present

see

blue

outer

circle

in

Fig.

-RRB-

Figure

shows

the

distribution

among

the

four

E.

coli

genomes

represented

on

the

microarray

of

these

transcripts

expressed

in

E.

coli

Not

surprisingly

the

UTI

strain

shows

highest

similarity

with

the

UPEC

isolate

of

the

four

genomes

on

the

array

the

large

majority

of

the

transcripts

found

present

-LRB-

-RRB-

can

be

found

in

corresponding

to

of

the

genome

E.

coli

expressed

genes

that

do

not

exist

in

of

these

can

be

found

in

and

the

remaining

genes

can

be

found

exclusively

in

one

or

both

of

the

two

EHEC

strains

present

on

the

array

-LRB-

Fig.

-RRB-

Thirty

of

the

genes

homologous

to

EHEC

genes

are

encoding

proteins

of

cryptic

prophages

whereas

the

large

majority

of

the

remaining

genes

encode

unknown

or

hypothetical

proteins

The

genes

that

can

be

found

in

but

not

in

includes

the

fec

cluster

encoding

an

iron

citrate

transport

system

-LRB-

fecABCDEIR

-RRB-

In

total

genes

were

expressed

in

strain

this

could

be

compared

with

genes

present

in

the

UPEC

-LRB-

cystitis

-RRB-

isolate

-LRB-

Lloyd

et

al.

-RRB-

Fig.

diagrams

showing

the

distribution

of

the

genes

filtered

present

in

strain

The

percentages

indicated

below

each

strain

show

how

large

part

of

the

genome

of

the

corresponding

strain

was

filtered

present

in

strain

E.

coli

core

genome

There

is

a

large

diversity

in

size

of

the

chromosome

of

E.

coli

in

all

E.

coli

-LRB-

and

Shigella

-RRB-

genomes

that

have

been

fully

sequenced

or

at

least

with

an

expected

coverage

of

greater

than

the

size

of

the

chromosome

ranges

from

to

Mbp

The

genomes

show

a

considerable

amount

of

diversity

and

the

estimated

size

of

the

current

pan-genome

was

estimated

to

contain

different

genes

-LRB-

Willenbrock

et

al.

-RRB-

Several

studies

have

identified

sets

of

core

genes

found

in

most

E.

coli

genomes

However

the

number

of

these

core

genes

tends

to

decrease

as

the

full

genomic

sequences

of

new

E.

coli

strains

become

available

The

size

of

the

E.

coli

core

genome

has

recently

been

predicted

to

contain

genes

for

an

infinite

number

of

E.

coli

strains

and

the

number

of

new

genes

predicted

from

each

new

E.

coli

genome

that

is

sequenced

is

-LRB-

Willenbrock

et

al.

-RRB-

In

our

analysis

-LRB-

-RRB-

of

the

genes

found

present

in

strain

were

common

in

all

the

four

E.

coli

genomes

on

the

array

-LRB-

Fig.

-RRB-

which

is

well

above

the

estimated

E.

coli

core

genome

and

also

above

the

common

genes

conserved

among

the

sequenced

E.

coli

strains

-LRB-

Willenbrock

et

al.

-RRB-

Furthermore

considering

the

fact

that

the

microarray

contains

only

four

E.

coli

genomes

the

total

number

of

genes

detected

present

-LRB-

genes

-RRB-

in

seems

reasonable

comparing

the

size

of

other

sequenced

UTI

E.

coli

genomes

The

genome

size

of

strain

has

been

reported

to

be

Mbp

-LRB-

Zdziarski

et

al.

-RRB-

indicating

that

the

strain

contains

roughly

an

additional

genes

not

identified

in

the

present

analysis

Of

the

transcripts

on

the

chip

that

are

present

in

all

the

four

strains

represented

on

the

microarray

transcripts

were

below

detection

limit

on

all

microarrays

and

filtered

as

absent

in

strain

These

included

genes

encoding

hypothetical

proteins

Several

of

the

absent

genes

were

found

in

clusters

many

of

which

are

involved

in

surface

structure

elements

and

chemotaxis

These

included

genes

involved

in

flagellar

biosynthesis

-LRB-

flgABCDEFGHIJKL

flhABE

fliACDEFGHIJKLNOPQRSTZ

and

motAB

-RRB-

curli

production

-LRB-

csgABCEFG

-RRB-

colanic

acid

synthesis

-LRB-

wcaABCDEFGHI

and

wza

-RRB-

and

chemotaxis

-LRB-

cheBRWYZ

and

tap

-RRB-

Other

whole

cluster

of

genes

that

were

not

expressed

in

the

ABU

strain

but

found

in

all

the

four

E.

coli

present

on

the

chip

were

hyaBCDEF

-LRB-

hydrogenase

I

-RRB-

hycACD

-LRB-

hydrogenase

-RRB-

tauABCD

-LRB-

responsible

for

taurine

uptake

in

E.

coli

-RRB-

and

-LRB-

containing

the

fimbrial-like

genes

ydeQRST

-RRB-

as

well

as

the

fimEAIC

genes

which

previously

have

been

shown

to

be

absent

in

strain

-LRB-

Klemm

et

al.

-RRB-

UPEC-associated

genes

present

in

strain

The

four

UPEC

isolates

that

have

been

sequenced

and

contain

and

genes

respectively

on

the

chromosome

and

are

both

strains

and

yet

show

a

large

diversity

the

genome

of

is

almost

kb

smaller

than

that

of

-LRB-

Brzuszkiewicz

et

al.

-RRB-

The

genomic

differences

are

mainly

restricted

to

large

pathogenicity

islands

the

additional

DNA

in

are

genes

of

five

cryptic

prophages

which

are

absent

in

-LRB-

Brzuszkiewicz

et

al.

-RRB-

The

genes

that

are

present

only

in

the

strain

and

the

genes

present

only

in

the

two

UPEC

-LRB-

compared

with

other

sequenced

E.

coli

-RRB-

are

scattered

all

over

the

genome

-LRB-

Brzuszkiewicz

et

al.

-RRB-

Over

of

the

transcripts

were

present

in

strain

compared

with

of

the

transcripts

found

in

strain

Figure

shows

the

homology

of

sequenced

E.

coli

and

Shigella

isolates

including

the

three

sequenced

UPEC

strains

-LRB-

and

-RRB-

pasted

on

the

genome

the

outer

red

circle

in

the

figure

shows

the

results

from

the

presence/absence

analysis

on

strain

Many

virulence-associated

genes

are

located

on

the

large

pathogenicity

islands

-LRB-

PAIs

-RRB-

found

in

different

UPEC

strains

The

large

pathogenicity

island

at

pheV

in

-LRB-

also

called

PAI

-RRB-

encodes

haemolysin

-LRB-

hlyCABD

-RRB-

aerobactin

biosynthesis

proteins

-LRB-

iutA

and

iucABCD

-RRB-

antigen

-LRB-

-RRB-

and

the

secreted

autotransporter

toxin

-LRB-

sat

-RRB-

these

were

all

filtered

present

in

our

analysis

suggesting

that

strain

harbours

a

similar

island

on

its

chromosome

Interestingly

the

aerobactin

system

is

missing

in

the

other

three

UPEC

isolates

Furthermore

this

PAI

contains

genes

encoding

the

uropathogenic-associated

P

fimbriae

-LRB-

papIBAHCDJKEFG

-RRB-

The

pap

gene

cluster

of

has

been

sequenced

-LRB-

Klemm

et

al.

-RRB-

the

pap

genes

are

all

present

and

show

sequence

homology

with

the

corresponding

genes

in

The

results

of

the

microarray

analysis

corresponded

very

well

to

the

observed

sequence

homology

of

the

different

genes

in

the

cluster

-LRB-

i.e.

if

a

specific

gene

on

the

microarray

is

represented

with

probes

that

contain

a

non-homologous

region

compared

with

the

corresponding

gene

in

the

hybridised

sample

that

gene

will

not

hybridise

and

will

be

filtered

absent

-RRB-

the

six

genes

with

highest

sequence

homology

were

filtered

present

-LRB-

i.e.

papHCDJKF

with

and

homology

respectively

-RRB-

and

the

four

with

least

sequence

homology

were

filtered

absent

-LRB-

i.e.

papIAEG

with

and

homology

-RRB-

Fig.

atlas

comparing

the

absent

-LRB-

-RRB-

and

present

-LRB-

-RRB-

genes

in

strain

with

other

sequenced

E.

coli

and

Shigella

strains

including

the

three

sequenced

UPEC

isolates

and

The

UPEC

genome

is

used

as

reference

The

outer

blue

circle

represents

the

calculated

absence/presence

in

followed

by

the

three

UPEC

isolates

the

six

inner

circles

represent

Shigella

strains

The

seven

PAIs

of

are

indicated

in

red

The

blow-up

shows

the

presence/absence

of

the

fim

cluster

-LRB-

-RRB-

in

strain

The

employed

microarray

contains

probes

for

all

ten

known

and

putative

fimbriae-encoding

gene

clusters

in

Together

with

the

pap

cluster

two

other

fimbrial

clusters

that

have

been

associated

with

UPEC

virulence

are

known

to

be

present

in

strain

and

have

been

sequenced

i.e.

the

fim

and

sfa/foc

clusters

As

for

the

pap

cluster

the

filtering

of

absent

genes

corresponded

very

well

to

the

actual

presence

and

sequence

homology

of

the

genes

strain

contains

a

large

deletion

in

the

fim

cluster

but

shows

high

sequence

homology

with

the

present

genes

and

all

the

genes

in

the

deleted

part

of

the

cluster

i.e.

fimEAIC

were

filtered

absent

-LRB-

see

blow-up

in

Fig.

-RRB-

Also

the

sfa/foc

cluster

in

shows

high

homology

with

that

in

-LRB-

-RRB-

and

eight

of

nine

genes

were

filtered

present

the

putative

regulatory

gene

sfaC

was

filtered

absent

Regarding

the

other

fimbrial

clusters

present

on

the

microarray

of

the

genes

encoding

fimbriae

which

appear

to

be

common

in

UPEC

and

plays

a

role

in

biofilm

formation

-LRB-

Ulett

et

al.

-RRB-

and

another

putative

fimbriae

-LRB-

yehABCD

-RRB-

were

expressed

and

might

be

absent

in

strain

-LRB-

Table

-RRB-

Table

of

fimbriae-encoding

genes

in

strain

numberGenesNo

of

genesNo

-LRB-

-RRB-

of

absent

genesAbsentPutative

chaperone-usher

fimbrial

-LRB-

-RRB-

ecpD

-LRB-

-RRB-

-LRB-

-RRB-

AllPutative

chaperone-usher

fimbrial

-LRB-

-RRB-

AllPutative

chaperone-usher

fimbrial

-LRB-

-RRB-

yfcQRSUVP

-LRB-

-RRB-

papIAEGbPutative

chaperone-usher

fimbrial

-LRB-

-RRB-

ygiLAuf

-LRB-

-RRB-

aufBCDEFGP

fimbriae

-LRB-

-RRB-

-LRB-

papAD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

fimEAICaFimbrial

operons

present

on

PAIs

The

two

pap

clusters

share

the

same

probes

on

the

array

with

exception

for

papA

and

papD

which

are

represented

by

two

separate

probe

sets

eachbNone

of

these

pap

genes

are

absent

in

strain

but

papIAEG

were

filtered

absent

in

the

microarray

analysis

due

to

non-homologous

sequence

regions

compared

with

the

pap

probes

present

on

the

array

Presence

of

other

pathogenicity

islands

in

Strain

seems

to

carry

most

of

the

pathogenicity

islands

of

-LRB-

or

PAIs

similar

to

the

ones

in

-RRB-

according

to

our

present/absent

analysis

-LRB-

Table

-RRB-

The

only

PAI

of

in

which

most

genes

-LRB-

i.e.

-RRB-

were

filtered

absent

in

strain

is

PAI-pheU

-LRB-

PAI

-RRB-

the

island

that

contains

a

second

pap

cluster

The

three

genes

filtered

present

in

this

PAI

are

present

in

several

of

the

other

sequenced

E.

coli

and

Shigella

strains

indicating

that

these

three

genes

not

are

unique/characteristic

for

this

island

wherefore

this

PAI

is

most

probably

absent

in

strain

Table

of

presence

of

pathogenicity

islands

in

strain

nameCommon

namec

numberNo

of

genesaAbsent

-LRB-

-RRB-

Virulence-associated

III

-LRB-

-RRB-

cdiA

-LRB-

-RRB-

picU

-LRB-

-RRB-

-LRB-

-RRB-

mchBCDEF

-LRB-

-RRB-

sfa/foc

-LRB-

-RRB-

iroNEDCB

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

sitDCBA

-LRB-

-RRB-

-LRB-

-RRB-

fyuA

-LRB-

-RRB-

-LRB-

-RRB-

hcp

-LRB-

-RRB-

clpB

-LRB-

-RRB-

I

-LRB-

-RRB-

hlyA

-LRB-

-RRB-

pap

-LRB-

-RRB-

iha

-LRB-

-RRB-

sat

-LRB-

-RRB-

iutA

iucDCBA

-LRB-

-RRB-

-LRB-

-RRB-

kpsTM

-LRB-

-RRB-

II

-LRB-

-RRB-

-LRB-

-RRB-

aNo

of

genes

in

the

PAI

that

were

present

on

the

array

with

unique

probes

-LRB-

i.e.

genes

that

are

not

orthologues

to

any

other

E.

coli

transcripts

present

on

the

array

-RRB-

bBoldface

indicates

genes

filtered

present

in

strain

Insertion

of

the

high

pathogenicity

island

-LRB-

HPI

-RRB-

of

Yersinia

pestis

has

been

suggested

to

be

one

of

the

earliest

events

in

the

evolution

of

extraintestinal

E.

coli

strains

-LRB-

Welch

et

al.

-RRB-

The

genes

of

HPI

encoding

yersiniabactin

-LRB-

Ybt

-RRB-

were

all

expressed

in

strain

The

HPI

genes

have

been

found

up-regulated

during

urine

biofilm

growth

of

indicating

that

Ybt-mediated

iron-uptake

might

play

an

important

role

in

biofilm

growth

-LRB-

Hancock

and

Klemm

-RRB-

and

a

deletion

mutant

in

the

Ybt

uptake

receptor

-LRB-

FyuA

-RRB-

exhibits

reduced

biofilm

formation

-LRB-

Hancock

et

al.

-RRB-

The

HPI

genes

have

also

been

found

up-regulated

in

vivo

in

two

of

the

three

patients

-LRB-

particularly

in

see

Fig.

-RRB-

infected

with

this

strain

-LRB-

Roos

and

Klemm

-RRB-

The

pks

island

a

recently

characterised

and

widely

spread

genomic

island

found

in

for

example

meningitis

strains

and

the

uropathogenic

strain

encodes

a

machinery

for

the

synthesis

of

peptide

polyketides

hybrid

compounds

-LRB-

Nougayrede

et

al.

-RRB-

The

presence

of

the

island

is

associated

with

the

accumulation

of

double-strand

DNA

breaks

in

host

cells

and

has

genotoxic

activity

-LRB-

Nougayrede

et

al.

-RRB-

This

island

was

expressed

in

strain

and

up-regulated

in

urine

and

in

vivo

-LRB-

Table

Fig.

-RRB-

The

pks

island

is

widely

distributed

within

E.

coli

phylogenetic

group

and

has

been

found

in

both

pathogenic

and

commensal

isolates

in

commensal

strains

the

cell-cycle-blocking

activity

might

slow

the

turnover

of

the

intestinal

epithelium

and

therefore

prolong

colonisation

Presence

of

positively

selected

UPEC

genes

A

recent

paper

comparing

the

UPEC

isolates

and

with

six

other

finished

E.

coli

genome

sequences

presented

genes

that

are

under

positive

selection

only

in

UPEC

strains

-LRB-

Chen

et

al.

-RRB-

These

genes

are

involved

in

various

aspects

of

cell

surface

structure

DNA

metabolism

nutrient

acquisition

and

UTI

Of

these

genes

were

filtered

present

in

our

ABU

strain

many

of

these

genes

are

represented

by

more

than

one

transcript

on

the

array

due

to

sequence

differences

among

the

four

strains

present

on

the

array

in

all

cases

the

gene

filtered

present

in

corresponded

to

the

transcript

Four

genes

were

filtered

absent

agaI

yjiL

recC

and

yegO

they

encode

a

putative

isomerase

a

hypothetical

protein

exodeoxyribonuclease

V

gamma

subunit

and

a

hypothetical

transport

protein

respectively

The

genes

in

the

two

COG

categories

that

were

significantly

enriched

in

the

two

UPEC

strains

i.e.

cell

wall/membrane

biogenesis

-LRB-

amiA

cutE

fepE

ompC

ompF

and

yfaL

-RRB-

and

secondary

metabolites

biosynthesis

transport

and

metabolism

-LRB-

entD

entF

and

yojI

-RRB-

-LRB-

Chen

et

al.

-RRB-

were

all

present

in

strain

Functional

analysis

of

transcripts

of

ABU

E.

coli

To

gain

more

information

concerning

what

type

of

genes

were

absent

the

genes

were

grouped

into

functional

categories

defined

by

the

clusters

of

orthologous

groups

-LRB-

COGs

-RRB-

of

proteins

-LRB-

Tatusov

et

al.

-RRB-

Previous

studies

have

in

attempts

to

identify

essential

genes

and

the

E.

coli

core

genome

found

that

groups

with

genes

involved

in

metabolism

and

various

cellular

processes

-LRB-

excluding

cell

motility

-RRB-

contain

a

substantially

higher

percentage

of

conserved

and

essential

genes

while

COGs

with

genes

of

unknown

function

and

external

origin

as

well

as

genes

involved

in

signalling

and

motility

contain

fewer

essential

genes

-LRB-

Anjum

et

al.

Gerdes

et

al.

-RRB-

Classification

of

the

absent

genes

of

strain

revealed

that

the

groups

cell

motility

defence

mechanisms

and

not

in

COGs

had

a

significant

overrepresentation

of

absent

genes

-LRB-

Table

-RRB-

A

significantly

lower

proportion

of

absent

genes

were

found

in

the

groups

cell

cycle

control

posttranslational

modification

and

translation

This

is

in

agreement

with

a

previously

published

study

of

pathogenic

E.

coli

Anjum

et

al.

-LRB-

-RRB-

studied

strains

of

E.

coli

and

found

that

the

two

groups

with

largest

proportion

of

absent

genes

were

not

in

COGs

and

cell

motility

while

the

six

groups

with

the

lowest

proportion

of

absent

genes

were

translation

cell

division

posttranslational

modification

coenzyme

metabolism

nucleotide

transport

and

metabolism

and

energy

production

and

conversion

which

all

with

exception

for

the

last

group

contained

significantly

fewer

absent

genes

in

strain

-LRB-

Table

-RRB-

This

suggests

that

strain

utilises

a

similar

set

of

core

genes

as

other

E.

coli

strains

Table

of

absent

genes

in

functional

categoriesFunctional

categoryAbsentTotalZ

testNo

P

valueAmino

acid

transport

and

Carbohydrate

transport

and

Cell

cycle

control

cell

division

and

chromosome

Cell

Cell

wall/membrane/envelope

Coenzyme

transport

and

Defense

Energy

production

and

Function

General

function

prediction

Inorganic

ion

transport

and

Intracellular

trafficking

secretion

and

vesicular

Lipid

transport

and

Nucleotide

transport

and

Posttranslational

modification

protein

turnover

Replication

recombination

and

Secondary

metabolites

biosynthesis

transport

and

Signal

transduction

Translation

ribosomal

structure

and

Not

in

genes

absent

in

strain

There

were

genes

that

could

not

be

detected

according

to

our

expression

profiling

in

ABU

strain

of

these

genes

are

exclusively

found

in

i.e.

not

present

in

the

other

three

strains

represented

on

the

array

The

majority

genes

corresponded

to

hypothetical

putative

or

unknown

proteins

Considering

the

very

different

patient

symptom

profiles

of

strains

and

-LRB-

one

being

a

true

pathogen

while

the

latter

is

a

commensal-like

strain

-RRB-

genes

that

are

present

in

UPEC

isolate

but

not

expressed

in

ABU

strain

can

be

considered

as

virulence

factor

candidates

However

most

genes

associated

with

UPEC

pathogenesis

were

expressed

in

strain

and

up-regulated

during

growth

in

urine

e.g.

all

iron-related

genes

encoding

uptake

and

transport

of

aerobactin

salmochelin

yersiniabactin

and

haem/haemoglobin

-LRB-

Table

-RRB-

Two

exceptions

were

the

ireA

gene

encoding

an

iron-regulated

outer-membrane

protein

that

was

filtered

absent

as

well

as

the

tsx

gene

encoding

a

nucleoside-binding

outer-membrane

protein

Although

the

tsx

gene

has

not

previously

been

associated

with

UPEC

virulence

it

has

just

recently

been

identified

together

with

more

well-known

UPEC

genes

as

involved

in

movement

from

the

intestinal

tract

to

the

bladder

and

vagina

-LRB-

i.e.

occurred

significantly

more

often

in

multiple-site

isolates

than

in

rectal

site-only

isolates

-RRB-

-LRB-

Xie

et

al.

-RRB-

furthermore

Tsx

was

also

recently

identified

together

with

other

outer-membrane

proteins

from

cells

grown

under

conditions

mimicking

the

urinary

tract

-LRB-

Hagan

and

Mobley

-RRB-

Table

of

ABU

isolate

compared

with

UPEC

isolates

and

in

U

BF

PatEnt

U

BF

PatFep

Pl

U

BF

PatFeo

BF

PatFhu

Pl

U

BF

PatIro

U

PatIuc

Pl

U

BF

PatIutA

U

BF

PatSit

U

BF

PatFyuA

U

BF

PatIha

U

PatIreA

Pks

island

U

PatRfaH

BFd-serine

PatPap

Fim

Foc/sfa

Vat

BF

PatSat

UTsx

Biofilm

aBoldface

indicates

genes

that

were

filtered

absent

in

strain

in

urine

-LRB-

U

-RRB-

biofilm

-LRB-

BF

-RRB-

plates

-LRB-

Pl

-RRB-

and

patients

-LRB-

Pat

-RRB-

compared

with

MOPS

minimal

mediumcUndefined

Extensive

electron

microscopy

analysis

of

the

strain

has

never

reported

any

capsule

Type

IV

fimbriae

are

assembled

by

the

type

II

general

secretory

pathway

They

occur

in

a

wide

range

of

species

and

frequently

are

associated

with

diseases

The

ppdD

and

hofBC

genes

-LRB-

-RRB-

which

encode

type

IV

prepilin

and

are

present

in

and

were

filtered

absent

in

strain

genes

present

in

strain

but

not

found

in

other

UPEC

strains

The

majority

of

the

genes

that

are

absent

in

the

other

three

UPEC

isolates

-LRB-

i.e.

and

-RRB-

were

filtered

absent

in

strain

as

well

-LRB-

gaps

in

Fig.

-RRB-

However

there

are

a

few

exceptions

where

a

gene

that

is

not

found

in

any

of

the

other

UPEC

strains

is

filtered

present

in

strain

The

aerobactin

system

belongs

to

one

of

the

exceptions

indicating

that

strain

is

particularly

well

equipped

with

iron

uptake

systems

The

other

exceptions

are

all

but

one

located

on

PAIs

and

they

all

encode

hypothetical

proteins

-LRB-

on

PAI-serX

-RRB-

-LRB-

on

PAI-icdA

-RRB-

-LRB-

on

PAI-metV

-RRB-

-LRB-

on

PAI-pheV

where

the

aerobactin

genes

also

are

found

-RRB-

and

and

are

not

present

in

any

of

the

sequenced

E.

coli

and

Shigella

strains

represented

in

Fig.

indicating

that

some

genes

unique

to

can

be

found

in

strain

as

well

Discussion

Bacterial

genomes

are

under

constant

change

New

genes

are

acquired

by

horizontal

transfer

and

old

ones

are

lost

by

mutations

It

is

generally

believed

that

commensal

E.

coli

can

become

pathogenic

through

the

acquisition

of

novel

genes

encoding

virulence

factors

and

niche-adaptation

factors

-LRB-

Kaper

et

al.

-RRB-

In

contrast

to

organisms

that

have

acquired

genes

for

pathogenesis

E.

coli

is

an

example

of

an

organism

that

has

adapted

to

a

commensal-like

existence

through

gene

deletions

and

point

mutations

Using

primarily

the

as

a

scaffold

we

used

presence/absence

data

from

seven

sets

of

different

gene

expression

profiles

-LRB-

in

total

microarrays

-RRB-

to

model

the

gene

pool

of

strain

Given

the

limitations

of

the

approach

i.e.

genes

not

present

on

the

employed

chip

have

been

ignored

a

substantial

body

of

information

was

gathered

concerning

the

genomic

content

of

the

strain

As

it

turned

out

the

strain

was

highly

similar

to

-LRB-

-RRB-

of

the

genes

found

to

be

expressed

on

the

employed

microarray

by

are

also

found

in

and

genes

on

six

of

the

seven

pathogenicity

islands

of

were

expressed

by

furthermore

genes

not

found

in

any

other

UPEC

isolate

were

expressed

by

An

estimated

genes

are

not

expressed

by

Arguably

in

the

light

of

the

difference

in

patient

symptoms

invoked

by

encounters

with

the

two

strains

this

list

represents

virulence

gene

candidates

Although

strain

seems

to

be

a

deconstructed

uropathogen

and

does

not

provoke

symptoms

in

the

human

host

it

grows

fast

in

urine

and

is

an

excellent

colonizer

of

the

human

bladder

-LRB-

Roos

and

Klemm

Roos

et

al.

Klemm

et

al.

-RRB-

It

can

do

so

because

it

has

kept

a

large

assortment

of

fitness

factors

required

for

this

particular

ecological

niche

Among

the

genes

expressed

under

realistic

environmental

conditions

such

as

in

the

human

bladder

are

candidates

for

fitness

factor

genes

e.g.

the

many

iron

acquisitions

systems

expressed

by

the

strain

and

many

genes

involved

in

sugar

acid

and

amino

acid

metabolism

Interestingly

many

of

the

known

and

putative

virulence

factors

of

the

urinary

tract

are

expressed

by

strain

and

might

therefore

be

considered

as

fitness

factors

rather

than

virulence

factors

these

include

of

positively

selected

UPEC

genes

as

well

as

the

newly

characterised

pks

island

inducing

breaks

in

double-stranded

DNA

in

host

cells

Also

virulence-associated

genes

such

as

cdiA

mchBCDEF

flu

hcp

rfaH

sat

picU

and

vat

were

all

expressed

by

strain

Very

few

of

the

known

or

putative

virulence

factors

were

absent

in

-LRB-

or

not

expressed

by

-RRB-

strain

The

pap

fim

and

foc/sfa

clusters

encoding

UPEC-class

fimbriae

are

dysfunctional

in

strain

and

the

clpB

ireA

and

tsx

genes

were

not

expressed

in

the

ABU

strain

These

stand

out

as

potential

virulence

candidates

together

with

a

number

of

uncharacterised

genes

encoding

hypothetical

proteins

Thus

from

the

analyses

performed

here

we

can

make

predictions

about

several

gene

categories

such

as

potential

virulence

genes

fitness

genes

and

household-class

genes

It

is

also

noteworthy

that

the

information

reported

herein

complements

a

potential

genome

sequence

of

strain

Whole

genome

sequencing

can

identify

the

presence

of

genes

but

is

unable

to

reveal

if

they

are

transcribed

Genes

can

be

silenced

not

only

due

to

lesions

in

the

actual

gene

and

its

promoter

but

also

due

to

mutations

of

genes

encoding

regulatory

factors

The

methodology

employed

in

the

present

work

reveals

the

active

genome

of

strain

ABU

strain

is

closely

related

to

fully

virulent

uropathogenic

strains

All

evidences

suggest

that

the

strain

is

a

deconstructed

pathogen

This

study

dispels

the

commonly

held

idea

that

ABU

strains

are

commensals

that

have

picked

up

niche-adaptation

genes

by

horizontal

gene

transfer

Rather

strain

was

originally

a

true

pathogenic

strain

that

has

lost

whole

or

part

of

operons

that

contribute

to

virulence

O O O O O O B I O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

asymptomatic bacteriuria
global gene expression
microarray
urinary tract infections
virulence factors