The

effect

of

the

chemical

biological

and

physical

environment

on

quorum

sensing

in

structured

microbial

communities

As

researchers

attempt

to

study

quorum

sensing

in

relevant

clinical

or

environmental

settings

it

is

apparent

that

many

factors

have

the

potential

to

affect

signaling

These

factors

span

a

range

of

physical

chemical

and

biological

variables

that

can

impact

signal

production

stability

and

distribution

Optimizing

experimental

systems

to

natural

or

clinical

environments

may

be

crucial

for

defining

when

and

where

quorum

sensing

occurs

These

points

are

illustrated

in

our

case

study

of

S.

aureus

signaling

in

biofilms

where

signal

stability

may

be

affected

by

the

host

environment

The

basic

signaling

schemes

have

been

worked

out

at

the

molecular

level

for

a

few

of

the

major

quorum-sensing

systems

As

these

studies

continue

to

refine

our

understanding

of

these

mechanisms

an

emerging

challenge

is

to

identify

if

and

when

the

local

environment

can

affect

signaling

Introduction

As

illustrated

by

many

articles

in

this

special

issue

the

diversity

of

mechanisms

bacteria

use

to

communicate

with

one

another

is

remarkable

Quorum

sensing

is

widespread

among

different

bacterial

species

emphasizing

the

importance

of

coordinating

behavior

as

a

group

When

considering

the

relevant

context

of

quorum

sensing

it

is

clear

that

the

environment

can

be

profoundly

important

Many

bacteria

exist

in

spatially

structured

multi-species

communities

such

as

biofilms

on

submerged

surfaces

or

flocs

in

the

water

column

of

aquatic

environments

-LSB-

-RSB-

Within

these

communities

bacteria

may

achieve

the

high

cell

numbers

capable

of

generating

the

local

signal

concentrations

required

to

produce

a

quorum

sensing

response

Quorum

sensing

undoubtedly

occurs

in

situations

where

bacteria

are

growing

planktonically

for

example

Vibrio

harveyi

quorum

sensing-controlled

bioluminescence

in

the

ocean

thought

to

produce

the

milky

seas

phenomenon

-LSB-

-RSB-

An

argument

could

however

be

made

that

quorum

sensing

is

more

likely

to

occur

in

structured

communities

If

the

reader

accepts

this

point

further

considerations

lead

to

some

interesting

questions

for

example

Are

certain

signals

better

suited

for

signaling

in

different

types

of

environments?What

are

the

important

parameters

influencing

signaling

in

different

environments

Take

as

an

example

the

well-studied

environmentally

ubiquitous

Pseudomonas

aeruginosa

This

bacterium

has

been

isolated

from

a

range

of

soil

and

aquatic

samples

-LSB-

-RSB-

The

nature

of

signaling

in

these

environments

could

be

quite

different

because

signal

production

and

diffusion

dynamics

may

change

This

review

will

discuss

factors

that

may

affect

signaling

in

structured

microbial

communities

In

addition

quorum

sensing

in

Staphylococcus

aureus

a

peptide-based

signaling

system

will

be

briefly

reviewed

and

its

role

in

biofilm

formation

will

be

discussed

Factors

affecting

signal

diffusion

stability

and

distribution

The

traditional

way

of

studying

quorum

sensing

in

the

laboratory

is

in

shaken

liquid-batch

cultures

The

quorum

sensing

response

occurs

at

a

specific

point

in

the

growth

curve

coinciding

with

a

threshold

concentration

of

signal

Volume

is

fixed

in

a

culture

flask

and

signal

molecules

produced

by

bacteria

usually

increase

in

concentration

with

time

-LRB-

unless

they

are

degraded

-RRB-

Batch

cultures

represent

a

closed

system

in

which

only

gaseous

exchange

occurs

Most

structured

communities

are

in

open

systems

in

which

exchange

occurs

with

the

surrounding

liquid

-LSB-

-RSB-

In

an

open

flowing

system

signals

may

be

washed

away

by

the

overlying

fluid

In

open

systems

the

concentration

of

signal

molecules

within

structured

communities

is

primarily

a

function

of

signal-production

rate

the

degradation

rate

or

half-life

of

the

signal

the

diffusion

properties

of

the

signal

andthe

external

hydrodynamic

or

mass-transfer

conditions

The

prevailing

environmental

conditions

and

resident

biology

are

to

different

degrees

important

for

each

of

these

processes

The

effects

of

signal

and

environmental

chemistry

on

quorum

sensing

Some

of

the

best-studied

quorum-sensing

signals

include

the

acyl-homoserine

lactones

-LRB-

AHL

-RRB-

used

by

many

Gram-negative

bacterial

species

small

peptides

used

by

some

Gram-positive

species

and

the

furans

of

the

phylogenetically

widely

spread

signaling

-LRB-

reviews

are

available

elsewhere

-LSB-

-RSB-

-RRB-

Signal

chemistry

varies

widely

in

different

quorum-sensing

systems

and

is

important

for

determining

its

stability

and

diffusion

characteristics

in

different

environments

All

AHL

signals

have

a

characteristic

homoserine

lactone

moiety

The

homoserine

lactone

is

derived

from

S-adenosylmethionine

one

of

the

substrates

for

AHL

synthesis

-LSB-

-RSB-

Although

stable

at

neutral

and

slightly

acidic

pH

the

lactone

ring

is

subject

to

chemical

hydrolysis

under

basic

conditions

-LSB-

-RSB-

The

resulting

product

-LRB-

e.g.

butyryl-homoserine

lactone

would

become

butyryl-homoserine

-RRB-

lacks

biological

signaling

activity

and

is

capable

of

spontaneously

re-lactonizing

when

the

pH

is

lowered

The

formula

describing

the

relationship

between

pH

and

the

lactone

ring

stability

is

-LRB-

-LSB-

OH

-RSB-

-RRB-

-LSB-

-RSB-

which

gives

the

half

life

in

days

For

example

at

pH

the

homoserine

lactone

ring

would

be

stable

for

hours

whereas

at

pH

the

stability

would

be

of

the

order

of

minutes

-LRB-

Fig.

-RRB-

In

many

environments

this

might

be

insignificant

In

some

alkaline

environments

however

like

the

photosynthetic

mats

at

Octopus

Springs

in

Yellowstone

National

Park

the

pH

is

high

enough

to

potentially

affect

signal

gradients

Kaufmann

et

al.

recently

demonstrated

that

in

addition

to

lactone

hydrolysis

the

AHL

of

P.

aeruginosa

is

capable

of

spontaneously

undergoing

a

Claisen-like

condensation

reaction

in

aqueous

environments

forming

a

tetramic

acid

product

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

This

compound

was

shown

to

have

significant

antimicrobial

activity

against

other

species

and

a

high

affinity

for

iron

It

was

proposed

this

iron-binding

activity

constituted

an

alternative

means

of

acquiring

iron

by

P.

aeruginosa

Fig.

AHL

half-lives

in

different

alkaline

environmentsFig

of

quorum-sensing

signals

and

their

derivatives

Letter

designations

for

the

Gram-positive

peptide

signals

indicate

amino

acids

For

the

L.

lactis

signal

nisin

the

structural

abbreviations

were

Bu

dehydrobutirine

with

a

lanthionine

bridge

Ha

dehydroalanine

Hb

dehydrobutirine

Structured

communities

can

create

highly

heterogeneous

localized

niches

where

chemistry

can

vary

drastically

over

very

small

distances

For

example

consumption

of

dissolved

oxygen

by

laboratory

biofilm

cells

can

result

in

completely

anaerobic

regions

within

the

biofilm

only

μm

from

the

fully

oxygen

saturated

overlying

liquid

-LSB-

-RSB-

Similarly

pH

can

vary

dramatically

as

demonstrated

by

pH

microelectrodes

positioned

in

biofilms

formed

from

the

dental

pathogen

Streptococcus

mutans

On

addition

of

the

growth

substrate

sucrose

the

pH

dropped

from

outside

the

biofilm

to

approximately

μm

deep

within

the

biofilm

-LRB-

Gieseke

A

Nguyen

D

von

Ohle

C

Stoodley

P

unpublished

results

-RRB-

Clearly

if

the

half-life

of

signaling

molecules

is

a

function

of

local

chemistry

it

is

reasonable

to

expect

that

degradation

rates

will

vary

in

the

community

The

acyl

side-chains

of

AHLs

range

in

length

from

to

carbon

atoms

-LSB-

-RSB-

The

substitution

chemistry

of

the

acyl

side-chain

is

also

highly

variable

Although

most

AHLs

have

a

ketone

group

at

the

position

some

AHLs

also

have

either

a

ketone

or

hydroxyl

group

at

the

position

In

addition

the

acyl

side-chain

can

vary

in

saturation

Although

most

AHLs

have

fully

saturated

acyl

side-chains

some

for

example

the

homoserine

lactone

of

Rhizobium

leguminosarum

are

unsaturated

-LSB-

-RSB-

All

these

features

of

AHL

acyl

side-chain

chemistry

in

particular

acyl

chain

length

affect

the

relative

hydrophobicity

of

the

signal

Pearson

et

al.

observed

this

for

P.

aeruginosa

which

produces

two

major

AHL

signals

a

-LRB-

i.e.

four-carbon

side-chain

-RRB-

and

AHL

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

They

found

that

the

signal

diffused

freely

across

the

cell

membrane

whereas

the

signal

partitioned

to

the

cell

membrane

where

its

extrusion

was

aided

by

the

activity

of

the

MexAB-OprM

efflux

pump

The

hydrophobicity

of

the

local

environment

could

have

a

large

effect

on

AHL

diffusion

properties

For

example

several

AHL-utilizing

bacterial

species

are

also

capable

of

producing

highly

hydrophobic

secreted

polysaccharides

-LRB-

e.g.

the

pel/psl

polysaccharides

of

P.

aeruginosa

-RRB-

-LSB-

-RSB-

In

a

structured

community

such

as

a

biofilm

secreted

polysaccharides

can

serve

as

the

scaffolding

holding

the

community

together

-LSB-

-RSB-

Under

such

conditions

these

polysaccharides

might

also

act

as

an

AHL-sequestering

matrix

Charlton

et

al.

provided

evidence

of

this

indicating

that

the

signal

of

P.

aeruginosa

partitioned

into

the

extracellular

biofilm

matrix

-LSB-

-RSB-

Perhaps

the

signal

has

reduced

reactivity

with

the

biofilm

matrix

making

it

a

more

effective

signal

in

a

biofilm

and

explaining

why

it

is

produced

in

greater

amounts

than

by

P.

aeruginosa

growing

as

a

biofilm

-LSB-

-RSB-

Another

factor

affecting

AHL

stability

in

the

environment

is

biologically-driven

signal

degradation

Emphasizing

this

point

Wang

et

al.

demonstrated

that

radiolabled

AHLs

fed

to

soil

microbial

communities

were

almost

instantly

mineralized

-LSB-

-RSB-

Several

bacterial

species

have

the

ability

to

use

AHLs

as

carbon

and

nitrogen

sources

These

include

species

that

produce

their

own

AHLs

and

those

known

not

to

make

them

Why

some

organisms

both

make

and

break

down

their

own

AHLs

is

a

mystery

AHL

degradation

occurs

as

a

result

of

two

primary

types

of

enzymatic

activity

lactonases

which

break

the

homoserine

lactone

ring

and

acylases

which

cleave

the

amide

bond

linking

the

acyl

side-chain

to

the

homoserine

lactone

ring

-LSB-

-RSB-

Biological

AHL

degradation

is

a

key

consideration

in

multi-species

environments

Leadbetter

proposed

the

concept

of

insulation

in

which

AHL-degrading

organisms

could

prevent

AHLs

produced

by

bacteria

spatially

fixed

in

one

location

from

reaching

other

bacteria

of

the

same

species

-LSB-

-RSB-

Environmental

chemistry

also

plays

a

key

role

in

signaling

The

paradigm

for

this

type

of

quorum

sensing

is

the

luxS

system

of

Vibrio

harveyi

-LSB-

-RSB-

One

of

the

key

steps

in

signal

synthesis

is

the

LuxS-catalyzed

conversion

of

S-ribosyl

homocysteine

to

homocysteine

and

-LRB-

DPD

-RRB-

-LSB-

-RSB-

DPD

is

believed

to

be

the

key

precursor

to

all

signals

DPD

undergoes

non-enzymatic

cyclization

and

a

number

of

spontaneous

reversible

rearrangements

to

form

the

active

signal-a

furanosyl

borate

diester

and

other

furans

-LSB-

-RSB-

The

signal

for

Salmonella

typhimurium

was

recently

solved

and

found

to

be

-LRB-

S

-RRB-

-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran

a

molecule

related

to

but

structurally

distinct

from

the

V.

harveyi

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Spontaneous

extracellular

chemical

rearrangements

of

S.

typhimurium

can

produce

the

V.

harveyi

signal

the

reverse

is

also

true

-LSB-

-RSB-

Current

thinking

is

that

the

local

chemical

environment

can

affect

the

ratio

of

the

different

furanosyl

esters

In

particular

the

presence

of

borate

may

shift

the

equilibrium

toward

formation

of

the

V.

harveyi

-LSB-

-RSB-

The

chemical

structures

of

the

Gram-positive

quorum-sensing

peptides

vary

greatly

in

the

number

of

residues

and

the

types

of

modification

-LRB-

Fig.

-RRB-

Because

of

the

post-translational

modifications

on

these

peptides

and

their

inability

to

diffuse

across

membranes

their

biosynthetic

pathways

are

more

complex

than

those

of

the

AHLs

and

dedicated

signal-export

systems

are

usually

required

Many

also

induce

their

own

biosynthesis

and

are

thus

frequently

termed

autoinducing

peptides

Several

signals

are

simple

linear

peptides

for

example

the

competence-stimulating

peptide

-LRB-

CSP

-RRB-

which

regulates

competence

in

Streptococcus

pneumoniae

-LSB-

-RSB-

The

corresponding

linear

competence

pheromone

in

Bacillus

subtilis

called

ComX

is

more

unusual

in

that

an

internal

tryptophan

residue

has

been

cyclized

and

isoprenylated

with

a

geranyl

moiety

-LSB-

-RSB-

a

modification

that

is

catalyzed

by

ComQ

-LSB-

-RSB-

Perhaps

the

largest

and

most

complex

peptide

signals

are

the

lantibiotics

which

also

have

antimicrobial

activity

-LSB-

-RSB-

These

molecules

for

example

nisin

produced

by

Lactococcus

lactis

are

known

to

positively

regulate

their

own

biosynthesis

in

a

density-dependent

manner

They

also

have

extensive

post-translation

modifications

including

dehydrated

residues

such

as

dehydroalanine

and

dehydrobutyrine

and

thioether

bridges

called

lanthionines

-LRB-

Fig.

depicts

the

base

structure

of

the

lantibiotic

nisin

of

L.

lactis

-RRB-

An

emerging

class

of

peptide

signals

are

the

cyclic

lactones

and

thiolactones

-LSB-

-RSB-

The

first

of

this

class

to

be

discovered

was

the

type

I

autoinducing

peptide

-LRB-

AIP

-RRB-

of

Staphylococcus

aureus

an

eight-residue

thiolactone-containing

peptide

with

the

C-terminus

constrained

through

linkage

with

a

cysteine

side-chain

-LSB-

-RSB-

Since

the

discovery

of

AIPs

related

signals

have

been

identified

in

Enterococcus

faecalis

-LSB-

-RSB-

Listeria

monocytogenes

-LSB-

-RSB-

and

other

Staphylococci

-LSB-

-RSB-

Environmental

conditions

can

have

a

significant

effect

on

peptide

signal

stability

The

linear

peptides

are

likely

to

have

short

half-lives

as

they

are

rapidly

metabolized

by

the

action

of

secreted

proteases

Indeed

this

issue

has

led

some

to

question

their

role

as

a

means

of

communication

raising

the

possibility

they

serve

as

a

general

strategy

for

a

single

cell

to

time

regulatory

events

-LSB-

-RSB-

In

contrast

the

lantibiotics

have

multiple

lanthionine

bridges

that

block

the

activity

of

many

proteases

-LSB-

-RSB-

greatly

improving

the

longevity

of

these

molecules

Like

AHLs

lantibiotics

are

pH-sensitive

with

solubility

and

stability

dropping

rapidly

at

higher

pH

those

with

dehydrated

residues

are

also

reactive

with

thiols

-LSB-

-RSB-

Because

of

these

chemical

constraints

the

lantibiotic

nisin

has

a

half-life

of

only

h

in

mouse

serum

-LSB-

-RSB-

the

pH

of

which

is

approximately

For

L.

lactis

in

the

environment

however

growth

and

production

of

nisin

are

optimum

at

lower

pH

at

which

stability

is

greater

facilitating

autoinduction

In

a

similar

fashion

the

thiolactone-containing

peptides

are

resistant

to

proteolysis

and

sensitive

to

high

pH

and

reactive

thiols

-LRB-

chemistry

of

the

S.

aureus

AIP

is

discussed

in

more

detail

below

-RRB-

and

some

have

increased

sensitivity

to

oxidative

damage

-LSB-

-RSB-

Although

it

remains

to

be

demonstrated

protease

resistance

may

be

one

of

the

most

significant

environmental

constraints

faced

by

bacteria

using

peptide

communication

signals

and

extensive

post-translational

modifications

could

have

evolved

to

overcome

this

problem

The

effect

of

the

hydrodynamic

environment

on

quorum

sensing

Mass

transfer

is

the

physical

process

by

which

molecules

are

transported

in

a

system

and

it

has

the

potential

to

affect

cell-to-cell

signaling

in

many

ways

Delivery

of

nutrients

to

the

active

biomass

in

the

structured

community

is

one

example

This

could

affect

the

relative

metabolic

activity

of

the

community

which

in

turn

has

the

potential

to

affect

signal-production

rates

Mass

transfer

is

affected

by

the

hydrodynamics

of

the

bulk

fluid

and

the

geometry

of

the

structured

community

These

two

factors

affect

each

other

because

a

microbial

community

both

shapes

and

is

shaped

by

its

external

environment

If

liquid

flow

is

a

feature

of

the

environment

it

will

wash

signal

away

diluting

its

concentration

within

the

community

In

these

circumstances

the

system

can

be

divided

into

three

zones

-LRB-

Fig.

-RRB-

First

the

biomass

of

the

community

which

is

producing

signaling

molecules

second

the

viscous

sublayer

-LRB-

δ

-RRB-

located

at

the

biomass-bulk

fluid

interface

and

third

the

well

mixed

turbulent

core

of

the

overlying

fluid

In

laminar

flow

there

is

no

turbulent

core

and

δ

essentially

extends

to

either

the

center

of

the

channel

if

flow

is

in

a

closed

conduit

-LRB-

e.g.

pipe

or

catheter

-RRB-

or

to

the

fluid

surface

if

flow

is

in

an

open

channel

-LRB-

e.g.

river

-RRB-

Using

this

simplified

system

we

have

constructed

a

one-dimensional

model

based

on

a

flat

biofilm

to

illustrate

how

different

values

of

δ

may

result

in

different

concentration

profiles

while

the

thickness

of

the

biofilm

the

cell

density

in

the

biofilm

and

the

rate

of

signal-production

in

the

biofilm

remain

constant

The

model

is

based

on

coupling

the

diffusion

of

signal

through

the

viscous

sublayer

with

the

simultaneous

diffusion

and

production

of

signal

that

occurs

within

the

biofilm

itself

In

the

fluid

outside

the

biofilm

the

concentration

of

signal

changes

linearly

where

Cz

is

the

signal

concentration

-LRB-

mg

L

-RRB-

at

depth

z

is

the

signal

concentration

outside

the

viscous

sublayer

-LRB-

mg

L

-RRB-

is

the

signal

production

rate

-LRB-

mg

cell

s

-RRB-

Xb

is

the

active

cell

density

in

the

biofilm

-LRB-

CFU

cm

-RRB-

Lf

is

the

biofilm

thickness

-LRB-

μm

-RRB-

δ

is

the

thickness

of

the

viscous

sublayer

-LRB-

or

μm

-RRB-

Daq

is

the

diffusion

coefficient

of

the

signal

in

water

-LRB-

s

-RRB-

and

δz

is

the

proportional

depth

within

δ

-LRB-

μm

-RRB-

Within

the

biofilm

the

concentration

of

the

signal

changes

quadratically

with

depth

where

De

is

the

effective

diffusion

coefficient

of

signal

in

the

biofilm

-LRB-

s

-RRB-

and

Lz

is

the

proportional

depth

within

the

biofilm

-LRB-

μm

-RRB-

Fig.

diagram

illustrating

how

external

flow

may

affect

the

concentration

of

a

cell

signal

produced

by

biofilm

cells

The

biofilm

is

μm

thick

-LRB-

so

there

is

no

oxygen

limitation

and

we

can

assume

equal

signal

production

throughout

the

depth

of

the

biofilm

-RRB-

and

the

relative

concentration

profiles

are

for

three

flow

conditions

-LRB-

a

-RRB-

Very

high

shear

negligible

diffusion

boundary

layer

-LRB-

δ

-RRB-

-LRB-

b

-RRB-

Lower

flow

with

a

diffusion

boundary

layer

of

μm

-LRB-

δB

-RRB-

-LRB-

c

-RRB-

Lower

flow

again

with

a

diffusion

boundary

layer

of

μ

-LRB-

δC

-RRB-

The

model

is

steady-state

Fickian

diffusion

for

flat-slab

geometry

with

a

uniform

density

of

bacteria

producing

signals

at

a

constant

rate

with

no

degradation

There

is

no

convection

within

the

biofilm

and

no

flux

at

the

substratum

The

concentration

of

signal

in

the

turbulent

core

of

the

flow

is

zero

The

effective

diffusion

coefficient

in

the

biofilm

is

assumed

to

be

times

that

of

the

bulk

fluid

based

on

the

molecular

weight

of

AHL

-LSB-

-RSB-

For

simplification

the

viscous

sublayer

is

shown

as

the

diffusion

boundary

layer

In

reality

the

transition

-LRB-

the

buffer

region

of

the

boundary

layer

-RRB-

from

the

viscous

sublayer

to

the

turbulent

core

is

not

a

sharp

line

as

depicted

but

gradual

If

we

assume

an

inducing

AHL

concentration

of

mg

L

-LRB-

μmol

-RRB-

depicted

by

the

vertical

dashed

line

biofilm

A

is

not

induced

at

all

in

biofilm

B

the

bottom

is

induced

and

in

biofilm

C

the

bottom

is

induced

as

shown

by

the

horizontal

dashed

lines

where

the

inducing

concentration

intersects

the

concentration

profile

Where

known

we

used

data

as

best

we

could

based

on

the

signal

AHL

unknown

model

data

were

hypothetical

All

data

were

based

on

those

used

for

illustrative

purposes

by

Stewart

-LSB-

-RSB-

These

considerations

led

us

to

hypothesize

that

external

flow

conditions

can

affect

quorum

sensing

in

many

ways

For

example

in

a

somewhat

closed

static

system

-LRB-

e.g.

a

biofilm

in

a

mud

puddle

or

in

the

well

of

a

microtitre

plate

-RRB-

signals

produced

by

a

large

biofilm

cell

aggregate

may

induce

a

quorum-sensing

response

in

neighboring

bacteria

that

are

not

part

of

the

aggregate

In

contrast

in

an

open

system

subject

to

convective

flow

-LRB-

e.g.

a

biofilm

in

a

river

or

the

channel

of

a

flow

cell

biofilm

reactor

-RRB-

signals

might

be

continuously

removed

from

the

system

Under

these

conditions

mass

transfer

may

prevent

signals

produced

by

a

large

biofilm

cell

aggregate

from

inducing

cells

in

the

vicinity

of

the

aggregate

Experimental

results

on

interspecies

communications

in

dental

plaque

biofilms

by

Egland

et

al.

-LSB-

-RSB-

support

this

idea

and

suggest

that

diffusible

signals

are

adapted

to

function

over

short

distances

At

higher

flows

the

higher

flux

of

signal

molecules

from

structured

communities

may

reduce

signal

concentration

in

the

interior

or

core

of

the

community

A

subsequent

consequence

is

that

more

biomass

would

be

necessary

to

produce

an

inducing

concentration

of

signal

Few

studies

have

directly

addressed

the

effect

of

hydrodynamics

on

quorum

sensing

in

structured

communities

Purevdorj

et

al.

-LSB-

-RSB-

found

that

at

relatively

high

flow

rates

flow

velocity

was

a

stronger

determinant

of

P.

aeruginosa

biofilm

structure

than

quorum-sensing-required

functions

Yarwood

et

al.

-LSB-

-RSB-

grew

wild

type

-LRB-

WT

-RRB-

and

accessory

gene

regulator

-LRB-

agr

-RRB-

mutant

biofilms

of

S.

aureus

by

batch

culture

under

static

conditions

batch

culture

on

spinning

disks

and

in

flow

cells

The

QS

mutation

had

the

greatest

effect

under

static

conditions

resulting

in

an

increase

in

biofilm

formation

consistent

with

the

study

of

Vuong

et

al.

-LSB-

-RSB-

who

hypothesized

that

agr

was

a

repressor

of

biofilm

formation

Interestingly

there

was

no

difference

between

WT

and

agr

-LRB-

quorum

sensing

-RRB-

mutant

biofilms

when

grown

in

flow

cells

Care

must

however

be

taken

in

correlating

growth

system

with

phenotype

because

media

type

and

concentration

were

also

varied

in

this

study

Nevertheless

Fux

et

al.

-LSB-

-RSB-

obtained

similar

results

for

flow

cell-grown

biofilms

After

h

the

WT

S.

aureus

biofilms

had

less

biomass

than

a

TRAP

mutant

but

were

significantly

rougher

After

days

however

there

was

little

difference

between

the

biofilms

as

measured

by

COMSTAT

confocal

image-analysis

software

-LSB-

-RSB-

In

the

same

study

the

opposite

trend

as

measured

by

viable

plate

counts

was

observed

for

filter

colony

biofilms

-LSB-

-RSB-

grown

under

static

conditions

on

nutrient

agar

The

WT

biofilms

contained

an

order

of

magnitude

more

cells

than

the

TRAP

mutant

Interestingly

a

regulatory

network

mathematical

model

has

predicted

that

at

intermediate

concentrations

of

bacteria

can

suddenly

switch

between

induced

and

noninduced

states

-LSB-

-RSB-

This

effect

may

help

explain

experimental

observation

of

different

biofilm

phenotypes

under

what

are

assumed

to

be

similar

or

even

replicate

growth

conditions

Quorum

sensing

in

the

gas

phase

with

volatile

signals

Quorum

sensing

is

usually

discussed

and

studied

in

the

context

of

aqueous

environments

Some

environmental

conditions

however

for

example

those

found

in

soil

may

not

always

be

ideal

for

signaling

in

the

liquid

phase

Soil

is

subject

to

wetting

and

drying

cycles

and

even

somewhat

dry

soils

are

teeming

with

metabolically

active

bacteria

-LSB-

-RSB-

Under

these

conditions

it

may

be

advantageous

to

some

bacterial

species

to

engage

in

quorum

sensing

Local

cell

numbers

may

be

quite

high

but

not

linked

to

one

another

through

the

liquid

phase

The

production

of

signals

able

to

act

through

the

gas

phase

would

circumvent

these

issues

There

is

precedence

for

volatile

signaling

in

the

microbial

world

An

interesting

example

is

the

signaling

molecule

palmitic

acid

methyl

ester

-LRB-

PAME

-RRB-

of

Ralstonia

solanacearum

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

The

signal

of

this

quorum-sensing

system

can

act

in

the

gas

phase

This

plant

pathogen

regulates

the

production

of

secreted

polysaccharides

-LRB-

an

important

virulence

factor

-RRB-

by

use

of

PAME

-LSB-

-RSB-

the

synthesis

of

which

is

catalyzed

by

the

phcB

gene

product

-LSB-

-RSB-

When

PAME

concentrations

reach

nmol

L

PAME-regulated

genes

are

induced

presumably

because

of

interaction

of

the

signal

with

the

membrane-associated

PhcS

sensor

kinase

Although

homologs

of

phcB

are

not

found

on

many

of

the

microbial

genomes

yet

sequenced

signaling

in

the

gas

phase

may

be

one

of

the

next

important

frontiers

in

quorum

sensing

A

case

study

of

signaling

in

structured

communities-Quorum-sensing

in

Staphylococcus

aureus

biofilms

Continuing

with

the

themes

of

this

review

a

case

study

will

be

presented

with

a

discussion

on

environmental

chemical

and

biological

factors

discussed

above

that

have

the

potential

to

affect

quorum-sensing

in

S.

aureus

biofilms

S.

aureus

is

a

human

commensal

that

resides

in

a

non-pathogenic

state

in

the

nasal

airways

When

there

is

a

breach

in

the

host

defenses

enabling

access

S.

aureus

can

convert

to

a

pathogenic

state

and

secrete

an

impressive

array

of

toxins

hemolysins

and

degratory

enzymes

-LSB-

-RSB-

causing

damage

to

host

tissues

This

lifestyle

switch

is

mediated

in

a

cell-density-dependent

manner

by

the

action

of

the

AIP

molecule

Early

in

the

growth

phase

surface

adhesins

and

antigens

are

produced

and

when

AIP

reaches

a

critical

concentration

the

quorum-sensing

cascade

is

activated

and

these

surface

proteins

are

down-regulated

and

invasive

factors

are

secreted

Microarray

studies

have

revealed

that

genes

are

up-regulated

and

are

down-regulated

by

the

action

of

this

quorum-sensing

molecule

-LSB-

-RSB-

this

represents

almost

five

percent

of

the

genome

Intriguingly

there

are

four

specific

classes

of

AIP

molecule

that

approximately

correlate

with

the

type

of

disease

caused

by

the

producing

S.

aureus

strain

In

a

fascinating

mechanism

of

bacterial

cross-talk

these

different

classes

of

AIP

cross-inhibit

quorum-sensing

in

other

S.

aureus

groups

and

other

staphylococcal

species

-LSB-

-RSB-

this

may

serve

to

isolate

specific

sub-populations

for

cooperative

action

The

locus

responsible

for

the

quorum-sensing

regulation

in

S.

aureus

is

the

accessory

gene

regulator

or

Agr

locus

and

is

known

to

contain

two

divergent

transcripts

named

RNAII

and

RNAIII

-LSB-

-RSB-

The

RNAII

transcript

is

an

operon

of

four

genes

agrBDCA

that

encode

factors

required

to

synthesize

AIP

and

activate

the

regulatory

cascade

-LRB-

Fig.

-RRB-

The

biosynthetic

pathway

leading

to

functional

AIP

is

not

clear

but

it

is

known

that

AgrD

is

the

peptide

precursor

of

AIP

and

AgrB

is

a

membrane

protease

involved

in

its

processing

-LSB-

-RSB-

AgrC

and

AgrA

form

a

typical

two-component

regulatory

pair

and

the

binding

of

AIP

to

a

surface

receptor

on

AgrC

activates

this

phosphoryl-transfer

cascade

-LSB-

-RSB-

When

phosphorylated

AgrA

is

known

to

bind

and

induce

expression

of

the

RNAIII

transcript

-LSB-

-RSB-

which

encodes

a

regulatory

RNA

molecule

that

acts

as

the

primary

effector

of

S.

aureus

quorum-sensing

-LSB-

-RSB-

AgrA

also

induces

expression

of

the

Agr

proteins

through

the

RNAII

transcript

triggering

the

autoinduction

phenomenon

Fig.

diagram

of

quorum-sensing

systems

of

S.

aureus

The

gene

locus

for

the

agr

system

is

shown

in

black

and

contains

two

divergent

transcripts

RNAII

and

RNAIII

driven

by

the

and

promoters

respectively

The

RNAII

transcript

encodes

the

agrBDCA

operon

which

encodes

the

signal

processing

and

detection

components

for

quorum-sensing

in

S.

aureus

-LRB-

see

text

-RRB-

The

RNAIII

transcript

is

a

regulatory

RNA

that

up-regulates

and

down-regulates

all

genes

related

to

quorum-sensing

This

transcript

also

encodes

the

amphipathic

peptide

δ-hemolysin

RAP

and

TRAP

are

part

of

a

second

quorum-sensing

system

thought

to

be

a

precursor

of

the

agr

system

RAP

is

secreted

and

induces

TRAP

phosphorylation

which

in

turn

induces

expression

of

the

RNAII

transcript

RIP

is

a

heptapeptide

known

to

inhibit

the

ability

of

RAP

to

induce

TRAP

phosphorylation

A

second

quorum-sensing

cascade

is

thought

to

serve

as

a

precursor

to

the

Agr

system

-LSB-

-RSB-

setting

the

stage

for

AIP

regulation

As

S.

aureus

cells

multiply

the

RNAIII-activating

protein

-LRB-

RAP

-RRB-

is

secreted

and

accumulates

outside

the

cell

and

at

a

threshold

concentration

RAP

triggers

the

phosphorylation

of

the

cytoplasmic

protein

TRAP

-LSB-

-RSB-

which

induces

expression

of

the

RNAII

transcript

-LRB-

Fig.

-RRB-

Intriguingly

a

linear

heptapeptide

called

RIP

is

known

to

block

the

activity

of

RAP

enabling

small-molecule

control

over

S.

aureus

pathogenicity

-LSB-

-RSB-

Chemistry

of

the

S.

aureus

AIP

signal

The

reasons

for

evolving

a

cyclic

thiolactone

structure

as

a

quorum-sensing

signal

are

still

not

clear

The

thioester

bond

and

the

presence

of

other

labile

amino

acids

reduce

stability

because

of

the

potential

for

oxidative

damage

base-catalyzed

hydrolysis

and

thioester

exchange

Indeed

the

methionine

residue

in

AIP

is

rapidly

oxidized

in

vitro

to

a

methionyl

sulfoxide

converting

the

signal

to

an

inactive

byproduct

-LSB-

-RSB-

Similarly

oxidants

produced

in

vivo

by

the

phagocyte

NADPH

oxidase

are

known

to

accelerate

this

inactivation

-LSB-

-RSB-

Despite

these

issues

the

AIP

lifetime

in

host

tissues

is

reported

to

be

h

-LSB-

-RSB-

which

is

more

than

adequate

for

regulation

bearing

in

mind

that

S.

aureus

doubles

every

min

in

the

host

-LSB-

-RSB-

Structure-function

studies

have

shown

that

the

thiolactone

can

not

be

replaced

with

a

more

stabile

lactone

suggesting

that

the

signal

receptor

might

require

acylation

to

activate

the

cascade

-LSB-

-RSB-

although

a

lactam

substitution

does

activate

at

high

concentrations

-LSB-

-RSB-

As

discussed

above

constraining

the

peptide

will

improve

metabolic

stability

to

proteases

-LSB-

-RSB-

by

protecting

the

C-terminus

and

impeding

access

to

endoproteases

which

probably

increases

the

half-life

compared

with

that

of

the

linear

AIP

counterpart

Clearly

further

study

is

necessary

to

determine

the

physiological

benefit

of

this

thiolactone

structure

The

relationship

between

S.

aureus

quorum-sensing

and

biofilm

communities

Biofilm

formation

is

increasingly

being

recognized

as

an

important

virulence

factor

in

S.

aureus

pathogenesis

Several

biofilm-associated

diseases

including

osteomyelitis

-LSB-

-RSB-

endocarditis

-LSB-

-RSB-

medical

device

infections

-LSB-

-RSB-

and

potentially

even

skin

infections

-LSB-

-RSB-

have

much

clinical

relevance

The

formation

of

S.

aureus

biofilms

progresses

in

a

similar

fashion

to

the

Gram-negative

biofilms

with

attachment

followed

by

development

into

a

highly-structured

cell

community

Several

attachment

factors

for

example

the

microbial

surface

components

recognizing

adhesive

matrix

molecules

-LRB-

MSCRAMMs

-RRB-

and

the

surface-attached

Atl

protein

have

been

implicated

in

this

initial

stage

of

biofilm

formation

-LRB-

reviewed

elsewhere

-LSB-

-RSB-

-RRB-

Secretion

of

a

polysaccharide

adhesin

is

thought

to

be

critical

for

development

of

a

structured

biofilm

-LSB-

-RSB-

although

some

biofilm-forming

S.

aureus

strains

can

not

produce

this

polymer

-LSB-

-RSB-

Unlike

some

well-studied

Gram-negative

bacterial

species

S.

aureus

are

non-motile

which

leads

to

significantly

reduced

biofilm

architecture

in

flowing

systems

The

significance

of

this

difference

is

not

clear

because

simple

alterations

of

growth

conditions

for

P.

aeruginosa

are

known

to

eliminate

higher-ordered

biofilm

structure

-LSB-

-RSB-

Because

the

development

of

a

robust

P.

aeruginosa

biofilm

under

some

conditions

requires

an

active

quorum-sensing

system

-LSB-

-RSB-

one

might

assume

the

behavior

of

S.

aureus

is

similar

the

opposite

seems

to

be

true

however

because

inactivation

of

the

Agr

system

tends

to

enhance

attachment

-LSB-

-RSB-

Agr

is

known

to

down-regulate

surface

attachment

factors

providing

an

explanation

of

this

observation

The

Agr

cascade

also

up-regulates

both

the

secretion

of

proteases

that

can

degrade

these

attachment

factors

and

the

secretion

of

amphipathic

peptides

-LRB-

phenol-soluble

modulins

-RRB-

that

facilitate

detachment

-LSB-

-RSB-

suggesting

quorum-sensing

may

play

a

role

in

biofilm

turnover

Despite

these

intriguing

observations

deciphering

the

literature

on

this

topic

has

been

challenging

because

of

non-uniform

biofilm

culturing

methods

and

strain-to-strain

differences

-LSB-

-RSB-

To

emphasize

this

point

enhanced

attachment

by

Agr

mutants

in

static

biofilm

systems

is

not

observed

in

flowing

biofilm

reactor

systems

-LSB-

-RSB-

An

elegant

study

by

Yarwood

et

al.

addressed

some

of

the

discrepancies

in

the

literature

and

suggested

an

alterative

role

for

S.

aureus

quorum-sensing

in

biofilms

-LSB-

-RSB-

Under

static

conditions

attachment

of

Agr

mutants

was

markedly

better

than

that

of

wild-type

as

shear

force

increased

with

increased

flow

rate

however

this

advantage

was

lost

and

under

some

experimental

conditions

became

a

disadvantage

As

in

P.

aeruginosa

biofilm

studies

the

method

and

type

of

experiment

seemed

to

dictate

the

requirement

for

the

Agr

system

By

following

a

quorum-sensing

promoter

in

a

flow

cell

biofilm

Yarwood

et

al.

observed

that

only

patches

of

surface

cells

activated

the

Agr

cascade

Surprisingly

these

cells

detached

from

the

biofilm

in

periodic

waves

suggesting

an

alternative

role

for

the

Agr

system

in

the

detachment

and

recycling

phase

of

biofilm

development

In

a

biofilm

infection

of

the

host

it

seems

probable

that

S.

aureus

will

be

subjected

to

increased

flow

and/or

shear

force

in

addition

to

many

other

factors

which

will

affect

the

quorum-sensing

phenomenon

Increased

flow

will

probably

dictate

the

local

AIP

concentration

potentially

deactivating

quorum-sensing

by

washing

away

the

signal

whereas

increased

shear

force

could

also

perform

this

function

while

also

accelerating

cell

detachment

We

can

only

speculate

on

how

this

interplay

will

affect

S.

aureus

in

the

context

of

an

infection

because

studies

in

this

area

are

limited

but

given

the

medical

importance

of

these

biofilms

it

warrants

further

exploration

Complicating

these

biofilm

studies

are

the

frequent

occurrence

of

quorum-sensing

negative

isolates

in

clinical

models

In

one

study

of

S.

epidermidis

isolates

from

joint

prostheses

infections

were

Agr

mutants

-LSB-

-RSB-

these

mutants

can

also

be

isolated

from

chronic

infections

or

through

extended

passage

in

vitro

-LSB-

-RSB-

It

has

been

proposed

that

the

metabolic

burden

of

the

Agr

cascade

leads

to

the

mutations

in

this

locus

-LSB-

-RSB-

with

parallels

to

the

in

vitro

conversion

of

mucoid

P.

aeruginosa

strains

to

non-mucoid

-LSB-

-RSB-

The

frequent

occurrence

of

Agr

mutants

suggests

it

may

be

advantageous

for

some

portion

of

an

S.

aureus

population

to

dispense

with

the

quorum-sensing

system

By

inactivating

Agr

a

more

heterogeneous

robust

biofilm

is

likely

to

form

with

attachment

of

the

mutants

improved

by

the

higher

level

of

surface

factor

expression

Although

it

remains

to

be

investigated

this

heterogeneity

could

mirror

findings

for

P.

aeruginosa

biofilms

in

which

variants

arise

at

a

greater

frequency

than

in

planktonic

cultures

-LSB-

-RSB-

As

studies

on

S.

aureus

progress

a

plausible

picture

of

the

role

of

quorum-sensing

in

biofilm

development

is

beginning

to

emerge

As

S.

aureus

attaches

and

develops

an

initial

biofilm

heterogeneity

arising

as

a

result

of

generation

of

Agr

mutants

may

lead

to

a

more

robust

structure

with

improved

adherence

properties

Communication

between

cells

will

be

dictated

by

the

secretion

and

sensing

of

AIP

molecules

which

will

enable

cross-activation

or

inhibition

of

quorum-sensing

depending

on

the

staphylococcal

subspecies

present

in

close

proximity

When

a

quorum

is

reached

a

portion

of

the

biofilm

will

slough

off

by

down-regulation

of

adherence

factors

which

could

potentially

be

dictated

by

the

action

of

secreted

proteases

or

amphipathic

peptides

The

detached

S.

aureus

cells

will

have

an

activated

quorum-sensing

system

that

leads

to

the

secretion

of

numerous

invasive

factors

enabling

spread

through

the

host

tissues

and

the

development

of

an

infection

This

model

is

in

direct

contrast

to

the

well-studied

P.

aeruginosa

paradigm

for

which

quorum

sensing-regulated

functions

are

important

for

maintaining

the

structural

integrity

of

the

biofilm

Clearly

more

studies

are

required

to

confirm

or

alter

this

hypothetical

view

of

the

S.

aureus

lifecycle

but

as

this

model

is

refined

it

may

play

a

significant

role

in

the

development

of

treatment

for

staphylococcal

diseases

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

clinical/biomedical analysis
cell systems/single cell analysis
biosensors