More on BILOFILM

[Guest guest]

Chemistry and Industry

February 26, 2007

Killer dialogue: when bacteria are told to do so they form biofilms

on solid surfaces which reduce the effectiveness of antibiotics.

Corfield looks at ways of inhibiting these instructions;

Bacteria

BYLINE: Corfield,

SECTION: Pg. 24(2) No. 4 ISSN: 0009-3068

LENGTH: 1564 words

The idea of alien intelligences calmly watching our every move

anddiscussing

the right time to take the Earth from us is the stuff of books,

movies and nightmares. But how would you feel if you knew thatevery

time you sat in the dentist's chair your dentist was fighting a

losing battle with exactly such afoe?

One of the most startling discoveries to come out of the

burgeoning world of microbiology in the past two decades is the fact

that bacteria can communicate.

In fact, not just communicate but converse on alevel where they can

induce each other to switch on dormant genes that then have the

capacity to do you harm.

Dental plaque, it turns out, is the least of our worries. The

superbug MRSA is part of the same phenomenon, called 'quorum sensing'.

Quorum sensing is the ability of bacteria to communicate and

coordinate behaviour using signalling molecules called autoinducers.

Autoinducers are continuously produced by bacteria but when their

concentration reaches a certain threshold--that is to say, when the

bacteria producing it have a quorum--they switch on transcription

genes withinthe bacteria's DNA telling it to do two things: to

produce more autoinducer and, crucially, to change behaviour.

It is the behaviour change that does the damage. Most bacteria

spend their lives as free-swimming, planktonic organisms but when

mandated to do so by the autoinducer will switch to a sessile

lifestyle, dropping out of the swimming phase and anchoring to the

nearest solid surface, be it a tooth, contact lens or the newly-

minted plastic ball-joint of a hip replacement. There they form

biofilms--bacterial mats that reduce the effectiveness of antibiotics

and where the local concentration of autoinducer goes through the

roof. Once a biofilm is established it is very difficult to get rid

of--witness the ubiquity of dental plaque and the dogged resistance

of MRSA to treatment.

Dietrich Mack at the University of Swansea and his group have

beenactive in discovering just how quorum sensing works. In

Staphylococcus aureus and S. epidermidis--the microbes responsible

for both MRSA and implantation rejects--they have identified at least

two major gene expression pathways responsible for initiating biofilm

formation, one based on a polysaccharide and the other on a peptide.

But as Mack acknowledges, 'Our research shows that the expression of

these pathways is not straightforward. In certain cases where

weinhibit a gene responsible involved in quorum sensing it can

actually increase the amount of biofilm formed'.

Quorum sensing, however, does more than merely initiate biofilm

formation. It is a potent weapon of war between bacteria. For

example, the four most common strains of S. aureus, including MRSA,

use four slightly different autoinducers to initiate biofilm

formation, all of which also aggressively inhibit the receptor sites

of the other strains.

The strain that reaches its critical quorum level first not only gets

to put down its biofilm inducing roots first, it also gets to silence

its competitors, preventing them from building up more of theirown

autoinducer.

Production of orthopaedic implants--from artificial hips to hip

and knee joints--is an expanding industry worth $2.5bn in 2005 in

Europe, according to Frost & Sullivan. Eighty per cent of hospital-

acquired infections are associated with implants or other 'in-

dwelling' medical devices, while 60% of hospital infections generally

involve biofilms. Since MRSA and other biofilm-infections are

frequently fatal, there are compelling financial and ethical reasons

to find ways of preventing biofilm formation.

Just how biofilms heighten resistance to antibiotics is not

straightforward.

It may be simply because the ability of antibiotics to penetrate the

biofilm to the bacterial cells themselves is impaired, or it may be

that the life-style change from planktonic to sessile changes the

metabolic state of the bacterial cell and therefore its resistance to

antibiotics. A more extreme suggestion is that the bacteria are

fundamentally altered in some way so that they behave more like a

multicellular tissue than a loose agglomeration of co-operating single

cells.

For years, the approach to tackle biofilms has been incorporate

antimicrobial agents in biomaterials that are to be used within the

body, but the problem is the incredible ability of bacteria to

develop antibiotic resistance. Because of their short generation time

and their uncomplicated genomes, the fact is that bacteria can mutate

and develop resistance faster than we can develop drugs to combat

them. Simply killing bacteria in situ can lead to dead microbial cells

and associated detritus fouling the surfaces of crucial implants.

Defeating biomaterial biofilm formation requires a different

approach, one that does not result in the death of the bacterium but

rather in the neutralisation of its malevolence.

There are several possibilities: coating the biomaterial with

substances that prevent bioadhesion, developing responsive surfaces

that react to bacterial invasion, controlling the orientation of

surface-tethered adhesion molecules, or interfering with receptor-

ligand specific adhesion. But as Llinos and Geoff s of

the AO Foundation in Davos, Switzerland, point out: 'no surface

modification or coating fully prevents bacterial adhesion'.

This leaves perhaps the most exciting possibility of all:

disabling their quorum sensing mechanisms so that the bacteria cannot

form biofilms in the first place.

Several signal molecule families involved in quorum sensing have

been identified in Gram-negative bacteria--those with two sets of

cellmembranes--like Pseudomonas aeruginosa, but the most intensively

studied is the N-acylhomoserine lactone (AHL) family. AHLs contain a

homoserine lactone ring attached via an amide bond to an acyl side

chaincontaining anything from four to 14 carbon atoms.

Once the AHL reaches a critical threshold, concentration members

of the LuxR and LuxN family of transcriptional activator genes are

switched on, forming proteins that start binding the bacteria to the

substrate, thereby beginning biofilm formation. Variations in the

chain length and oxidation at the 3-position provide different Gram-

negative bacteria with species-specific languages with which they can

communicate with their own kind.

Yet, since 75 Gram-negative bacterial species are known to use

AHL, and only 25 AHL varieties have been found, it must also be the

casethat some of these species share a common tongue and can

therefore talk across species boundaries.

Since biofilms usually consist of a multitude of different bacteria

species--which have different niches and therefore do not compete

with each other--this implies that different bacteria co-operate in

biofilm formation.

It is a frightening thought.

There is some good news, however. Some natural molecules have

beenfound to interfere with AHL-mediated quorum sensing and the most

important of these are halogenated furanones produced by the large

marine alga Delisea pulchra.

Halogenated furanones are structurally similar to AHLs and interfere

with the ability of AHLs to bond to biomaterial surfaces. An analogy

would be the way that carbon monoxide interferes with oxygen's

ability to bond with haemoglobin

y occupying the haemoglobin's receptor sites first.

The Australian firm Biosignal is leading the way in the

application of antibiofilm agents to contact lenses. Biosignal's

compounds are based on the naturally occurring furanones from Delisea

pulchra. As the trend toward long-wearing disposable contact lenses

gathers momentum it is increasingly important to make sure that the

lenses do not grow a biofilm and cause eye infection. An initial

human safety trial of their furanone-based coating was completed last

year and the results look positive. 'The potential market is

enormous', says Oredsson, Biosignal's ceo, 'somewhere between

$5bn and $6bn per year. We plan to levy a small but meaningful

royalty on the use of Biosignal's proprietary technology--we aim for

around 5%.'

Gram-positive bacteria like S. aureus and Staphylococcus

epidermidis--the major cause of implant biofilm infections--use

peptides rather than AHLs as signal molecules. In S. epidermidis a

single peptide, once it has reached its critical level, activates an

accessory gene regulator (agr) operon that results in the synthesis

of PolysaccharideIntercellular Adhesin (PIA), a molecular glue that

starts the process of biofilm formation.

An inhibiting peptide, appropriately known as RIP, can inhibit

biofilm formation in both S. epidermidis and S. aureus and is under

investigation as a potential treatment for Staphylococcus-induced

infections.

As yet, however, there is no magic bullet for preventing Gram-

positive, quorum sensing-induced, biofilm formation, and scaling up

thesetechniques to clinical level offers substantial technical

challenges. When asked exactly how inhibition of quorum sensing can

be used to stop MRSA biofilm formation,

Dietrich Mack responded: 'That is a verygood question. I would like

to know the answer to that too.'

But there is everything left to play for. Oredsson acknowledges

that contact lenses are just the tip of the iceberg, and that the

impetus behind quorum sensing remains a cure for serious bacterial

infections, including those caused y MRSA.

Corfield is a science writer based in Oxfordshire

LOAD-DATE: March 27, 2007

McGiffert

Campaign Manager

www.StopHospitalInfections.org

512-477-4431 ext 115

512-477-8934 fax