Positively

Selected

Codons

in

Immune-Exposed

Loops

of

the

Vaccine

Candidate

of

Haemophilus

influenzae

The

high

levels

of

variation

in

surface

epitopes

can

be

considered

as

an

evolutionary

hallmark

of

immune

selection

New

computational

tools

enable

analysis

of

this

variation

by

identifying

codons

that

exhibit

high

rates

of

amino

acid

changes

relative

to

the

synonymous

substitution

rate

In

the

outer

membrane

protein

of

Haemophilus

influenzae

a

vaccine

candidate

for

nontypeable

strains

we

identified

four

codons

with

this

attribute

in

domains

that

did

not

correspond

to

known

or

assumed

B

and

T-cell

epitopes

of

These

codons

flank

hypervariable

domains

and

do

not

appear

to

be

false

positives

as

judged

from

parsimony

and

maximum

likelihood

analyses

Some

closely

spaced

positively

selected

codons

have

been

previously

considered

part

of

a

transmembrane

domain

which

would

render

this

region

unsuited

for

inclusion

in

a

vaccine

Secondary

structure

analysis

three-dimensional

structural

database

searches

and

homology

modeling

using

FadL

of

E.

coli

as

a

structural

homologue

however

revealed

that

all

positively

selected

codons

are

located

in

or

near

extracellular

looping

domains

The

spacing

and

level

of

diversity

of

these

positively

selected

and

exposed

codons

in

suggest

that

vaccine

targets

based

on

these

and

conserved

flanking

residues

may

provide

broad

coverage

in

H.

influenzae

Introduction

Vaccination

with

Haemophilus

influenzae

type

b

polysaccharide

-LRB-

in

the

form

of

Hib

conjugate

vaccines

-RRB-

has

proven

to

be

highly

effective

in

preventing

invasive

H.

influenzae

disease

and

has

effectively

reduced

serotype

b

in

many

parts

of

the

world

-LRB-

Black

et

al.

-RRB-

Nontypeable

H.

influenzae

strains

however

remain

a

serious

problem

-LRB-

Zeckel

et

al.

-RRB-

These

strains

are

generally

less

invasive

than

their

encapsulated

type

b

counterpart

but

are

a

major

cause

of

ear

infections

-LRB-

otitis

media

-RRB-

and

sinusitis

in

children

They

are

also

associated

with

respiratory

tract

infections

such

as

pneumonia

in

infants

children

and

adults

Ongoing

efforts

to

develop

a

vaccine

against

nontypeable

H.

influenzae

have

mainly

focused

on

immunogenic

surface-exposed

proteins

-LRB-

Bolduc

et

al.

-RRB-

This

approach

clearly

has

potential

as

immune

responses

against

surface

proteins

have

been

shown

to

aid

the

recovery

from

otitis

media

-LRB-

Shurin

et

al.

-RRB-

and

vaccination

with

surface-exposed

domains

of

a

major

outer

membrane

protein

-LRB-

-RRB-

provides

protection

in

animal

models

-LRB-

Bolduc

et

al.

-RRB-

However

this

effect

is

strain-specific

-LRB-

Gonzales

et

al.

-RRB-

and

hence

not

sufficient

to

provide

broad

protection

This

problem

may

be

overcome

by

the

inclusion

of

less

variable

immunogenic

protein

regions

in

the

vaccine

Identification

of

candidate

vaccine

antigens

is

classically

achieved

via

immunization

studies

with

isolated

or

recombinant

antigen

and

via

epitope

mapping

often

in

combination

with

analysis

of

correlates

of

protection

Vaccine

development

is

often

hampered

by

antigenic

diversity

within

the

surface-exposed

regions

This

limits

a

broad

cross-reactivity

of

the

elicited

immune

response

This

holds

also

for

proteins

such

as

in

which

large

conserved

domains

separate

relatively

small

variable

domains

-LRB-

Munson

and

Grass

Chong

et

al.

Bolduc

et

al.

-RRB-

On

the

other

hand

the

genetic

diversity

of

vaccine

candidates

can

be

used

to

choose

variants

for

use

in

immunological

assays

-LRB-

Bolduc

et

al.

-RRB-

Nowadays

instead

of

an

arbitrary

selection

of

vaccine

candidates

novel

and

rigorous

computational

approaches

can

be

used

to

identify

codons

whose

diversity

is

driven

by

the

immune

system

These

codons

are

characterized

by

a

higher

rate

of

nonsynonymous

substitutions

-LRB-

dN

-RRB-

relative

to

the

synonymous

substitution

rate

-LRB-

dS

-RRB-

-LRB-

Yang

et

al.

Fitzpatrick

and

McInerney

-RRB-

The

codons

are

referred

to

as

positively

selected

although

they

may

typically

reflect

immune

selection

-LRB-

Fitzpatrick

and

McInerney

-RRB-

To

avoid

confusion

with

the

results

of

immunological

studies

we

refer

here

to

codons

with

a

higher

rate

of

nonsynonymous

substitution

than

synonymous

substitution

-LRB-

dN

dS

-RRB-

as

positively

selected

Knowledge

of

the

position

of

positively

selected

codons

in

a

protein

would

constitute

an

excellent

starting

point

for

immunization

studies

and

epitope

mapping

not

only

because

of

their

biological

function

but

also

because

the

number

of

variable

sites

of

proteins

that

are

candidates

for

inclusion

in

a

vaccine

can

be

reduced

In

the

present

study

we

identified

codons

that

evolved

more

rapidly

through

nonsynonymous

than

through

synonymous

substitutions

in

a

sample

of

sequences

We

compared

the

location

of

these

codons

with

the

location

of

peptides

that

were

used

in

epitope

mapping

and

with

B

and

T-cell

antigens

to

examine

the

congruence

among

these

techniques

and

to

identify

regions

that

could

be

important

for

vaccine

design

Finally

we

localized

stretches

with

positively

selected

codons

in

secondary

structures

and

three-dimensional

-LRB-

-RRB-

models

of

Our

computational

approach

led

to

the

identification

of

several

novel

domains

with

positive

selected

codons

within

the

protein

that

may

be

attractive

targets

in

future

vaccine

design

Materials

and

Methods

Evolutionary

Analysis

of

the

Selection

Pressure

on

We

used

the

nr

database

for

BLASTP

searches

under

standard

settings

to

collect

closely

related

sequences

of

H.

influenzae

using

accession

as

a

query

The

GenBank

accession

numbers

and

the

data

set

of

sequences

are

provided

as

Supplementary

Information

We

aligned

the

sequences

using

the

standard

alignment

parameters

using

Clustalx

-LRB-

Thompson

et

al.

-RRB-

and

checked

the

amino

acid

alignment

with

DNASP

version

-LRB-

Rozas

et

al.

-RRB-

Codons

comprised

in

insertions

and

deletions

and

in

extremely

variable

stretches

were

removed

from

the

alignment

and

are

referred

to

in

this

work

as

hypervariable

domains

A

phylogenetic

tree

was

reconstructed

using

PAUP

-LRB-

Swofford

-RRB-

with

the

maximum

likelihood

algorithm

-LRB-

random

additions

of

taxa

TBR

branch

swapping

-RRB-

under

the

optimal

nucleotide

substitution

model

based

on

modeltest

version

-LRB-

Posada

and

Crandall

-RRB-

Bootstrapping

based

on

maximum

likelihood

was

used

to

assess

support

for

internodes

using

random

additions

SPR

branch

swapping

steepest

descent

chuckscore

and

nchuck

We

used

the

alignment

and

tree

topology

of

one

of

the

maximum

likelihood

-LRB-

ML

-RRB-

trees

as

input

for

PAML

-LRB-

Yang

-RRB-

This

program

implements

the

currently

most

widely

used

algorithms

to

detect

selection

on

codons

which

take

into

account

the

transition

transversion

ratio

and

codon

usage

bias

and

allow

identification

of

codons

that

accumulate

nonsynonymous

mutations

more

rapidly

than

synonymous

ones

To

detect

such

sites

the

rates

of

nonsynonymous

and

synonymous

substitutions

are

determined

through

ML

analyses

using

the

model

of

Goldman

and

Yang

-LRB-

-RRB-

For

immune-exposed

proteins

of

pathogens

a

dN/dS

ratio

indicates

that

nonsynonymous

substitutions

are

driven

by

selection

pressures

exerted

by

the

immune

system

-LRB-

Derrick

et

al.

Jiggins

et

al.

Fitzpatrick

and

McInerney

-RRB-

Because

it

is

most

likely

that

only

a

subset

of

codons

in

any

protein-coding

gene

evolves

under

positive

selection

several

distributions

of

dN/dS

that

reflect

neutral

-LRB-

-RRB-

and

selection

-LRB-

-RRB-

models

can

be

fitted

to

the

data

using

PAML

-LRB-

Yang

-RRB-

The

neutral

models

assume

a

single

estimate

of

ω

-LRB-

ω

-RRB-

and

a

discrete

beta

distribution

of

ω

with

categories

-LRB-

ω

-RRB-

The

dN/dS

ratio

under

the

neutral

models

is

constrained

to

values

-LRB-

Yang

et

al.

Yang

and

Swanson

-RRB-

The

selection

models

and

have

an

additional

class

of

ω

values

that

exceed

unity

This

latter

codon

category

is

believed

to

comprise

codons

that

code

for

amino

acids

that

are

affected

by

the

immune

system

-LRB-

Yang

et

al.

-RRB-

and

that

consequently

evolve

rapidly

at

the

amino

acid

level

The

cross-category

ω

's

under

and

were

estimated

by

averaging

the

ω

estimates

of

individual

ω

classes

weighted

by

their

frequency

We

used

multiple

starting

values

for

the

selection

models

and

because

these

models

may

have

multiple

local

optima

-LRB-

e.g.

Wong

et

al.

-RRB-

The

fit

of

and

to

the

data

was

compared

with

selection

models

-LRB-

and

respectively

-RRB-

The

ratio

of

the

fit

of

neutral

and

selection

models

mentioned

above

follows

a

chi-square

distribution

with

two

degrees

of

freedom

i.e.

for

significance

at

the

significance

level

two

times

the

difference

of

the

likelihood

of

neutral

and

selection

models

should

be

We

examined

the

evidence

for

positive

selection

in

the

entire

alignment

-LRB-

excluding

codons

with

ambiguity

codes

and

gaps

see

above

-RRB-

in

a

data

set

comprising

nontypeable

isolates

and

in

a

data

set

of

sequences

from

which

five

variable

domains

were

deleted

The

latter

data

set

enables

the

detection

of

positively

selected

codons

in

highly

conserved

domains

The

selection

models

may

give

false-positive

results

in

the

case

of

a

poor

fit

of

the

beta

distribution

to

the

true

distribution

of

ω

in

the

interval

between

and

-LRB-

Swanson

et

al.

-RRB-

The

test

advocated

by

Swanson

et

al.

-LRB-

-RRB-

to

guard

against

such

false

positives

of

the

ML

methods

implemented

in

PAML

involves

the

comparison

of

-LRB-

ω2

-RRB-

and

-LRB-

ω2

-RRB-

This

likelihood

ratio

test

-LRB-

LRT

-RRB-

is

asymptotically

distributed

as

a

mixture

of

a

point

mass

at

and

a

chi-square

with

one

degree

of

freedom

which

corresponds

to

units

difference

between

the

likelihoods

of

and

at

the

level

If

the

fit

of

the

selection

models

is

significantly

better

than

that

of

the

corresponding

neutral

models

the

selection

models

can

be

used

to

identify

individual

codons

under

positive

selection

-LRB-

Yang

et

al.

-RRB-

For

inferring

which

sites

are

under

positive

selection

the

Bayes

theorem

was

used

-LRB-

Yang

et

al.

-RRB-

which

assumes

that

given

the

proportion

of

sites

across

ω

classes

as

estimated

from

the

ML

analyses

assigns

priors

to

model

parameters

to

integrate

over

their

uncertainties

The

Bayes

Empirical

Bayes

-LRB-

BEB

-RRB-

approach

used

here

was

specifically

advocated

for

small

data

sets

because

it

takes

into

account

sampling

errors

of

the

proportions

and

ω

ratios

of

site

classes

-LRB-

Yang

et

al.

-RRB-

The

rigorous

identification

of

codons

under

positive

selection

is

an

important

step

when

linking

evolutionary

and

structural

data

and

when

testing

individual

codons

p-values

should

ideally

be

confirmed

by

independent

tests

and

corrected

for

multiple

tests

To

these

ends

we

applied

the

conservative

tests

of

positive

selection

based

on

parsimony

-LRB-

Suzuki

and

Gojobori

-RRB-

implemented

as

the

Single

Likelihood

Ancestor

Counting

-LRB-

SLAC

-RRB-

analysis

in

the

datamonkey

web

server

-LRB-

Kosakovsky

Pond

and

Frost

-RRB-

In

contrast

to

the

original

version

of

the

Suzuki-Gojobori

test

the

modified

version

allows

selection

of

a

nucleotide

substitution

model

We

also

used

a

new

ML

method

developed

by

Massingham

and

Goldman

-LRB-

-RRB-

which

does

not

assume

an

underlying

distribution

of

ω

Instead

their

sitewise

likelihood

ratio

-LRB-

SLR

-RRB-

procedure

which

uses

the

same

codon

substitution

model

as

in

Goldman

and

Yang

-LRB-

-RRB-

performs

a

LRT

on

a

sitewise

basis

testing

the

model

-LRB-

neutrality

ω

-RRB-

against

an

alternative

model

-LRB-

ω

-RRB-

In

the

SLR

method

tree

topology

branch

lengths

equilibrium

codon

frequencies

and

transition/transversion

rate

ratio

are

assumed

to

be

common

to

all

sites

in

an

alignment

Under

the

model

all

parameters

except

ω

are

allowed

to

vary

freely

Under

the

alternative

model

all

parameters

vary

freely

The

SLR

method

applies

Hochberg

's

step-up

procedure

to

correct

for

multiple

tests

-LRB-

Massingham

and

Goldman

-RRB-

Both

the

parsimony

method

and

the

SLR

method

also

allow

the

identification

of

negatively

selected

sites

Secondary

Structure

of

Following

the

localization

of

positively

selected

codons

in

the

primary

sequence

we

determined

the

higher-order

structure

of

amino

acid

stretches

in

using

TMHMM

version

-LRB-

Krogh

et

al.

-RRB-

to

determine

whether

stretches

with

positively

selected

codons

take

secondary

structures

typical

of

transmembrane

domains

The

exposition

of

regions

of

was

assessed

by

means

of

the

solvent

accessibility

algorithm

as

implemented

in

Jpred

-LRB-

Cuff

et

al.

-RRB-

We

identified

exposed

residues

using

the

secondary

structure

algorithm

implemented

in

SSPRO

-LRB-

Cheng

et

al.

-RRB-

and

-LRB-

Cuff

and

Barton

-RRB-

These

attributes

may

suggest

whether

regions

with

positively

selected

codons

are

exposed

to

the

immune

system

Tertiary

Structure

of

We

examined

the

structure

of

using

x-ray

crystallography

or

NMR

data

on

structurally

related

proteins

We

used

the

precompiled

Vector

Alignment

Search

Tool

-LRB-

VAST

-RRB-

and

NCBI

's

structural

database

to

identify

closely

related

structural

variants

that

could

serve

as

a

template

for

homology

modeling

of

The

entries

in

VAST

contain

experimentally

verified

information

on

secondary

structure

elements

such

as

type

relative

orientation

and

connectivity

Structurally

related

proteins

can

be

fitted

to

each

other

on

a

residue-by-residue

basis

using

VAST

's

transformation

matrices

and

a

Gibbs

sampling

algorithm

In

addition

alternative

alignments

can

be

generated

which

take

into

account

the

information

in

secondary

structure

thereby

increasing

the

confidence

one

can

have

in

amino

acid

alignments

of

relatively

divergent

proteins

The

VAST

program

was

used

for

one

randomly

chosen

H.

influenzae

sequence

-LRB-

GenBank

accession

no.

of

the

nontypeable

strain

-RRB-

Superimposed

models

of

template

and

target

protein

were

constructed

to

illustrate

the

position

of

gaps

and

insertions

hypervariable

domains

and

positively

selected

codons

As

an

alternative

to

the

identification

of

structural

neighbors

as

implemented

in

VAST

homology

modeling

can

be

used

This

always

starts

by

looking

for

close

relatives

using

standard

PSI-BLAST

or

other

similarity

searches

We

used

homology

modeling

as

implemented

in

-LRB-

Combet

et

al.

-RRB-

and

SWISS-MODEL

-LRB-

Peitsch

Schwede

et

al.

-RRB-

to

assess

the

congruence

with

the

VAST

searches

and

to

collect

additional

data

on

liability

of

portions

of

the

models

of

and

its

structurally

closest

neighbors

The

models

of

were

visualized

using

version

a

free

model

viewing

and

selection

program

To

judge

the

quality

of

the

reconstruction

we

collected

the

percentage

identity

between

template

and

target

the

VAST

P-value

the

score

reported

by

this

program

and

the

squared

root

of

mean

square

deviations

-LRB-

RMSD

-RRB-

of

the

backbone

atoms

between

the

template

and

the

target

proteins

in

angstroms

-LRB-

Å

-RRB-

using

a

sliding

window

approach

To

assess

whether

hypervariable

domains

were

robustly

assigned

to

their

exact

coordinates

in

the

structures

we

also

examined

the

reports

of

loop

reconstructions

by

the

CSP

-LRB-

Constrained

Space

Programming

-RRB-

routine

as

implemented

in

Swiss-Model

-LRB-

Schwede

et

al.

-RRB-

To

further

quantify

the

degree

of

similarity

in

β-strands

between

and

a

structural

template

we

compared

the

distribution

of

amino

acids

maintaining

β-barrels

of

transmembrane

proteins

using

the

TBB-PRED

program

-LRB-

Liu

et

al.

-RRB-

which

uses

atomic

data

to

assign

the

most

likely

amino

acid

stretches

that

contribute

to

β-barrels

We

used

the

Support

Vector

Machines

-LRB-

SVM

-RRB-

algorithm

to

determine

the

location

of

β-barrels

Results

Alignment

of

Sequences

BLASTP

searches

used

cutoffs

of

-LRB-

E-value

-RRB-

and

-LRB-

score

-RRB-

to

identify

closely

related

sequences

Thirty-six

hits

were

obtained

including

protein-coding

sequences

-LRB-

nontypeable

a

b

e

and

f

-RRB-

that

have

previously

been

analyzed

as

part

of

a

population

genetics

study

of

-LRB-

Bolduc

et

al.

six

sequences

were

left

out

because

they

were

not

among

the

top

hits

of

the

BLASTP

search

-RRB-

The

average

pairwise

amino

acid

similarity

in

the

entire

data

set

was

The

sequences

of

the

nontypeable

and

typeable

protein

variants

comprised

three

hypervariable

portions

with

many

insertions

and

deletions

-LRB-

nucleotides

plus

and

plus

-RRB-

and

a

few

codons

with

ambiguity

codes

or

gaps

-LRB-

and

-RRB-

In

addition

codons

missed

from

the

end

of

the

gene

of

isolate

leading

to

a

total

of

codons

that

were

deleted

to

ensure

a

correct

alignment

The

amino

acid

and

the

nucleotide

alignments

of

the

five

most

variable

domains

that

remained

after

removal

of

the

codons

mentioned

above

can

be

viewed

as

Supplementary

Information

The

nucleotide

alignment

revealed

that

many

of

the

underlying

codons

in

the

variable

domain

frequently

shared

nucleotides

at

two

of

three

positions

indicating

that

the

alignment

of

these

regions

was

correct

Identification

of

Positively

Selected

Codons

The

computational

approaches

implemented

in

PAML

version

-LRB-

Yang

-RRB-

determine

which

if

any

codons

may

have

evolved

under

positive

selection

On

the

basis

of

a

single

phylogenetic

estimate

using

the

preferred

nucleotide

substitution

model

derived

from

MODELTEST

-LRB-

the

transversion

model

TVM

which

has

variable

base

frequencies

variable

transversion

rates

and

equal

transition

rates

-RRB-

with

a

proportion

of

invariant

sites

-LRB-

-RRB-

and

gamma

distributed

substitution

rates

-LRB-

shape

parameter

-RRB-

we

found

ML

trees

one

of

which

is

shown

in

Fig.

-LRB-

ML

-RRB-

Bootstrapping

indicated

that

four

branches

received

support

-LRB-

Fig.

-RRB-

Support

for

the

other

branches

was

-LRB-

not

shown

in

Fig.

-RRB-

Comparison

of

neutral

and

selection

models

using

the

data

set

and

the

tree

in

Fig.

indicated

that

both

selection

models

-LRB-

likelihood

-RRB-

and

-LRB-

-RRB-

fitted

the

data

significantly

better

than

the

neutral

models

-LRB-

-RRB-

and

-LRB-

-RRB-

respectively

-LRB-

Table

-RRB-

Different

starting

values

of

ω

did

not

lead

to

different

parameter

estimates

after

optimization

-LRB-

not

shown

-RRB-

Both

comparisons

and

exceeded

by

far

the

difference

required

for

significance

at

the

level

-LRB-

see

Materials

and

Methods

-RRB-

This

suggested

that

some

codons

had

a

higher

nonsynonymous

than

synonymous

substitution

rate

Similarly

the

Swanson

test

indicated

significant

evidence

for

the

occurrence

of

positive

selection

under

-LRB-

Table

-RRB-

Both

selection

models

and

suggested

that

the

same

codons

were

under

positive

selection

i.e.

codons

and

and

possibly

also

codon

-LRB-

under

model

-RRB-

The

alignment

-LRB-

Supplementary

Information

-RRB-

further

showed

that

in

this

sample

of

sequences

positively

selected

codons

mostly

flank

hypervariable

domains

Limitation

of

the

analyses

to

the

nontypeable

strains

resulted

in

very

similar

findings

In

these

analyses

both

-LRB-

likelihood

-RRB-

and

-LRB-

-RRB-

fitted

the

data

significantly

better

than

-LRB-

-RRB-

and

-LRB-

-RRB-

respectively

-LRB-

not

shown

-RRB-

Thus

both

model

comparisons

were

again

significant

This

held

also

true

for

the

positively

selected

codons

For

relative

to

the

analyses

in

Table

one

additional

positively

selected

codon

-LRB-

-RRB-

was

found

For

no

additional

codons

were

found

After

deletion

of

the

five

variable

domains

no

evidence

for

positive

selection

was

left

-LRB-

-RRB-

Fig.

Maximum

likelihood

tree

based

on

the

TVM

model

of

nucleotide

substitution

with

a

proportion

of

invariant

sites

and

rate

heterogeneity

-LRB-

see

text

-RRB-

The

error

bar

indicates

the

number

of

substitutions

per

nucleotide

Asterisks

indicate

branches

with

bootstrap

support.Table

Positively

selected

codons

in

OMP

Heamophilus

influenza

according

to

neutral

models

-LRB-

and

-RRB-

and

selection

models

-LRB-

and

-RRB-

LikelihoodTree

lengthKappa

κParametersCodonAmino

acidProbability

positive

selection

-LRB-

BEB

-RRB-

dN/dS

codon

ω0

ω1

ω0

ω1

ω2

B

-LRB-

p

q

-RRB-

B

-LRB-

p

q

-RRB-

ω2

B

-LRB-

p

q

-RRB-

ω2

The

likelihood

indicates

the

fit

of

the

data

to

the

models

of

Yang

-LRB-

-RRB-

Tree

length

is

measured

as

the

number

of

mutations

per

codon

Kappa

is

the

transversion

transition

ratio

pi

denotes

the

proportion

of

sites

falling

in

site

class

ωi

The

parameters

p

and

q

are

the

shape

parameters

of

the

beta

distribution

which

underlies

The

probability

that

codons

were

under

positive

selection

was

determined

using

Bayes

Empirical

Bayes

-LRB-

Yang

et

al.

-RRB-

with

the

proportion

ω

and

standard

error

indicated

per

codon

Model

refers

to

the

model

of

Swanson

et

al.

-LRB-

-RRB-

in

which

ω2

is

fixed

at

one

under

The

reference

sequence

is

The

modified

Suzuki-Gojobori

method

indicated

that

four

sites

evolved

under

positive

selection

-LRB-

substitution

model

codons

-LSB-

P

-RSB-

-LSB-

P

-RSB-

-LSB-

P

-RSB-

and

-LSB-

P

-RSB-

likelihood

tree

length

per

site

dN/dS

over

all

codons

middle

row

in

Fig.

-RRB-

Using

the

SLR

method

we

found

eight

positively

selected

codons

Relative

to

the

results

of

PAML

-LRB-

top

row

in

Fig.

-RRB-

all

but

one

positively

selected

codon

found

by

the

SLR

method

remained

significant

upon

correction

for

multiple

tests

-LRB-

except

for

codon

P

bottom

row

in

Fig.

and

Table

-RRB-

Overall

the

strongest

evidence

for

positive

selection

based

on

the

parsimony

and

ML

methods

was

found

for

residues

and

Fig.

Codons

under

positive

selection

as

inferred

from

the

site

models

in

PAML

-LRB-

top

panel

-RRB-

the

modified

Suzuki-Gojobori

method

implemented

as

the

SLAC

procedure

in

the

datamonkey

server

-LRB-

middle

panel

-RRB-

and

the

sitewise

likelihood

ratio

test

-LRB-

bottom

panel

-RRB-

of

sequences

of

H.

influenzae

Codons

with

significant

evidence

for

positive

selection

have

probabilities

-LRB-

PAML

and

SLR

methods

-RRB-

or

-LRB-

modified

Suzuki-Gojobori

method

-RRB-

The

spacing

between

codons

is

adjusted

for

the

insertions

and

deletions

in

Topographic

Localization

of

the

Amino

Acids

Encoded

by

Putatively

Positively

Selected

Codons

and

Their

Relation

to

Immunological

Data

of

A

priori

positively

selected

codons

in

B-cell

and

T-helper

determinants

in

the

protein

are

suitable

vaccination

targets

because

these

sites

can

be

expected

to

be

highly

immunogenic

and

involved

in

the

clearance

of

variants

The

most

promising

broadly

protective

regions

of

based

on

epitope

mapping

studies

are

mainly

located

outside

the

stretch

of

closely

spaced

positively

selected

codons

-LRB-

Fig.

-RRB-

Peptide-specific

antisera

directed

against

these

regions

did

not

invoke

protective

immunity

although

strong

IgG

and

T-cell

responses

were

obtained

-LRB-

Proulx

et

al.

Chong

et

al.

-RRB-

Only

in

rare

cases

was

targeting

of

the

conserved

regions

associated

with

protection

-LRB-

one

epitope

located

on

peptide

and

recognized

by

MAb

was

protective

-LSB-

Panezutti

et

al.

-RSB-

-RRB-

Furthermore

two

of

five

positively

selected

sites

are

present

in

a

synthetic

peptide

-LRB-

-RRB-

that

has

been

used

in

immunization

studies

-LRB-

Proulx

et

al.

Panezutti

et

al.

Chong

et

al.

-RRB-

The

peptide

which

was

not

identified

as

a

conserved

B-cell

epitope

across

typeable

and

nontypeable

H.

influenzae

isolates

did

not

carry

an

immunodominant

epitope

in

animal

models

Another

peptide

that

was

used

for

epitope

mapping

and

immunological

assays

-LRB-

Proulx

et

al.

Chong

et

al.

-RRB-

fully

overlaps

with

the

stretch

with

positively

selected

amino

acids

This

-LRB-

Chong

et

al.

-RRB-

or

-LRB-

Proulx

et

al.

-RRB-

peptide

covers

amino

acids

of

in

H.

influenzae

serotype

b

strain

MinnA

-LRB-

Munson

and

Grass

-RRB-

which

correspond

to

residues

in

the

reference

sequence

However

antisera

isolated

from

rabbits

guinea

pigs

and

humans

did

not

react

with

-LRB-

Chong

et

al.

-RRB-

and

vice

versa

specific

sera

did

not

recognize

the

intact

protein

Despite

these

negative

immunological

data

our

computational

analysis

suggests

that

the

stretches

with

positively

selected

codons

are

likely

under

immune

selection

in

vivo

It

may

be

worthwhile

to

study

these

stretches

in

more

detail

at

the

immunological

level

Fig.

Diagram

of

different

immunological

and

genetic

attributes

of

of

Haemophilus

influenzae

From

top

to

bottom

linear

B-cell

epitopes

in

different

hosts

-LRB-

murine

guinea

pig

and

rabbit

-RRB-

B-cell

epitopes

conserved

among

H.

influenzae

strains

murine

T-helper

determinants

positively

selected

codons

distribution

of

amino

acids

with

a

potentially

extended

-LRB-

black

-RRB-

or

a

helical

configuration

-LRB-

gray

Jpred

-RRB-

typical

of

α-helices

and

β-sheets

respectively

and

distribution

of

amino

acids

with

solvent

accessibility

-LRB-

-RRB-

location

of

hypervariable

domains

and

location

of

synthetic

peptides

against

which

were

used

for

immunological

studies

-LRB-

Proulx

et

al.

Chong

et

al.

-RRB-

The

approximate

locations

of

peptides

and

are

boxed

and

dotted

respectively

-LRB-

see

text

-RRB-

Five

of

the

nine

codons

for

which

significant

evidence

for

positive

selection

was

obtained

in

both

PAML

selection

models

and

cluster

tightly

in

the

primary

sequence

of

-LRB-

amino

acids

-RRB-

and

four

of

these

are

confirmed

as

positively

selected

sites

using

the

other

types

of

analyses

of

positive

selection

-LRB-

Fig.

-RRB-

Chong

et

al.

-LRB-

-RRB-

found

that

some

of

these

codons

were

part

of

a

transmembrane

domain

based

on

secondary

structure

analysis

hydrophobicity

and

reactivity

with

monoclonal

antibodies

The

nature

of

the

variability

of

the

first

hypervariable

domain

has

also

been

questioned

on

the

basis

of

a

lack

of

hydrophilic

residues

-LRB-

Munson

et

al.

-RRB-

TMHMM

version

was

used

to

determine

whether

stretches

with

positively

selected

codons

involved

a

transmembrane

domain

or

adopted

secondary

structures

typical

of

such

domains

-LRB-

Krogh

et

al.

-RRB-

No

secondary

structures

such

as

α-helices

and

β-sheets

typically

associated

with

transmembrane

domains

were

found

in

this

stretch

-LRB-

not

shown

-RRB-

Investigation

of

the

exposition

of

this

region

using

the

solvent

accessibility

algorithm

as

implemented

in

Jpred

-LRB-

Cuff

et

al.

-RRB-

classified

this

portion

of

as

an

exposed

stretch

with

high

solvent

accessibility

-LRB-

Fig.

-RRB-

which

exceeded

for

the

entire

stretch

with

the

five

positively

selected

codons

Thus

the

positively

selected

codons

are

likely

to

be

exposed

The

above

findings

deviate

from

previous

suggestions

that

these

amino

acids

are

part

of

a

putative

transmembrane

domain

-LRB-

Chong

et

al.

-RRB-

which

would

render

this

stretch

unsuitable

for

vaccine

purposes

Localization

of

the

Amino

Acids

Encoded

by

Putative

Positively

Selected

Codons

as

Assessed

by

Modeling

The

apparent

ambiguity

about

the

putative

transmembrane

domain

of

led

us

to

examine

the

topology

of

the

protein

BLASTP

searches

identified

one

structure

with

high

similarity

to

of

H.

influenzae

the

FadL

outer

membrane

protein

of

E.

coli

-LRB-

similar

in

primary

amino

acid

sequence

-RRB-

This

protein

has

been

analyzed

at

a

resolution

of

and

Å

using

two

x-ray

crystallography

methods

-LRB-

van

den

Berg

et

al.

-RRB-

Apart

from

its

role

in

the

transport

of

long-chain

fatty

acids

across

the

outer

membrane

FadL

is

also

a

receptor

for

the

bacteriophage

and

FadL

are

both

classified

in

which

also

includes

TodX

from

Pseudomonas

putida

and

TbuX

from

Ralstonia

pickettii

The

latter

are

membrane

proteins

of

uncertain

function

that

are

involved

in

toluene

catabolism

The

VAST

database

identified

the

same

structural

neighbors

-LRB-

-RRB-

as

the

BLASTP

search

and

the

homology

modeling

programs

and

SWISS-MODEL

-LRB-

not

shown

-RRB-

There

are

many

indications

that

the

reconstructed

model

of

-LRB-

Fig.

-RRB-

is

reliable

The

percentage

identity

of

the

sequences

to

the

target

based

on

secondary

elements

was

approximately

which

in

principle

may

allow

confident

models

as

evidenced

by

the

lower

boundary

for

which

homology

modeling

is

used

-LRB-

-RRB-

Although

VAST

provides

no

easily

interpretable

confidence

parameters

-LRB-

the

score

of

this

structure

pair

was

using

an

alignment

length

of

residues

and

the

VAST

P-value

was

-RRB-

the

level

of

similarity

and

the

root-mean-square

deviation

-LRB-

RSMD

-RRB-

of

the

fit

across

and

indicate

a

high

degree

of

structural

similarity

-LRB-

RMSD

Å

Fig.

-RRB-

-LRB-

T.

Madej

NCBI

personal

communication

-RRB-

A

large

number

of

secondary

structures

such

as

helices

strands

and

loops

are

shared

between

and

FadL

as

evident

from

VAST

searches

-LRB-

Figs.

and

B

-RRB-

This

interpretation

is

supported

by

the

congruence

among

models

of

secondary

structure

to

assign

coiled

regions

that

are

exposed

as

determined

through

the

use

of

SSPRO

version

-LRB-

not

shown

-RRB-

and

by

the

agreement

of

regions

with

a

β-barrel

structure

between

FadL

and

as

judged

from

the

results

of

the

TBB-PRED

server

based

on

atomic

data

of

β-barrel

proteins

-LRB-

Fig.

-RRB-

Clearly

the

fit

of

the

overall

structure

of

the

reconstructed

model

of

does

not

imply

that

the

model

is

stable

and

robust

for

every

region

of

the

protein

The

hypervariable

domains

introduce

many

differences

in

exposed

portions

of

the

models

between

model

and

target

in

the

superimposed

models

-LRB-

Fig.

bottom

row

-RRB-

Further

it

is

important

to

note

that

only

a

single

sequence

was

used

to

model

the

structure

based

on

FadL

Slightly

different

loops

may

be

obtained

when

using

different

sequences

Not

surprisingly

the

CSP

algorithm

implemented

in

SWISS-MODEL

had

considerable

difficulties

in

finding

and

optimally

position

loops

-LRB-

not

shown

-RRB-

Many

of

the

loops

examined

by

the

program

could

not

be

confidently

reconstructed

and

in

many

cases

the

accepted

loops

-LRB-

by

the

Spare

Part

procedure

-RRB-

were

found

only

after

shorter

ones

had

not

been

accepted

These

regions

in

many

instances

corresponded

to

hypervariable

and

looping

regions

-LRB-

Fig.

-RRB-

that

had

much

higher

RMSD

values

than

other

regions

of

the

molecule

However

as

judged

from

the

secondary

structure

analyses

these

regions

likely

do

not

contribute

much

to

the

overall

stability

of

the

β-barrel

structure

of

-LRB-

Fig.

-RRB-

Fig.

model

of

of

H.

influenzae

-LRB-

A

-RRB-

FadL

of

E.

coli

-LRB-

B

-RRB-

and

superimposed

models

of

both

proteins

-LRB-

C

-RRB-

Brown

arrows

and

threads

mark

strands

yellow

threads

mark

regions

with

positively

selected

codons

green

regions

mark

helical

regions

and

blue

regions

mark

coiled

regions

E

extracellular

side

M

membrane

P

periplasm

In

the

superimposed

models

a

splitting

of

the

two

models

indicates

gaps

or

insertions.Fig

Alignment

and

structural

analysis

of

OMP

of

H.

influenzae

and

FadL

of

E.

coli

based

on

the

VAST

structural

database

The

deviation

-LRB-

Å

-RRB-

of

the

model

of

OMP

relative

to

the

template

FadL

is

shown

for

windows

of

five

amino

acids

-LRB-

above

alignment

-RRB-

The

contribution

of

individual

residues

to

the

β-barrel

structure

of

OMP

as

analyzed

with

TBB-PRED

is

marked

by

b

-LRB-

barrel

-RRB-

and

n

-LRB-

nonbarrel

-RRB-

Asterisks

above

the

alignment

mark

conserved

amino

acid

positions

asterisks

below

the

alignment

mark

positively

selected

amino

acid

positions

The

alignment

of

OMP

starts

at

residue

The

most

relevant

conclusion

of

the

reconstruction

was

that

the

regions

that

comprised

positively

selected

codons

-LRB-

yellow

in

Fig.

-RRB-

were

always

on

the

extracellular

side

of

the

membrane

indicating

that

the

positively

selected

codons

are

potential

candidates

for

vaccine

development

The

positions

of

insertions

and

deletions

between

FadL

and

that

are

longer

than

a

few

amino

acids

occurred

in

agreement

with

expectation

mostly

in

these

exposed

regions

of

Looping

regions

with

insertions

and

deletions

are

indicated

by

the

splitting

of

the

two

molecules

in

superimposed

models

-LRB-

Fig.

-RRB-

Overall

the

superimposed

models

agree

with

the

models

based

on

individual

structures

in

that

as

expected

for

a

β-barrel

protein

particularly

the

main

chain

hydrogen-bonding

patterns

characteristic

of

strands

contribute

to

the

structural

maintenance

of

Discussion

Positive

Selection

on

Identification

of

positive

selection

on

OMPs

is

generally

hampered

by

extremely

high

levels

of

sequence

divergence

and

the

frequent

occurrence

of

insertions

and

deletions

in

hot

spots

These

domains

are

however

potentially

interesting

for

vaccine

studies

as

they

may

be

the

targets

of

immune

selection

-LRB-

Smith

et

al.

-RRB-

To

enable

identification

of

positively

selected

codons

we

focused

on

codons

directly

adjacent

to

hypervariable

and

unalignable

regions

-LRB-

cf.

Supplementary

Information

-RRB-

The

hypervariable

regions

were

excluded

as

it

is

simply

not

possible

to

identify

homologous

nucleotide

positions

for

these

domains

Our

strategy

to

detect

positively

selected

codons

is

still

feasible

as

the

inference

of

positive

selection

is

not

very

sensitive

to

the

use

of

slightly

different

trees

resulting

from

for

example

the

deletion

of

hypervariable

codons

if

sufficient

phylogenetic

signal

remains

-LRB-

Derrick

et

al.

-RRB-

Our

results

indeed

indicate

that

after

deletion

of

the

hypervariable

regions

a

substantial

number

of

substitutions

remains

for

-LRB-

see

Supplementary

Information

-RRB-

which

allows

the

identification

of

positively

selected

codons

Another

important

issue

to

consider

in

this

type

of

study

is

how

recombination

affects

the

inferences

of

positive

selection

Recombination

generates

different

tree

topologies

when

based

on

different

regions

of

a

gene

and

it

has

been

shown

that

depending

on

the

level

of

recombination

the

LRT

may

be

affected

-LRB-

Anisimova

et

al.

-RRB-

In

general

recombination

is

considered

most

problematic

for

analysis

of

selection

pressures

using

divergent

sequence

data

-LRB-

cf.

viral

sequence

data

-LSB-

Anisimova

and

Yang

-RSB-

-RRB-

but

it

has

generally

been

difficult

to

determine

its

quantitative

importance

Simulation

studies

provide

information

on

the

circumstances

in

which

recombination

may

affect

evolutionary

analysis

of

selection

pressures

For

example

in

spite

of

the

congruent

results

between

and

in

this

study

these

models

are

not

equally

affected

by

recombination

-LRB-

Anisimova

et

al.

-RRB-

In

addition

the

Bayesian

analysis

that

was

used

to

identify

positively

selected

residues

is

less

affected

by

the

presence

of

recombination

than

the

LRTs

presumably

because

it

does

not

depend

strongly

on

the

entire

gene

tree

topology

but

instead

reconstructs

the

numbers

of

synonymous

and

nonsynonymous

mutations

of

individual

codons

Finally

the

proportion

of

correctly

classified

sites

for

the

BEB

procedure

in

simulations

under

positive

selection

and

recombination

increases

with

selection

pressure

-LRB-

Anisimova

et

al.

-RRB-

The

higher

ω

values

of

positively

selected

codons

in

this

study

-LRB-

Table

-RRB-

relative

to

those

of

the

simulation

study

mentioned

above

-LRB-

ω2

-RRB-

and

the

large

number

of

positively

selected

sites

in

suggest

that

the

inference

of

positive

selection

and

sites

under

positive

selection

is

valid

Use

and

Value

of

Reconstruction

of

The

alternation

of

relatively

conserved

and

hypervariable

stretches

in

and

other

OMPs

also

impacts

the

reconstruction

Hypervariable

amino

acid

stretches

may

render

a

structure

unstable

despite

the

fact

that

more

conserved

parts

may

share

considerable

similarity

between

target

and

template

Thus

although

the

level

of

congruence

between

the

experimental

structure

of

FadL

and

the

computational

structure

of

may

vary

considerably

the

most

probable

and

approximate

location

of

stretches

with

positively

selected

codons

can

be

determined

if

the

conserved

portions

of

can

be

confidently

reconstructed

With

this

goal

in

mind

the

identification

of

β-barrel

regions

and

the

degree

of

fit

of

stranded

regions

between

model

and

template

are

most

important

These

attributes

leave

little

doubt

that

the

fit

between

FadL

and

is

sufficient

to

pinpoint

the

approximate

localization

of

positively

selected

codons

-LRB-

Fig.

-RRB-

This

is

not

surprising

as

more

divergent

structural

templates

and

targets

such

as

OmpF

and

PhoE

of

E.

coli

have

successfully

been

used

for

the

reconstruction

of

models

of

porins

in

Neisseria

-LRB-

Derrick

et

al.

-RRB-

In

sum

there

is

every

reason

to

believe

that

the

basic

barrel

structure

is

not

profoundly

affected

by

hypervariable

loops

even

if

these

experience

frequent

insertions

and

deletions

that

are

potentially

also

under

positive

selection

-LRB-

Smith

et

al.

Derrick

et

al.

-RRB-

Are

Positively

Selected

Sites

Useful

for

Vaccine

Formulations

The

question

arises

why

the

peptides

that

partially

-LRB-

-RRB-

and

fully

-LRB-

-RRB-

overlapped

with

the

stretch

of

positively

selected

sites

did

not

include

immunodominant

epitopes

-LRB-

Proulx

et

al.

Panezutti

et

al.

Chong

et

al.

-RRB-

The

stretches

however

were

very

long

-LRB-

and

amino

acids

respectively

-RRB-

and

they

extended

well

into

the

interior

of

-LRB-

not

shown

-RRB-

The

potentially

most

interesting

peptide

-LRB-

-RRB-

comprises

the

entire

hypervariable

domain

of

the

Eagan

strain

-LRB-

Chong

et

al.

-RRB-

including

residue

-LRB-

SASQRNV

is

the

C-terminus

Supplementary

Information

-RRB-

Our

analyses

suggest

that

relatively

short

peptides

starting

at

the

C-terminal

end

of

the

first

hypervariable

domain

would

be

suited

for

immunological

experiments

This

portion

of

has

not

yet

been

explored

Interestingly

one

of

the

positively

selected

residues

in

that

flank

the

first

hypervariable

domain

allows

examination

of

the

impact

and

prospect

of

positively

selected

residues

for

vaccine

formulation

in

a

considerable

fraction

of

the

H.

influenzae

population

The

positively

selected

codons

and

are

flanked

on

one

side

by

the

first

hypervariable

region

and

are

separated

from

codon

by

a

conserved

amino

acid

stretch

-LRB-

see

Supplementary

Information

-RRB-

The

latter

residue

in

turn

is

flanked

by

a

long

region

at

the

C-terminal

side

that

is

conserved

in

the

collection

of

H.

influenzae

strains

examined

by

Bolduc

et

al.

-LRB-

-RRB-

In

our

study

sample

only

three

amino

acids

are

encoded

by

residue

and

this

number

increases

only

marginally

in

the

larger

set

of

H.

influenzae

isolates

-LRB-

Bolduc

et

al.

-RRB-

Consequently

judging

from

the

spacing

of

positively

selected

and

conserved

residues

there

are

opportunities

to

combine

sites

under

purifying

and

positive

selection

in

immunological

experiments

of

If

it

can

be

demonstrated

by

for

example

epitope

mapping

that

these

positively

selected

and

exposed

residues

of

are

located

in

immunologically

relevant

domains

a

large

portion

of

the

H.

influenzae

population

can

be

targeted

by

short

peptides

from

this

region

of

Relative

to

traditional

approaches

to

vaccine

development

this

strategy

may

allow

a

much

broader

coverage

of

the

H.

influenzae

population

Another

question

is

to

what

extent

the

isolates

studied

here

reflect

the

evolutionary

diversity

of

H.

influenzae

The

isolates

analyzed

by

Bolduc

et

al.

-LRB-

-RRB-

were

selected

for

sequencing

because

they

were

phylogenetically

diverse

in

a

collection

of

more

than

H.

influenzae

isolates

Given

the

increase

in

multilocus

sequence

typing

-LRB-

MLST

-RRB-

of

human

pathogenic

bacteria

it

is

also

of

interest

to

compare

the

diversity

of

the

strains

of

Bolduc

et

al.

-LRB-

-RRB-

with

the

phylogenetic

relationships

based

on

housekeeping

genes

in

the

MLST

database

of

H.

influenzae

In

the

case

of

H.

influenzae

seven

housekeeping

genes

are

widely

used

to

characterize

phylogenetic

relationships

In

a

housekeeping

tree

of

isolates

-LRB-

Meats

et

al.

-RRB-

most

of

the

nontypeable

isolates

were

comprised

of

two

highly

divergent

clusters

and

in

contrast

to

the

housekeeping

genes

of

encapsulated

isolates

the

congruence

among

trees

based

on

single

housekeeping

genes

of

nontypeable

isolates

was

only

marginal

The

current

view

of

H.

influenzae

evolution

is

that

nontypeable

isolates

are

genetically

distinct

from

encapsulated

variants

and

that

nontypeable

isolates

form

distinct

and

diverse

populations

Among

the

five

strains

that

were

used

by

both

Bolduc

et

al.

-LRB-

-RRB-

and

Meats

et

al.

-LRB-

-RRB-

the

shared

isolates

-LRB-

d-Rd

and

-RRB-

were

scattered

over

the

MLST

tree

Apart

from

issues

of

sampling

the

congruence

of

gene

tree

topologies

based

on

different

housekeeping

genes

and

hinges

on

the

level

of

recombination

between

genes

It

is

currently

unknown

how

important

recombination

is

in

H.

influenzae

Although

information

on

the

clinical

symptoms

caused

by

the

isolates

with

sequences

is

extremely

limited

strains

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

caused

middle

ear

infection

pneumonia

and

nasopharyngeal

infection

in

children

In

spite

of

the

limited

amount

of

information

on

the

nontypeable

isolates

with

sequences

the

intermingling

of

nontypeable

and

typeable

isolates

on

the

housekeeping

tree

and

the

clinical

symptoms

of

some

of

the

nontypeable

isolates

suggest

that

in

principle

the

diversity

studied

here

might

be

a

suited

starting

point

for

vaccine

development

with

the

potential

of

providing

broad

coverage

Recent

technological

progress

in

vaccinology

offers

hope

that

epitopes

based

on

moderately

rapidly

evolving

portions

of

OMPs

may

turn

out

to

be

realistic

vaccine

material

Although

it

can

not

be

excluded

that

some

epitopes

will

not

be

stable

or

immunogenic

or

may

not

constitute

stable

constructs

extracellular

loops

in

OMPs

have

been

demonstrated

to

be

immunodominant

-LRB-

Easton

et

al.

-RRB-

Also

the

inclusion

of

multiple

OMPs

in

vaccine

formulations

is

possible

by

using

outer

membrane

vesicles

or

whole-cell

vaccines

based

on

recombinant

bacteria

-LRB-

de

Jonge

et

al.

-RRB-

and

the

delivery

of

antigens

based

on

OMPs

of

nontypeable

H.

influenzae

based

on

fusion

proteins

-LRB-

Riedmann

et

al.

-RRB-

offer

opportunities

to

target

interesting

vaccine

candidate

regions

of

OMPs

Finally

whole-cell

vaccines

of

LPS

mutants

with

strongly

reduced

toxicity

are

increasingly

suited

for

vaccination

purposes

-LRB-

Fisseha

et

al.

-RRB-

thereby

reducing

the

adverse

effects

of

immune

responses

against

these

types

of

vaccines

Impact

of

Computational

Analyses

on

Vaccine

Development

Positive

selection

on

codons

of

exposed

outer

membrane

proteins

of

pathogenic

viruses

and

bacteria

is

common

-LRB-

Urwin

et

al.

Yang

et

al.

Yang

Fitzpatrick

and

McInerney

-RRB-

The

lack

of

correspondence

between

interesting

sites

in

of

H.

influenzae

in

immunological

and

genetic

diversity

studies

highlights

the

difficulty

of

finding

the

most

promising

epitopes

even

among

closely

related

sequences

The

a

priori

identification

of

positively

selected

codons

will

normally

greatly

reduce

the

number

of

peptides

that

need

to

be

screened

using

epitope

mapping

or

immunization

Because

the

incorporation

of

antigenic

diversity

in

the

experimental

setup

of

immunological

and

protection

studies

is

a

major

bottleneck

in

terms

of

time

and

costs

the

identification

of

a

limited

number

of

positively

selected

codons

suggests

that

a

multidisciplinary

approach

comprising

evolutionary

and

structural

data

and

tools

can

greatly

improve

the

focus

of

ensuing

empirical

studies

Electronic

Supplementary

Material

Supplementary

material

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

haemophilus influenzae
immune selection
epitope
vaccine

outer membrane proteini
structural information


Microbiological evaluation of a new growth-based approach for rapid detection of methicillin-resistant Staphylococcus aureus Objectives Recently , a rapid screening tool for methicillin-resistant Staphylococcus aureus -LRB- MRSA -RRB- has been introduced that applies a novel detection technology allowing the rapid presence or absence of MRSA to be determined from an enrichment broth after only a few hours of incubation . To evaluate the reliability of this new assay to successfully detect MRSA strains of different origin and clonality , well-characterized S. aureus strains were tested in this study . Introduction The increasing numbers of multidrug-resistant Gram-positive pathogens have generated worldwide concern in the medical community . In particular , methicillin-resistant Staphylococcus aureus -LRB- MRSA -RRB- is a major cause of disease and healthcare expenditures in almost every continent . The emergence and spread of MRSA has been shown to be associated with both hospital - and community-acquired infections . Effective treatment options for these infections are limited and the situation may become more severe soon . For these reasons , a proactive management of MRSA in healthcare facilities is needed .1,2 Active screening and compliance to appropriate infection control activities have been shown to play an important role in the control of MRSA .1 Rapid diagnostic tests have the potential to make efforts even more effective . Thus , infection prevention has taken a step forward with the introduction of various tests for rapid identification of MRSA carriers .1,2 In 2006 , a new rapid method based on a novel bioluminescence detection technology for the rapid detection of MRSA directly from specimens was published .3 An improved version of this assay , the 3M ™ BacLite ™ Rapid MRSA Test -LRB- 3M Company , Maplewood , MN , USA -RRB- , was recently introduced , which allows the presence or absence of MRSA to be determined within 5 h. Although the clinical performance has been previously analysed ,4,5 so far no data are available on the detection of MRSA strains with highly diverse genetic backgrounds . The aim of this study was to assess the reliability of this assay to successfully detect MRSA strains circulating currently in Germany and other parts of Europe on the basis of several well-characterized S. aureus strain collections . For this purpose , S. aureus strains of different origin and clonality comprising 724 methicillin-susceptible and methicillin-resistant strains comprising > 90 % of all registered European MRSA spa types within the SeqNet network -LRB- www.SeqNet.org -RRB- were tested in this study . Materials and methods All staphylococcal strains were freshly isolated from clinical material at the University of Münster or during the course of various multicenter studies . One hundred and sixty-four strains isolated from patients with S. aureus bacteraemia -LRB- including 12 MRSA strains -RRB- as well as 50 isolates from the anterior nares of patients who did not subsequently develop S. aureus bacteraemia in a subsequent observation period -LRB- 4 MRSA strains -RRB- , were included into this study . Apart from 28 isolates from 14 patients -LRB- S. aureus first recovered from the anterior nares and subsequently from blood , one infected with MRSA -RRB- , only one isolate per patient was tested . In addition , 96 MRSA strains , each presenting a unique spa type , were selected from our institutional collection -LRB- Table 1 -RRB- . Furthermore , four hundred MRSA isolates were collected during the course of a recent multicenter study , also including community-acquired MRSA . In total , the 724 S. aureus strains tested comprised 211 methicillin-susceptible S. aureus -LRB- MSSA -RRB- and 513 MRSA strains . Table 1 Grouping of spa types tested in this study into spa clonal complexes -LRB- spa-CCs -RRB- spa-CCa spa typesb Putative MLST typesc 001 t001 , t002 , t003 , t010 , t035 , t039 , t041 , t045 , t055 , t057 , t066 , t105 , t106 , t109 , t110 , t143 , t149 , t151 , t264 , t265 , t422 , t820 , t892 , t1018 ST-5 , ST-45 , ST-46 , ST-222 , ST-225 , ST-228 , ST-231 , ST-111 , ST-228 004 t004 , t028 , t029 , t033 , t040 , t043 , t061 , t065 , t141 , t142 , t266 , t911 ST-45 , ST-45 , ST-46 015 t015 , t031 , t069 , t073 , t102 , t116 , t133 ST-45 024 t008 , t009 , t024 , t036 , t051 , t052 , t068 , t113 , t115 , t139 , t146 , t243 , t305 ST-8 , ST-235 , ST-247 , ST-250 , ST-254 , ST-247 , ST-250 032 t005 , t022 , t032 , t107 , t290 , t379 , t432 , t794 ST-22 , ST-23 , ST-60 037 t011 , t030 , t037 , t047 , t108 , t135 , t137 , t138 ST-30 , ST-239 , ST-240 , ST-241 , ST-246 038 t038 , t161 , t247 ST-45 044 t042 , t044 , t131 ST-80 Singletons t012 , t091 , t101 , t104 , t163 , t321 , t372 , t417 , t431 , t907 ST-7 , ST-30 , ST-22 aNaming of spa-CC was derived from the group founder . bt026 , t103 , t111 , t132 , t145 , t282 , t322 and t1007 were excluded from determination of the spa-CCs due to low number of repeats . cPutative MLST types were taken from the public SeqNet.org spa server database . If the biochemical identification of staphylococcal isolates using the ATB 32 Staph gallery -LRB- bioMérieux , Marcy l'Étoile , France -RRB- was ambiguous or categorized as unacceptable , partial 16S rRNA gene and RNA polymerase B -LRB- rpoB -RRB- gene sequencing was performed as described previously .6 Isolates were confirmed to be methicillin-resistant by detection of the mecA gene . To determine the clonal lineages of MRSA strains , the x region of the spa gene was amplified by PCR with primers 1095F -LRB- 5 ′ - AGACGATCCTTCGGTGAGC-3 ′ -RRB- and 1517R -LRB- 5 ′ - GCTTTTGCAATGTCATTTACTG-3 ′ -RRB- . DNA sequences were obtained with an ABI 377 sequencer -LRB- Applied Biosystems , Foster City , CA , USA -RRB- . spa types were determined using the Ridom StaphType software version 1.3 -LRB- Ridom GmbH , Würzburg , Germany -RRB- , and spa clonal complexes -LRB- spa-CCs -RRB- were assigned by using the BURP algorithm .7 The 3M ™ BacLite ™ Rapid MRSA Test is a novel culture-based test for the detection of MRSA performed on a semi-automated system comprising a sample processor and a luminometer . The assay consists of three successive selectivity steps : selective enrichment in a proprietary broth containing cefoxitin -LRB- 2 mg/L -RRB- and colistin -LRB- 50 mg/L -RRB- , immuno-magnetic extraction using a highly specific anti-S . aureus monoclonal antibody and selective lysis using lysostaphin . The selectivity steps are followed by a detection step using a highly sensitive cell marker , adenylate kinase -LRB- AK -RRB- . AK is an essential enzyme found in all living cells , which regulates energy provision by catalysing the equilibrium reaction ATP + AMP = 2 ADP . By supplying a continual source of purified ADP , this assay drives the AK reaction to generate up to 40 000 ATP molecules per min . These amplified levels of ATP are measured using the luminometer supplied with the system . For the present study , test isolates were streaked on sheep blood agar plates and grown at 37 °C to confirm purity . For each isolate , one to three colonies were picked and suspended in sterile saline -LRB- 0.9 % -RRB- to achieve a turbidity equivalent to that of a 0.5 McFarland standard . Usually , 43 isolates were processed in each run . The 3M ™ BacLite ™ Rapid MRSA Test was performed according to the instructions of the manufacturer . In brief , 10 µL of each prepared bacterial suspension was transferred into a vial containing 1 mL of selective enrichment broth . Vials were incubated at 37 °C for 2 h before 150 µL aliquots of each test sample were transferred into two adjacent wells of a 96-well assay plate , each containing 20 µL of capture reagent . MRSA cells bound to the monoclonal antibody on the capture reagent were then extracted from the sample matrix using the sample processor and concentrated in 100 µL of broth . One well for each sample was processed automatically for a baseline signal -LRB- T0 -RRB- in the BacLite luminometer . After a further incubation period of 2 h at 37 °C , the second well for each sample was processed in the same way -LRB- T2 reading -RRB- . Results are expressed in relative light units and interpreted as either a positive or negative screen result by a software embedded algorithm . Positive and negative controls as well as reagent and broth controls were included in each test run . A run took ∼ 5 h with a total hands-on time of 45 -- 50 min -LRB- < 1.5 min per tested isolate -RRB- . Results and discussion Analysing a total of 724 S. aureus strains , all 513 MRSA strains tested were recognized as MRSA , whereas none of the 211 MSSA strains was detected positive by the 3M ™ BacLite ™ Rapid MRSA Test . These results are particularly impressive as the institutional MRSA strain collection used in this study represents more than 90 different spa types covering > 90 % of all registered European MRSA spa types within the SeqNet network -LRB- Table 1 -RRB- . Beside several singletons , the MRSA enrolled in this study were grouped into eight spa clonal complexes -LRB- Table 1 -RRB- . Thus , a very large number of MRSA strains with different genetic backgrounds were recognized as MRSA using this novel method . Hospitals and other healthcare facilities across the world are faced with alarming rates of infections caused by MRSA . Continuous spread of these pathogens requires efficient strategies for infection control . Early identification of MRSA carriers among hospitalized patients is crucial to prevent its spread .1 Therefore , rapid availability of laboratory results is of utmost importance . However , conventional screening methods require prolonged incubation and confirmatory testing . During this time , MRSA-negative patients may be held in unnecessary isolation , whereas unidentified MRSA-positive individuals remain a hidden reservoir for cross-infection . A rapid negative result should allow more effective use of hospital isolation resources , whereas a rapid positive result should help reduce the spread of the infection and MRSA infection rates .2 In the past few years , several in-house and commercial rapid MRSA assays based on molecular techniques have been introduced for the detection of this pathogen directly from the specimen , mostly from the anterior nares . The first molecular assays developed were based on the detection of an S. aureus-specific sequence and the mecA gene , which encodes methicillin resistance .8 These tests are difficult to use for the direct detection of MRSA from non-sterile specimens , such as nasal samples , because of the likely co-presence of MSSA and methicillin-resistant coagulase-negative staphylococci .9 In a setting of low prevalence of MRSA , a molecular test targeting the mecA and an S. aureus-specific gene in parallel applied directly to clinical specimens would result in a high number of false positives and unacceptable performance .9 This technical limitation has been overcome in some assays , by linking detection of the presence of the mecA gene with detection of the neighbouring chromosome-borne orfX gene .10,11 In that approach , regions near the integration site of SCCmec were targeted as surrogate markers instead of the mecA gene itself . However , these flanking regions are known to be more heterogeneous than assumed so far .10 Thus , false-negative results due to variations within the primer binding sites may occur . Moreover , false-positive results due to the detection of DNA from non-viable MRSA , deletions or replacement of the mec region in vivo or ` ghost sequences ' such as partial SCCmec sequences in MSSA can occur , albeit their incidence in the routine clinical setting is as yet unclear .12 Despite the technical improvements in recent molecular-based assays , their high costs and relatively high operator skill requirement remain obstacles to their widespread routine use . The ability of a test to detect a broad range of MRSA clones is particularly important for an assay that may be used across a wide geographic region as tests with gaps in detection could potentially ` select out ' strains whose spread would be uncontrolled . Beside a variety of method-inherent limitations , rapid DNA-based methods amplify the nucleic acid and not the organism , which means the MRSA strain is unavailable for further characterization , such as determination of the resistance profile and strain typing . In contrast , applying the rapid growth-based assay tested here , any further examinations to characterize the respective strains will be possible from the enrichment broth . For the 3M ™ BacLite ™ Rapid MRSA Test , a diagnostic sensitivity of 94.6 % and diagnostic specificity of 96.9 % for nasal screening swabs and 95.9 % sensitivity and 88.8 % specificity for groin screening swabs , respectively , have been reported .4,5 The analytical limit of detection of the assay was shown to be ≤ 94 cfu -LRB- 3M ™ BacLite ™ Rapid MRSA Test , Instructions for Use -RRB- .4,5 Here , the new growth-based rapid MRSA assay was shown to detect without exception all MRSA strains of large collections of strains comprising highly diverse genetic backgrounds . Such a phenotypic test might be potentially more likely to cope with new strains . Further studies are warranted to evaluate this method using clinical specimens . Funding This work was supported in part by a grant from the Bundesministerium für Bildung und Forschung -LRB- BMBF -RRB- , Germany -LRB- Pathogenomic Plus Network PTJ-BIO/0313801B -RRB- . The device and the respective assay consumables were given to us by 3M for this study . Transparency declarations Two of the authors -LRB- A. W. F. and K. B. -RRB- delivered independent scientific presentations at 3M-supported symposia . C. v. E. is a member of 3M 's Scientific Advisory Board . All other authors : none to declare .

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