An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal: P. aeruginosa biofilm formation, tolerance, and dispersal

M I N I R E V I E W

Anupdate onPseudomonasaeruginosa bio¢lmformation,tolerance, and dispersalMorten Harmsen1, Liang Yang2, Sunje J. Pamp3 & Tim Tolker-Nielsen1

1Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark;2Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark; and 3Department of Microbiology and Immunology, Stanford

University, School of Medicine, Palo Alto, CA, USA

Correspondence: Tim Tolker-Nielsen,

Department of International Health,

Immunology and Microbiology, Faculty of

Health Sciences, University of Copenhagen,

Blegdamsvej 3B, DK-2200 Copenhagen N,

Denmark. Tel.: 145 353 26 656; fax: 145

353 27 853; e-mail: [email protected]

Received 5 January 2010; revised 16 March

2010; accepted 21 April 2010.

Final version published online 18 May 2010.

DOI:10.1111/j.1574-695X.2010.00690.x

Editor: Gianfranco Donelli

Keywords

biofilm formation; tolerance; dispersal;

Pseudomonas aeruginosa.

Abstract

We review the recent advances in the understanding of the Pseudomonas aeruginosa

biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers

and microtiter trays. Recent work sheds light on the role of nutrients, motility, and

quorum sensing in structure formation in P. aeruginosa biofilms. The second

messenger, c-di-GMP, is established as an important regulator of the synthesis of

polysaccharide and protein components of the biofilm matrix. Extracellular DNA

is shown to be an essential component of the biofilm matrix. It has become

apparent that biofilm formation involves interactions between different subpopu-

lations. The molecular mechanisms underlying the tolerance of biofilm bacteria to

antimicrobial agents are beginning to be unraveled, and new knowledge has been

obtained regarding the environmental cues and regulatory mechanisms involved

in biofilm dispersal.

Introduction

Microbial biofilms have been subject to intense study during

the last decade mainly for two reasons. First, it is of basic

scientific interest to understand how bacteria form and live

in multicellular communities. Second, biofilm formation

causes considerable problems in medical and industrial

settings, because bacteria in biofilms can resist antibiotic

treatment, host immune responses, and biocide treatment.

Knowledge of the environmental cues, genetic elements, and

molecular mechanisms that are involved in biofilm forma-

tion is necessary for a rational design of strategies to

eliminate biofilms or to prevent biofilm formation.

A substantial part of the studies of microbial biofilms

conducted during the last decade has involved in vitro

laboratory setups such as microtiter trays and flow cham-

bers. Although the model biofilms grown in these setups

most likely differ from biofilms formed in nature, experi-

ments with these biofilms have provided important insights

regarding the fundamental processes of biofilm formation,

tolerance development, and biofilm dispersal that may be of

relevance outside the laboratory systems. In the present

review, we present an update on biofilm formation, toler-

ance, and dispersal based on in vitro studies with the

opportunistic pathogen Pseudomonas aeruginosa, which is a

model organism for biofilm research. Of the numerous

factors involved in the biofilm formation process, we

presently focus on attachment, motility, matrix production,

quorum sensing, and subpopulation interactions.

Attachment

Transport of P. aeruginosa bacteria to a surface before

attachment is assumed to involve diffusive, convective, and

active flagellum-driven transport (van Loosdrecht et al.,

1990). A variety of components including flagella (O’Toole

& Kolter, 1998; Sauer et al., 2002), type IV pili (O’Toole &

Kolter, 1998; Deziel et al., 2001; Chiang & Burrows, 2003),

Cup fimbria (Vallet et al., 2001), extracellular DNA (eDNA)

(Whitchurch et al., 2002), and Psl polysaccharide (Ma et al.,

2009) have been shown to play a role in the attachment of P.

aeruginosa to surfaces in microtiter trays and flow chambers.

FEMS Immunol Med Microbiol 59 (2010) 253–268 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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mailto:[email protected]

However, conditions under which lack of flagella and/or

type IV pili did not affect surface attachment in flow

chambers have also been reported (Klausen et al., 2003b).

After P. aeruginosa has attached to a surface, it either

detaches, remains attached at the position of attachment,

or moves along the surface by means of type IV pili or

flagella (Singh et al., 2002; Klausen et al., 2003a, b; Singh,

2004; Shrout et al., 2006). The gene sadB has been shown to

be involved in the regulation of the frequency by which P.

aeruginosa cells attach to and detach from surfaces (Caiazza

& O’Toole, 2004). Synthesis of the attachment regulator

SadB is regulated by the intracellular level of the second

messenger molecule cyclic diguanosine-50-monophosphate

(c-di-GMP) (Merritt et al., 2007).

Motility

Experiments in flow-chamber setups have suggested that

surface-associated motility is an integrated part of P. aerugi-

nosa biofilm formation (e.g. Singh et al., 2002; Klausen et al.,

2003a, b; Singh, 2004; Shrout et al., 2006; Patriquin et al.,

2008). The pattern of motility occurring in P. aeruginosa

biofilms appears to be dependent on the prevailing condi-

tions. With glucose as a carbon source, for example, the

attached bacteria differentiate initially into a nonmotile

subpopulation and a motile subpopulation (Klausen et al.,

2003a). The nonmotile subpopulation forms microcolonies

that serve as the ‘stalks’ of ‘mushroom’-shaped multicellular

structures that are formed when bacteria from the motile

subpopulation colonize the stalks and subsequently form

mushroom ‘caps’ upon the stalks (Klausen et al., 2003a).

With citrate as a carbon source, the entire bacterial popula-

tion is motile in the initial phase of biofilm formation and

microcolonies that could serve as mushroom stalks are not

formed, and consequently, a flat biofilm is formed (Klausen

et al., 2003b). Pseudomonas aeruginosa pilA mutants (defi-

cient in the biogenesis of type IV pili) formed protruding

microcolonies in biofilms irrigated with a citrate medium,

indicating that the motility occurring in the initial phase of

biofilm formation in citrate-grown P. aeruginosa wild-type

biofilms is driven by type IV pili and thus most likely is

similar to twitching motility (Klausen et al., 2003b). As

described below, P. aeruginosa twitching motility is stimu-

lated by iron limitation. When citrate is used as a carbon

source, it is present at a much higher concentration than

iron in the medium. Because citrate chelates iron, it is

possible that the use of citrate as a carbon source in flow-

chamber systems reduces the level of iron available to the

bacteria, which may explain why the entire population is

motile initially in P. aeruginosa biofilms irrigated with citrate

medium.

Twitching motility has been shown to be induced in

P. aeruginosa biofilms by iron limitation (Singh et al., 2002;

Singh, 2004; Patriquin et al., 2008). In accordance, P.

aeruginosa wild-type bacteria, which formed mushroom-

shaped structures in flow cells irrigated with iron-replete

medium, formed flat biofilms in flow chambers irrigated

with an iron-depleted medium (Patriquin et al., 2008).

Evidence was provided that iron-limitation-promoted

twitching motility is dependent on a functional Rhl quor-

um-sensing system, and a P. aeruginosa rhlI mutant was

shown to form microcolonies (lacking mushroom caps) in

flow chambers irrigated with an iron-depleted medium

(Patriquin et al., 2008). A role of the Rhl quorum-sensing

system in an iron-limitation-induced phenotype is in accor-

dance with numerous studies showing that the quorum-

sensing systems in P. aeruginosa are induced under iron

limitation, but repressed in the presence of high levels of

iron (e.g. Bollinger et al., 2001; Kim et al., 2005; Duan &

Surette, 2007). Spent medium from iron-limited, but not

iron-replete, P. aeruginosa wild-type cultures could induce

iron-limitation-promoted twitching motility of P. aerugino-

sa rhlI mutants, suggesting that some soluble factor, which is

low-iron-inducible and RlhI dependent, positively influ-

enced twitching motility (Patriquin et al., 2008). The avail-

able evidence suggested that this soluble factor is the

biosurfactant rhamnolipid, but it was not investigated

whether spent medium from P. aeruginosa rhlA cultures

(deficient in the biogenesis of rhamnolipid) failed to induce

iron-limitation-promoted twitching motility of P. aerugino-

sa rhlI mutants (Patriquin et al., 2008). In agreement with

the suggestion that increased levels of rhamnolipid may

stimulate twitching motility in iron-limited biofilms, Pamp

& Tolker-Nielsen (2007) reported that in addition to the

well-known role of rhamnolipid in facilitating swarming

motility (e.g. Kohler et al., 2000), the biosurfactant can also

facilitate twitching motility. If increased levels of rhamnoli-

pid produced in iron-deplete P. aeruginosa biofilms stimu-

late twitching motility and thereby prevent microcolony

formation, it would be expected that a P. aeruginosa rhlA

mutant would be able to form microcolonies under iron-

limited conditions. However, Pamp & Tolker-Nielsen (2007)

provided evidence that rhamnolipid is necessary for initial

microcolony formation in P. aeruginosa biofilms. In support

of a role of rhamnolipid in initial microcolony formation, it

has been shown that low concentrations of rhamnolipid

enhance the hydrophobicity of P. aeruginosa bacteria by

causing a release of lipopolysaccharide (LPS) from the cell

surface (Zhang & Miller, 1994; Al-Tahhan et al., 2000). An

increase in cell surface hydrophobicity may increase the

adhesiveness of the bacteria to a level that is critical for

initial microcolony formation in biofilms. It appears that

rhamnolipid, depending on the quantity produced, plays

multiple roles in P. aeruginosa biofilm formation. It is

necessary for initial microcolony formation (Pamp & Tolk-

er-Nielsen, 2007); it may prevent initial microcolony

FEMS Immunol Med Microbiol 59 (2010) 253–268c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

254 M. Harmsen et al.

formation in iron-limited biofilms by stimulating twitching

motility (Patriquin et al., 2008); it facilitates bacterial

migration and thereby the formation of mushroom caps

(Pamp & Tolker-Nielsen, 2007); it prevents colonization of

the channels between the mushroom-shaped structures

(Davey et al., 2003); and it plays a role in biofilm dispersal

(Schooling et al., 2004; Boles et al., 2005).

Evidence has been presented that the formation of the

mushroom caps in P. aeruginosa biofilms requires both type

IV pili and flagella (Klausen et al., 2003a; Barken et al.,

2008). Cap formation evidently requires flagellum-driven

surface-associated motility, whereas the dependence of cap

formation on type IV pili may be due to the binding of these

pili to eDNA that is abundant on the microcolonies that

become colonized during mushroom-structure formation

(Whitchurch et al., 2002; Allesen-Holm et al., 2006; Barken

et al., 2008). Alternatively, a kind of migration that requires

both flagella and type IV pili might be occurring in the later

phase of P. aeruginosa biofilm formation. In support of this

suggestion, it has been reported that P. aeruginosa swarming

motility in addition to flagella and rhamnolipid may also

require type IV pili under some conditions (Kohler et al.,

2000).

As described in the next section, evidence is accruing that

the shift between migrating and sessile surface behavior is

regulated via proteins with diguanylate cyclase or phospho-

diesterase activities that control levels of the second messen-

ger molecule c-di-GMP (Hickman et al., 2005; Kuchma

et al., 2007; Merritt et al., 2007). Low intracellular levels of

c-di-GMP promote motility, whereas high intracellular c-

di-GMP levels induce the formation of cell–cell intercon-

necting matrix components and promote microcolony

formation.

Matrix production

The extracellular polymeric substance (EPS) matrix serves as

the ‘house for biofilm cells’. The matrix plays a role in

numerous processes including attachment, cell-to-cell inter-

connection, interactions between subpopulations, tolerance,

and exchange of genetic material (Molin & Tolker-Nielsen,

2003; Friedman & Kolter, 2004a; Jackson et al., 2004; Ma

et al., 2009; Yang et al., 2009). The P. aeruginosa EPS matrix

contains mainly polysaccharides, proteins, and nucleic acids

(e.g. Whitchurch et al., 2002; Friedman & Kolter, 2004a;

Jackson et al., 2004; Borlee et al., 2010). The composition of

the matrix depends on the environmental conditions, the

age of the biofilm, and the particular P. aeruginosa strain

forming the biofilm. Evidence has been provided that

P. aeruginosa induces the synthesis of matrix components

in response to environmental signals sensed by the sensor

kinase/response regulators LadS, RetS, and GacS (Goodman

et al., 2004; Ventre et al., 2006; Goodman et al., 2009).

In addition, sensor kinases and response regulators encoded

by the bfiSR, bfmSR, and mifSR genes are evidently involved

in further P. aeruginosa biofilm formation (Petrova & Sauer,

2009), although the functions affected by these signal

transduction systems have not been identified. Below, we

describe the involvement of the matrix components Pel, Psl,

alginate, CdrA protein, Cup fimbria, type IV pili, lectins,

and eDNA in P. aeruginosa biofilm formation.

The glucose-rich Pel polysaccharide, encoded by the pel

cluster, was initially reported to be required for the forma-

tion of P. aeruginosa liquid–air interface pellicles and sur-

face-associated biofilms (Friedman & Kolter, 2004b).

Evidence has been presented that the transmembrane pro-

tein PelD binds c-di-GMP, and that there is a strict correla-

tion between c-di-GMP binding and the synthesis of the Pel

polysaccharide (Lee et al., 2007; Lory et al., 2009). Hickman

& Harwood (2008) presented data showing that c-di-GMP

binds to the transcriptional regulator FleQ and thereby

derepresses the transcription of the pel genes. The pel locus

is furthermore regulated by the sensor kinase-response

regulator hybrids RetS and LadS, which repress and activate

its expression, respectively (Goodman et al., 2004; Ventre

et al., 2006). This regulation is mediated through the small-

RNAs, RsmZ/Y, which alter the level of the free RsmA post-

transcriptional regulator (Goodman et al., 2004; Ventre

et al., 2006). The two-component system GacA/S interacts

with the RetS and LadS regulatory network, and recent data

suggest that RetS can modulate the phosphorylation state of

GacS by forming heterodimers that block the GacS autop-

hosphorylation, leading to a reduced rsmZ expression, and

subsequently free RsmA, thereby repressing exopolysacchar-

ide synthesis and promoting the translation of genes neces-

sary for acute infection (Goodman et al., 2009). Recent work

showed that rugose small-colony variants (RSCVs) of

P. aeruginosa isolated from laboratory biofilms and cystic

fibrosis (CF) patients had increased expression of the pel

locus compared with clonally related wild types, and it was

suggested that RSCVs may partially contribute to increased

persistence of biofilms in the airways of CF lungs (Starkey

et al., 2009). It was further shown that c-di-GMP signaling

regulated the increased expression of the pel locus in the

RSCVs (Starkey et al., 2009).

The mannose-rich Psl polysaccharide, encoded by the psl

cluster, is highly conserved in many P. aeruginosa strains

(Friedman & Kolter, 2004a; Ryder et al., 2007). The Psl

polysaccharide mediates cell-to-surface and cell-to-cell

interactions, which are essential for P. aeruginosa biofilm

formation and maintenance (Overhage et al., 2005; Ma et al.,

2006, 2009). In a recent study, Ma et al. (2009) showed that

Psl forms a helical structure around P. aeruginosa cells that

increase cell-to-surface and cell-to-cell interactions neces-

sary for biofilm formation. By lectin staining, Psl was shown

to be located predominantly in the peripheries of the


255P. aeruginosa biofilm formation, tolerance, and dispersal

mushroom-shaped structures, whereas it was evenly distrib-

uted in flat biofilms (Ma et al., 2009). As for the Pel

polysaccharide, Psl expression is regulated by LadS and RetS

(Goodman et al., 2004; Ventre et al., 2006) and by c-di-GMP

(Hickman et al., 2005). A newly characterized protein,

CdrA, which shows increased expression with elevated c-

di-GMP levels, is suggested to either crosslink Psl polymers

and/or tether Psl to the P. aeruginosa cell wall (Borlee et al.,

2010). This protein is the first to be identified as a structural

component of the P. aeruginosa biofilm matrix. Borlee et al.

(2010) found that the cdrAB complex is elevated in biofilms,

and that overexpression of both proteins induces auto-

aggregation if Psl is present. The second protein in the

complex, CdrB, works as an outer-membrane transporter of

CdrA. Evidence was provided that CdrA binds directly to the

mannose part of Psl, and it was suggested that CdrA is a

multivalent adhesin with the ability to recognize multiple

carbohydrates (Borlee et al., 2010).

Alginate is an acetylated polysaccharide composed of

nonrepetitive monomers of b-1,4-linked L-guluronic and

D-mannuronic acids (Govan & Deretic, 1996), and is mainly

produced by P. aeruginosa in chronic infections of the lungs

of CF patients (Hoiby, 1974; Govan & Deretic, 1996). Its

special physical and chemical properties play multiple roles

in protecting P. aeruginosa cells (Govan & Deretic, 1996).

Biofilms formed by an alginate-overproducing strain were

shown to exhibit a highly structured architecture and were

significantly more resistant to the antibiotic tobramycin

than biofilms formed by an isogenic nonmucoid strain

(Hentzer et al., 2001). This might indicate that alginate acts

as a physical barrier for antibiotics because alginate lyase

treatment has been shown to enhance the diffusion of

aminoglycosides through the EPS of biofilms formed by

mucoid P. aeruginosa strains (Hatch & Schiller, 1998). A

recent study showed that a supermucoid P. aeruginosa strain

was strongly impaired in attachment compared with the

respective mucoid or nonmucoid strains and formed a

thicker biofilm with large extended mushroom-like micro-

colonies (Hay et al., 2009a). Recently, it has been found that

post-transcriptional regulation plays a role in the biosynth-

esis of alginate in P. aeruginosa. The membrane-anchored

protein, Alg44, which is essential for alginate biosynthesis,

contains a c-di-GMP-binding PilZ domain (Amikam &

Galperin, 2006; Remminghorst & Rehm, 2006b; Merighi

et al., 2007; Oglesby et al., 2008; Lory et al., 2009). Regula-

tion of Alg44 activity via the PilZ domain has recently been

suggested to occur through a new membrane-anchored

diguanylate cyclase, MucR, which has been identified as a

positive regulator of alginate biosynthesis (Hay et al.,

2009b). It was suggested that MucR possibly acts by the

localized production of c-di-GMP in the vicinity of Alg44.

The PilZ domain of Alg44 in turn responds to the high local

concentration of c-di-GMP and promotes the production of

alginate. The glycosyltransferase Alg8 was characterized

recently and shown to be critical for alginate production

(Remminghorst & Rehm, 2006a; Oglesby et al., 2008). It was

shown that the Alg44 and Alg8 proteins were required in

combination for the polymerization reactions leading to

alginate production (Oglesby et al., 2008).

Recently, eDNA was recognized as one of the major

matrix components of bacterial biofilms (Whitchurch et al.,

2002; Qin et al., 2007; Rice et al., 2007). It was shown that

DNAse treatment led to the dispersal of young P. aeruginosa

biofilms (Whitchurch et al., 2002). However, DNAse did not

disperse a flow-chamber-grown P. aeruginosa mature bio-

film, probably due to increasing amounts of other EPS

materials being produced during biofilm formation. South-

ern and RAPD PCR analysis provided evidence that the

eDNA in the P. aeruginosa biofilm matrix is similar to

chromosomal P. aeruginosa DNA (Allesen-Holm et al.,

2006). In addition to a small amount of eDNA that is

present and necessary in the initial phase of P. aeruginosa

biofilm formation (Whitchurch et al., 2002), the release of a

large amount of eDNA occurs during subsequent P. aerugi-

nosa biofilm formation, evidently through lysis of a small

subpopulation of the bacteria regulated via the P. aeruginosa

quinolone signal (PQS) quorum-sensing system (Allesen-

Holm et al., 2006). In agreement with a role of quorum

sensing in cell lysis, D’Argenio et al. (2002) reported that

mutants that could not produce the PQS quorum-sensing

signal molecule did not show autolysis, whereas mutants

that overproduced PQS displayed high levels of autolysis. In

addition, Heurlier et al. (2005) presented evidence that P.

aeruginosa quorum-sensing mutants, unlike the wild type,

did not undergo cell lysis in stationary-phase cultures.

Moreover, Yang et al. (2007) presented evidence that high

levels of iron suppressed P. aeruginosa pqs gene expression,

DNA release, and structural biofilm development. Quino-

lone compounds have previously been shown to induce

prophages in bacteria (Phillips et al., 1987; Froshauer et al.,

1996), and studies by Webb et al. (2003) and Hentzer et al.

(2004) have suggested that quorum-sensing-regulated DNA

release might be linked to bacteriophage induction in

biofilms. PQS was shown to be necessary for membrane

vesicle formation in P. aeruginosa (Mashburn & Whiteley,

2005; Nakamura et al., 2008), and membrane vesicles

produced by P. aeruginosa might also play a role in DNA

release. The membrane vesicles released by P. aeruginosa

have bacteriolytic effects and contain DNA (Kadurugamuwa

& Beveridge, 1996; Renelli et al., 2004). eDNA might be

released either from vesicles that eventually lyse or through

the bacteriolytic activity of the vesicles that might lyse a

small subpopulation of P. aeruginosa bacteria. The eDNA

appears to be organized in distinct patterns in P. aeruginosa

biofilms (Allesen-Holm et al., 2006). In flow-chamber-

grown P. aeruginosa biofilms, which contain mushroom-



shaped structures, the eDNA was located primarily in the

stalk portion of the mushroom-shaped structures, with the

highest concentration in the outer parts of the stalks

forming a border between the stalk subpopulation and the

cap subpopulation (see Fig. 1). eDNA is primarily generated

in the initial microcolonies before they are colonized by

the cap-forming subpopulation, and the concentration of

eDNA is highest in the outer layer of these microcolonies

(Allesen-Holm et al., 2006; Yang et al., 2007). It is currently

not understood how the migration of the motile cells is

coordinated so that they form mushroom caps. However,

type IV pili bind to DNA with a high affinity (Aas et al.,

2002; van Schaik et al., 2005), and evidence has been

presented that the high concentration of eDNA on the outer

parts of the mushroom stalks can cause accumulation of the

migrating bacteria, which, in combination with bacterial

growth, might result in the formation of the mushroom caps

(Barken et al., 2008). Type IV pili may therefore function as

niche-specific matrix-adhesins, and, as for many other

components of the P. aeruginosa matrix, c-di-GMP appears

to play a role in the regulation of type IV pili synthesis,

in this case through the proteins PilZ and FimX, which have

c-di-GMP-binding domains (Huang et al., 2003; Guzzo

et al., 2009). The eDNA in P. aeruginosa biofilms appears to

have a stabilizing effect, as mature P. aeruginosa PAO1

biofilms that were pretreated with DNAse I were more

susceptible to sodium dodecyl sulfate (SDS) treatment than

biofilms that were not pretreated with DNAse I (Allesen-

Holm et al., 2006).

The P. aeruginosa CupA, B, and C fimbria were shown to

be involved in bacterial attachment and/or subsequent

biofilm formation (Vallet et al., 2001; Kulasekara et al.,

2005). Recently, a new Cup-fimbriae gene cluster, CupD,

has been identified on the pathogenicity island, PAPI-I,

present in P. aeruginosa strain PA14 (Mikkelsen et al.,

2009). Overexpression of this gene cluster results in in-

creased biofilm formation and decreased motility as was

observed for overexpression of the other Cup-fimbrial gene

clusters (Mikkelsen et al., 2009). Cup fimbriae are regulated

by c-di-GMP (Kulasekara et al., 2005; Rao et al., 2008;

Mikkelsen et al., 2009) as are many of the other P. aeruginosa

matrix components. Expression of cupA was shown to be

dependent on proteins with diguanylate cyclase/c-di-GMP-

binding domains (WspR, MorA, and PA1120) (D’Argenio

et al., 2002; Meissner et al., 2007), whereas expression of

cupB and cupC was shown to be regulated by the RocR

phosphodiesterase protein (Kulasekara et al., 2005; Rao

et al., 2008), and expression of cupD was regulated by the

PvrR phosphodiesterase protein (Mikkelsen et al., 2009).

Overexpression of the PvrR protein has previously been

shown to reduce CupA fimbria expression likely as a

consequence of a reduction in the intracellular c-di-GMP

level (Meissner et al., 2007). CupA was recently shown to be

involved in SDS-induced autoaggregation dependent on

increased intracellular levels of c-di-GMP, and two newly

characterized proteins: SiaA and SiaD (Klebensberger et al.,

2009).

Evidence has been provided that the lectin LecB binds to

specific carbohydrate ligands located on the surface of

P. aeruginosa cells, and a P. aeruginosa lecB mutant was

shown to be impaired in biofilm formation in comparison

with the wild-type strain, suggesting an important role for

LecB in biofilm development (Tielker et al., 2005).

Quorum sensing

Quorum sensing in P. aeruginosa is mediated through three

interconnected systems: the Las system that senses 3-oxo-

C12-homoserine lactone, the Rhl system that senses C4-

homoserine lactone, and the Pqs system that senses

2-heptyl-3-hydroxy-4-quinolone referred to as PQS (Juhas

et al., 2005). Microarray analysis has suggested that several

hundred genes in P. aeruginosa are quorum-sensing regu-

lated (Schuster et al., 2003; Wagner et al., 2003; Hentzer

et al., 2005).

Shrout et al. (2006) provided evidence that the impact of

quorum sensing on motility in P. aeruginosa biofilms is

nutrition dependent, which may explain the different results

regarding the role of quorum sensing in P. aeruginosa

biofilm formation. Davies et al. (1998) found that a

P. aeruginosa wild type formed structured biofilms with

large mushroom-shaped structures in flow chambers

Fig. 1. Confocal scanning laser microscopy

micrographs acquired in a propidium iodide-

stained biofilm formed by Gfp-tagged Pseudo-

monas aeruginosa PAO1. (a) Vertical section

through a mushroom-shaped multicellular

structure; (b) horizontal section through a mush-

room-shaped multicellular structure. The bacteria

appear green fluorescent and the eDNA appears

red fluorescent. Size bar = 20 mm. Reproduced

from Allesen-Holm et al. (2006).



irrigated with a glucose medium, while the corresponding

lasI quorum-sensing mutant formed flat and undifferen-

tiated biofilms. The flat biofilms formed by the lasI mutant

were susceptible to treatment with the detergent SDS, while

the structured biofilms formed by the wild type were

tolerant. Heydorn et al. (2002) reported that a P. aeruginosa

wild type and lasI mutant both formed flat biofilms in flow

chambers irrigated with citrate medium. Purevdorj et al.

(2002) showed that in flow chambers irrigated with dilute

Luria–Bertani (LB) under high-flow conditions, both the P.

aeruginosa wild type and the lasI mutant formed biofilms

containing large aggregates, although the biofilms differed

slightly in microscopic appearance. Hentzer et al. (2002)

demonstrated that homoserine lactone signal analogues

called furanones, known to inhibit P. aeruginosa quorum

sensing, affected biofilm development when added to the

growth medium consisting of diluted LB. Patriquin et al.

(2008) showed that the P. aeruginosa wild type formed

mushroom-shaped structures in flow chambers irrigated

with dilute tryptic soy broth, whereas an rhlI quorum-

sensing mutant formed microcolonies lacking the mush-

room caps. Similarly, Yang et al. (2009) showed that the P.

aeruginosa wild type formed mushroom-shaped structures

in flow chambers irrigated with glucose medium, whereas a

pqsA quorum-sensing mutant could only form small micro-

colonies lacking the mushroom cap. In addition, evidence

was provided that chemical inhibition of the Pqs system

caused P. aeruginosa to form microcolonies on which mush-

room caps were not subsequently formed (Yang et al., 2007,

2009).

The above-mentioned studies indicate that quorum sen-

sing is necessary for the formation of the cap portion of the

mushroom-shaped structures in P. aeruginosa biofilms.

Interestingly, quorum sensing is, however, shown to occur

mainly in the stalk portion of the mushroom-shaped

structures in P. aeruginosa biofilms (de Kievit et al., 2001;

Yang et al., 2009). Pseudomonas aeruginosa uses quorum

sensing to regulate numerous factors including the produc-

tion of rhamnolipid (Ochsner & Reiser, 1995) and eDNA

(Allesen-Holm et al., 2006). As mentioned in the previous

section, Pamp & Tolker-Nielsen (2007) provided evidence

that rhamnolipid plays a role in mushroom cap formation

by promoting motility occurring in the later phase of P.

aeruginosa biofilm formation. As described in more detail in

the following section, evidence was recently presented that

PQS quorum sensing in the initial microcolonies leads to the

production of eDNA, which plays an important role in the

subsequent formation of the mushroom caps (Allesen-

Holm et al., 2006; Barken et al., 2008; Yang et al., 2009).

In addition to regulating the production of rhamnolipid

and eDNA, quorum sensing evidently regulates a number of

other factors involved in P. aeruginosa biofilm formation.

Sakuragi & Kolter (2007) presented evidence that transcrip-

tion of the pel operon is considerably reduced in P. aerugi-

nosa lasI and rhlI mutants, suggesting that quorum-sensing

signaling regulates the production of Pel polysaccharide

during P. aeruginosa biofilm formation. On the contrary,

however, Ueda & Wood (2009) recently reported that Las

quorum-sensing represses Pel production in P. aeruginosa.

The tyrosine phosphatase TpbA was shown to be positively

regulated by Las quorum sensing, and evidence was pre-

sented that TpbA activity results in decreased levels of

c-di-GMP and thereby a reduction in Pel production (Ueda

& Wood, 2009). Transcriptome analysis has indicated that

the cupA3 and cupB5 genes are subject to quorum-sensing

control in P. aeruginosa biofilms (Hentzer et al., 2004),

suggesting that the expression of CupA and CupB fimbria

may be quorum sensing regulated during P. aeruginosa

biofilm formation. In addition, expression of the lectins

LecA and LecB was shown to be regulated via quorum

sensing (Winzer et al., 2000). The production of both lectins

was found to be directly dependent on the rhl locus, while,

in a lasR mutant, the onset of lectin synthesis was delayed,

but not abolished.

Unlike the previous examples, the last example of an

effect of quorum sensing on P. aeruginosa biofilm develop-

ment is related to the central metabolism in the bacteria.

Evidence has been presented that anaerobic nitrate respira-

tion may play an important role in P. aeruginosa biofilm

development in clinical settings, and that the rhlRI system is

necessary to prevent accumulation of toxic nitric oxide

during the process (Worlitzsch et al., 2002; Yoon et al.,

2002). In agreement, the nirCMSQ and napEF genes, which

are required for respiratory nitrate reduction, were found to

be strongly upregulated in P. aeruginosa during biofilm

growth (Hentzer et al., 2005).

Subpopulation interactions

Pseudomonas aeruginosa bacteria release several compounds

into their surrounding environment, for example iron side-

rophores, biosurfactants, and EPS. A common feature of

these compounds is that they are costly to synthesize and

able to benefit the entire population and therefore can be

regarded as ‘public goods’ (West et al., 2007). Many extra-

cellular public goods play a role in P. aeruginosa biofilm

formation. For example, the iron siderophore pyoverdine is

necessary for the formation of structured P. aeruginosa

biofilms (Banin et al., 2005). The biosurfactant rhamnolipid

plays multiple roles in P. aeruginosa biofilm formation, and

is, among other things, required for biofilm channel main-

tenance as well as for migration of motile subpopulations

(Davey et al., 2003; Pamp & Tolker-Nielsen, 2007). eDNA

released via quorum sensing plays a role as EPS material

required for the formation of structured P. aeruginosa

biofilms (Allesen-Holm et al., 2006).



The bacteria in P. aeruginosa biofilms exist in various

physiological states dependent on their spatial localization.

Pseudomonas aeruginosa biofilms contain subpopulations

that produce public goods and subpopulations that do not

produce public goods. For example the pyoverdine synthesis

genes, rhamnolipid synthesis genes, and quorum-sensing

genes were all reported to be expressed specifically in the

stalk portion of the mushroom-shaped structures in P.

aeruginosa biofilms (De Kievit et al., 2001; Lequette &

Greenberg, 2005; Kaneko et al., 2007; Yang et al., 2007,

2009).

Interactions between producers and nonproducers of

public goods may play a role in P. aeruginosa biofilm

formation. Yang et al. (2009) studied subpopulation inter-

actions in mixed P. aeruginosa biofilms containing a non-

motile subpopulation (pilA mutants) and a motile

subpopulation (wild type). In this model system, the non-

motile bacteria form the stalk portion and the motile

bacteria form the cap portion of mushroom-shaped biofilm

structures. In the study, the role of pyoverdine in subpopu-

lation interactions during P. aeruginosa biofilm formation

was investigated. Using a pvdA<gfp fluorescent reporter, the

pyoverdine synthesis genes were found to be expressed

specifically in the pilA stalk-forming subpopulation of the

mushroom-shaped structures formed in pilA/wild-type

mixed biofilms. In agreement with the study of Banin et al.

(2005), it was found that a P. aeruginosa pvdA mutant could

form small microcolonies, but was unable to form mush-

room-shaped structures (see Fig. 2a), suggesting that pyo-

verdine production is necessary for mushroom structure

formation. In pilA/pvdA mixed biofilms, the pvdA mutant

was able to associate with stalks formed by the pilA mutant

and form the cap portion of the mushroom-shaped biofilm

structures (Fig. 2b), indicating that pyoverdine production

in the cap is not necessary for mushroom-structure forma-

tion. In contrast, in pilApvdA/wild-type mixed biofilms, the

wild type was not able to associate with the pilApvdA

microcolonies (Fig. 2c), suggesting that pyoverdine synth-

esis in the stalk is necessary for the cap to be formed.

Additional experiments provided evidence that even though

the cap-forming subpopulation in the mushroom-shaped

structures did not synthesize pyoverdine; it used the major

pyoverdine receptor FpvA for uptake of pyoverdine synthe-

sized by the stalk-forming subpopulation. Yang et al. (2009)

also studied the role of PQS-mediated DNA release in

subpopulation interactions during P. aeruginosa biofilm

formation. Use of a pqsA<gfp fluorescent reporter provided

evidence that the pqs genes are expressed specifically in the

pilA stalk-forming subpopulation of the mushroom-shaped

structures formed in pilA/wild-type mixed biofilms. A P.

aeruginosa pqsA mutant could form small microcolonies,

but was unable to form mushroom-shaped structures,

suggesting that pqs expression is necessary for mushroom

structure formation. In a pilA/pqsA mixed biofilm, the pqsA

mutant was able to associate with the pilA stalks and form

the caps of the mushroom-shaped structures, indicating that

pqs expression in the cap is not necessary for mushroom-

structure formation. In contrast, in a pilApqsA/wild-type

mixed biofilm, the wild type did not associate with the

pilApvdA microcolonies, but formed independent mush-

room-shaped structures, suggesting that pqs expression in

the stalk is necessary for the cap to be formed. Evidence was

presented that the stalk-forming subpopulation produces

eDNA via PQS quorum-sensing, and that the motile bacteria

bind to the eDNA using type IV pili and thereby initiate cap

formation. The PQS quorum-sensing system is probably

expressed in the stalk microcolonies in P. aeruginosa bio-

films due to the high cell concentration in this location.

Because PQS chelates iron and can thereby induce the

synthesis of pyoverdin and its receptors (Bredenbruch

Fig. 2. Confocal scanning laser microscopy (CSLM) micrographs acquired in biofilms formed by Pseudomonas aeruginosa pvdA (a), a pilA/pvdA mixture

(b), and a pilApvdA/wild-type mixture (c). The wild type and pvdA mutant were tagged with Yfp (yellow), whereas the pilA mutant and the pilApvdA

mutant were tagged with Cfp (blue). The central pictures show horizontal CLSM optical sections, and the flanking pictures show vertical CLSM optical

sections. Size bars = 20 mm. Reproduced from Yang et al. (2009).



et al., 2006; Diggle et al., 2007), it is possible that the

observed distributed expression of the Pvd system in P.

aeruginosa biofilms is linked to the distributed expression of

the PQS system.

The study by Yang et al. (2009) is one of the first to

indicate that the formation of heterogeneous biofilms by P.

aeruginosa might occur through mechanisms that involve

complex interactions between subpopulations. The pilA,

pvdA, and pqsA mutants, which alone are deficient in the

formation of mushroom-shaped biofilm structures, are

shown in this study to interact with each other and together

build mature mushroom-shaped biofilm structures.

Tolerance

One of the most important features of microbial biofilms is

their tolerance to antibiotics and components of the host

immune system. Although antimicrobial agents may de-

crease the number of bacteria in biofilms, they will not

completely eradicate the bacteria, which may have impor-

tant clinical consequences in the form of relapses of infec-

tions. Tolerance to antimicrobial agents is a physiological

condition that does not involve mutation and allows the

bacteria to survive, but not necessarily grow, in the presence

of the antimicrobial agent. Investigations of P. aeruginosa

biofilms have revealed that biofilm tolerance is multifactor-

ial. The mechanisms that contribute to tolerance include

restricted antimicrobial diffusion, differential physiological

activity, induction of specific tolerance mechanisms, and

persister cell formation.

The available evidence suggests that biofilm matrices in

general do not inhibit diffusion of antibiotics, but penetra-

tion of some antimicrobial compounds appears to be

delayed. Penetration of ciprofloxacin and levofloxacin

through P. aeruginosa flow-chamber biofilms was found

not to be significantly delayed (Vrany et al., 1997). In

support of this, Walters et al. (2003) found that penetration

of ciprofloxacin was also not significantly delayed in a P.

aeruginosa colony biofilm; however, penetration of tobra-

mycin was somewhat retarded, but eventually penetrated the

biofilm completely. Whereas most antimicrobials might

diffuse readily through biofilms formed by wild-type P.

aeruginosa strains, it appears that alginate produced by

mucoid P. aeruginosa strains can retard the diffusion of

some antimicrobials (e.g. piperacillin, amikacin, gentami-

cin), whereas others penetrate readily (e.g. ciprofloxacin,

levofloxacin, sparfloxacin, ofloxacin) (Hoyle et al., 1992;

Shigeta et al., 1997). Recently, evidence was provided that

the activity of an antibiotic on mucoid P. aeruginosa biofilms

can be significantly enhanced by addition of alginate lyase,

and DNase, suggesting that alginate and eDNA can function

as an antibiotic barrier (Alipour et al., 2009). Cochran et al.

(2000) found that wild-type P. aeruginosa cells attached to

alginate beads were significantly less susceptible to disinfec-

tion by hydrogen peroxide than planktonic cells of the same

microorganism, although diffusion of hydrogen peroxide

was not significantly delayed, indicating that mechanisms

other than diffusion barriers contribute to the tolerance.

Biofilms contain distinct subpopulations of cells that

exhibit differential physiological states. An increasing body

of evidence suggests that the prevailing physiological states

of biofilm cell subpopulations directly relate to their sus-

ceptibility and tolerance phenotypes dependent on the

antimicrobial compound used. Evidence has been provided

that P. aeruginosa flow-chamber biofilms, as well as colony

biofilms and biofilms established in drip flow reactors, and

capillary glass tubes, are composed of at least two distinct

physiological subpopulations: a cell subpopulation close to

the substratum (e.g. the stalk portion in biofilms with

mushroom-shaped structures) that exhibits low metabolic

activity and a cell subpopulation on top (e.g. the cap portion

in biofilms with mushroom-shaped structures) that exhibits

high metabolic activity (see Fig. 3a) (Xu et al., 1998; Walters

et al., 2003; Werner et al., 2004; Pamp et al., 2008). This

spatial distribution of bacteria with low and high metabolic

activity is prevailing due to microscale chemical gradients

that are generated by the relative diffusion and consumption

rates of chemicals as can be revealed by microelectrode

measurements (e.g. Xu et al., 1998; Werner et al., 2004). For

example, oxygen and nutrient concentrations are high in the

bulk liquid and top layer of the biofilms, whereas concentra-

tions are low in the deeper layers of the biofilm. Conven-

tional antimicrobial agents that are known to interfere with

fundamental physiological processes of bacterial cells, such

as replication (e.g. ciprofloxacin), or translation (e.g. tetra-

cycline, tobramycin, gentamicin), were found to specifically

kill the metabolically active cells in the top layer of biofilms,

whereas cells of low metabolic activity survived the treat-

ment (see Fig. 3c and d) (Hentzer et al., 2003; Walters et al.,

2003; Banin et al., 2006; Pamp et al., 2008). In contrast,

antimicrobial agents that interfere with bacterial membrane

structure/function, such as colistin, EDTA, and SDS, were

found to kill the cells in the deeper layer, whereas cells of

high metabolic activity in the top layer survived the treat-

ment (see Fig. 3b) (Banin et al., 2006; Haagensen et al., 2007;

Pamp et al., 2008). In addition, DFO-gallium (a post-

transition metal in complex with a siderophore), which is

known to interfere with cellular iron metabolism, was found

to preferentially kill bacteria in the deeper layers of P.

aeruginosa biofilm (Kaneko et al., 2007; Banin et al., 2008).

Systematic combined antimicrobial treatments simulta-

neously targeting physiologically distinct subpopulations,

for example using ciprofloxacin1colistin (see Fig. 3e),

tetracycline1colistin (see Fig. 3f), or gentamicin1DFO-

gallium, enables killing of almost all the bacteria in P.

aeruginosa biofilms (Banin et al., 2008; Pamp et al., 2008).



In addition to tolerance caused by the mechanisms

described above, certain gene products that are produced

specifically in biofilms may have unique functions that

enhance the antibiotic tolerance of the biofilm. One example

of a biofilm-specific factor is the synthesis of periplasmic

glucans that bind tobramycin and prevents cell death most

likely by sequestering the antibiotic (Mah et al., 2003).

Synthesis of the periplasmic glucans requires the ndvB gene

in P. aeruginosa PA14, and biofilms formed by a P. aerugino-

sa ndvB mutant were found to be much more sensitive to

tobramycin than wild-type biofilms. In contrast, the ndvB

mutant and wild type showed no difference in tobramycin

sensitivity when grown in planktonic culture. Reverse tran-

scriptase PCR provided evidence that the ndvB gene was

expressed specifically in P. aeruginosa PA14 biofilms and not

in planktonic cells (Mah et al., 2003). However, microarray

analysis has provided evidence that ndvB is expressed at the

same level in biofilm and planktonic cells of P. aeruginosa

PAO1 (Hentzer et al., 2005), and therefore the ndvB-

mediated mechanism appears to be restricted to specific P.

aeruginosa strains.

Upon antimicrobial exposure, some biofilm cells are able

to actively adapt by inducing the expression of specific genes

that confer tolerance. As described above, only the active

cells in the top layer of P. aeruginosa flow-chamber biofilms

survive colistin treatment, whereas the inactive cells in the

deeper layers are killed by colistin (Fig. 3b). Upon exposure

to colistin, the cells in the top layer induce the expression of

the pmr-LPS-modification system as well as the MexAB-

OprM efflux pump (Pamp et al., 2008). In contrast, the cells

in the deeper layers are unable to induce the pmr operon and

mexAB-oprM genes, and are hence killed by the action of

colistin (Pamp et al., 2008). In contrast to the findings in

biofilms, it was found that planktonic exponential-phase

cells (exhibiting high metabolic activity) and planktonic

stationary-phase cells (exhibiting low metabolic activity)

were equally sensitive to colistin, suggesting that the me-

chanisms resulting in the observed colistin tolerance are

biofilm specific (Pamp et al., 2008). The efflux-pump genes

mexAB-oprM and mexCD-oprJ genes were also found to be

induced in a stack reactor P. aeruginosa biofilm in the

presence of azithromycin and were required for tolerance

development, but the spatial distribution of the tolerant cells

was not investigated (Gillis et al., 2005). As an active

adaptation response to b-lactam antibiotics such as imipi-

nem and ceftazidime, the cells in the top layer of the P.

aeruginosa flow-chamber biofilms were found to specifically

induce the chromosomally encoded AmpC b-lactamase

(Bagge et al., 2004). Based on average transcriptional

measurements on biofilm cell populations in comparison

with planktonic cell populations, expression of a potential

novel efflux system (encoded by PA1874–1877) appeared to

be increased in biofilm cells and was described to be

Fig. 3. Metabolic activity and antibiotic tolerance in Pseudomonas

aeruginosa biofilms. (a) Visualization of metabolically active cells in

P. aeruginosa biofilms. Cells appear green when they synthesize the

unstable variant of the green fluorescent protein Gfp[AGA], which is expre-

ssed under the control of a ribosomal promoter. (b–f) Mature P. aeruginosa

Gfp biofilms exposed to either 25mg mL�1 colistin (b), 60mg mL�1 cipro-

floxacin (c), 200mg mL�1 tetracycline (d), 25mg mL�1 colistin 160mg mL�1

ciprofloxacin (e), or 200mg mL�1 tetracycline125mg mL�1 colistin (f). Live

cells appear green due to the expression of Gfp, and dead cells appear red

as a result of staining with the dead cell indicator propidium iodide. (g) The

number of cells that survived the antimicrobial treatment were determined

by plate counting of cells harvested from biofilms. The size bars are 50mm.

Reproduced from Pamp et al. (2008).



involved in the tolerance toward tobramycin, gentamicin,

and ciprofloxacin (Zhang & Mah, 2008). In addition,

planktonic P. aeruginosa cells appear to use an adaptation

strategy, which involves upregulation of c-di-GMP-depen-

dent genes that increase protective biofilm formation in the

presence of subinhibitory concentrations of antimicrobial

agents (Hoffman et al., 2005).

Mulcahy et al. (2008) presented evidence that eDNA may

create a cation-limited environment in P. aeruginosa bio-

films that result in induction of the pmr-LPS-modification

system conferring tolerance toward antimicrobial peptides

and aminoglycosides. However, Pamp et al. (2008) did not

observe induction of the pmr genes in regions of P. aerugi-

nosa flow-chamber biofilms shown by Allesen-Holm et al.

(2006) to have high concentrations of eDNA.

From the observations described above, it appears that

the majority of biofilm cells can be killed by combined

antimicrobial treatment targeting the major physiologically

distinct cell subpopulations. However, it has been reported

that even in combined treatments involving two antimicro-

bials, a small number of bacteria did survive (see Fig. 3g)

(Pamp et al., 2008). Offspring of the bacteria that survived

the combined antimicrobial treatment did not exhibit

increased resistance to the antimicrobial agents used (Pamp

et al., 2008). It might be speculated that these few surviving

cells represent so-called persister cells. Persister cells are

dormant or slowly dividing bacteria that are less vulnerable

to antibiotics than the majority of the cell population

(Brooun et al., 2000; Lewis, 2001; De Groote et al., 2009).

Mutant screens in P. aeruginosa have provided evidence that

a number of genes (e.g. rpoS, spoT, relA, dksA, dinG, spuC,

algR, pilH, ycgM, pheA) are involved in persister formation,

suggesting that the persister phenotype can be reached

through multiple pathways (Murakami et al., 2005; Viducic

et al., 2006; De Groote et al., 2009). In addition to the

various physiologically distinct cell subpopulations in bio-

films, the generation of genetic variant cells within biofilms

has been described. Rough/wrinkled and small colony

variants in P. aeruginosa biofilms can emerge in the absence

or presence of an antimicrobial agent, whereof some of the

variants can exhibit reduced susceptibility toward antimi-

crobial compounds (e.g. H2O2) (e.g. Drenkard & Ausubel,

2002; Boles et al., 2004; Kirisits et al., 2005).

Recent reports have demonstrated that rhamnolipid

production by P. aeruginosa plays a role in the tolerance of

P. aeruginosa biofilms toward immune cells (Bjarnsholt

et al., 2005; Jensen et al., 2007; Alhede et al., 2009). Purified

P. aeruginosa rhamnolipids were shown to be able to destroy

polymorphonuclear neutrophilic leukocytes (PMNs) via

necrosis (Jensen et al., 2007). Moreover, it was reported that

biofilm cells of P. aeruginosa respond to the presence of

PMNs by upregulating the synthesis of rhamnolipid (Alhede

et al., 2009). The available evidence suggests that the

rhamnolipids stick to the biofilm bacteria and function as a

shield that eliminates immune cells upon contact (Alhede

et al., 2009).

Dispersal

In addition to the mechanisms involved in biofilm forma-

tion, bacteria also possess mechanisms to disperse from

biofilms. These mechanisms involve a reduction of bacterial

adhesiveness, and breakdown or modulation of the biofilm

matrix. Emigration of cells from biofilm communities is

necessary to spawn novel communities at new locations, and

it may be induced if the biofilm cells face unfavorable

conditions (e.g. Tolker-Nielsen et al., 2000; Gjermansen

et al., 2005).

In P. aeruginosa biofilms grown in flow chambers irri-

gated with LB medium, local dispersal was observed as a

hollowing out of some microcolonies (Purevdorj-Gage

et al., 2005). In the initial phase of the dispersal process, a

wall-forming subpopulation of nonmotile cells constituted

the outer part of the microcolonies, whereas a motile rapidly

moving subpopulation was present inside the microcolo-

nies. The motile subpopulation eventually found its way out

of the microcolony, which resulted in microcolonies with a

central void. This dispersal phenomenon was shown to be

dependent on the microcolonies reaching a critical size.

Dispersal of P. aeruginosa biofilms in response to shifts in

carbon availability was reported by Sauer et al. (2004).

Pseudomonas aeruginosa biofilms grown in flow chambers

on glutamate medium responded to an abrupt upshift in

carbon availability by initiating a dispersion process that led

to the majority of the biomass being released from the

biofilm. The extent of dispersion was dependent on the

carbon source and was associated with increased expression

of flagella and downregulation of twitching motility. In a

subsequent study by Morgan et al. (2006), a gene product

involved in sensing environmental cues that trigger P.

aeruginosa biofilm dispersal was identified. The gene encod-

ing this sensor was denoted bdlA for biofilm dispersion

locus, and through sequence analysis and phenotypic com-

parison of the P. aeruginosa wild type and bdlA mutant, the

BdlA protein was suggested to be a chemotaxis regulator

that affects the intracellular level of c-di-GMP. As described

in a previous section, evidence is accruing that the produc-

tion of matrix components such as Pel/Psl polysaccharides

and Cup fimbriae is regulated via proteins that contain

diguanylate cyclase or phosphodiesterase activities and con-

trol the intracellular level of c-di-GMP (Hickman et al.,

2005; Gjermansen et al., 2006; Meissner et al., 2007). It

appears that in general, high intracellular c-di-GMP levels

upregulate matrix production and biofilm formation,

whereas low intracellular c-di-GMP levels downregulate

matrix production and induce a planktonic lifestyle. Carbon



starvation and nitric oxide signaling were shown to induce

the dispersal of P. aeruginosa biofilms via the induction of

phosphodiesterase activity, causing decreased intracellular

c-di-GMP levels (Barraud et al., 2009; Schleheck et al.,

2009). Evidence was presented that the above-mentioned

BdlA chemotaxis regulator is involved in nitric oxide-

mediated biofilm dispersal (Barraud et al., 2009).

As described in a previous section, rhamnolipid appears

to play multiple roles in the P. aeruginosa biofilm develop-

ment cycle, one of them being that the production of large

amounts may lead to the dispersal of cells from the biofilms

(Schooling et al., 2004; Boles et al., 2005). Ryan et al. (2009)

recently presented evidence that rhamnolipid-mediated dis-

persal in P. aeruginosa biofilms may involve c-di-GMP.

Evidence was provided that the PA2572 protein has a

degenerate inactive c-di-GMP phosphodiesterase domain

that may play a regulatory role, and a P. aeruginosa PA2572

mutant was found to overproduce rhamnolipid.

Recent work by Davies & Marques (2009) provides

evidence that the compound cis-2-decenoic acid produced

by P. aeruginosa is capable of inducing the dispersal of

established biofilms and of inhibiting biofilm development.

When added exogenously to P. aeruginosa biofilms at a

native concentration, cis-2-decenoic acid was shown to

induce the dispersal of biofilm microcolonies. This molecule

was also shown to induce the dispersal of biofilms, formed

by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,

Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aur-

eus, and the yeast Candida albicans. The authors suggested

that cis-2-decenoic acid is produced continuously by P.

aeruginosa during growth in biofilms, and that small micro-

colonies do not disperse because cis-2-decenoic acid is

removed through diffusive and advective transport; how-

ever, dispersal from larger microcolonies occurs because the

rate of production of cis-2-decenoic acid exceeds the rate of

diffusion.

A recent publication by Gjermansen et al. (2010) de-

scribes a mechanism involved in the dispersal of Pseudomo-

nas putida biofilms that may also be of relevance for P.

aeruginosa biofilms. In P. putida, the large adhesive outer-

membrane protein, LapA, mediates attachment to surfaces

and to matrix components. Release of LapA from the cell

surface results in biofilm dispersal and is mediated through

the activity of the periplasmic protease LapG. The activity of

the LapG protease is controlled by the transmembrane

protein LapD, which contains a c-di-GMP-binding domain,

and represses LapG when the concentration of c-di-GMP is

high, but derepressses LapG when the concentration of c-di-

GMP is low. The available evidence (Hinsa et al., 2003;

Hinsa & O’Toole, 2006; Monds et al., 2007; Newell et al.,

2009) suggests that a similar system is operating in Pseudo-

monas fluorescens. Pseudomonas aeruginosa encodes a num-

ber of large adhesive proteins, but it does not have a

homolog of lapA. However, P. aeruginosa does have homo-

logs of lapD and lapG, and therefore a mechanism similar to

the P. putida mechanism could potentially be involved in P.

aeruginosa biofilm dispersal.

Final remarks

Recent work has provided knowledge about the environ-

mental cues, genetic elements, and molecular mechanism

involved in biofilm formation, tolerance, and dispersal.

Based on this research, potential antibiofilm strategies can

be envisioned, for example enzymatic digestion of matrix

components, blocking of c-di-GMP regulated matrix synth-

esis, treatment with multiple antibiotics that target different

subpopulations, and blocking of quorum sensing. However,

the molecular understanding of the biofilm lifestyle ob-

tained from in vitro studies should be used in future in vivo

studies using animal models that mimic the complex inter-

actions between the biofilm and the host. A more detailed

understanding of the multicellular nature of microbial life

will ultimately enable us to develop efficient treatments

against biofilm-related infections.

Acknowledgement

This work was supported by grants from the Danish Council

for Independent Research.

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An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal: P. aeruginosa biofilm formation, tolerance, and dispersal