Aquaculture 240 (2004) 69 – 88

www.elsevier.com/locate/aqua-online

Disruption of bacterial quorum sensing:

an unexplored strategy to fight
infections in aquaculture
Tom Defoirdta,b, Nico Boona, Peter Bossierb, Willy Verstraetea,*
a
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653,
9000 Gent, Belgium
b
Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Gent, Belgium
Received 14 April 2004; accepted 18 June 2004

Abstract

Disease outbreaks—some of them caused by pathogenic bacteria—are considered to be one of

the largest constraints to development of the aquaculture sector. So far, antibiotics and disinfectants
have only had limited success in the prevention or cure of aquatic disease. Moreover, the frequent
use of biocides, especially in subtherapeutic doses, is leading to the rapid development of resistance.
Therefore, there is an urgent need to develop alternative ways to control infections caused by
bacterial pathogens in aquaculture. Many of these pathogens are found to control virulence factor
expression by a cell-to-cell communication system. Hence, disruption of bacterial quorum sensing
has been proposed as a new anti-infective strategy and several techniques that could be used to
disrupt quorum sensing have been investigated. These techniques comprise (1) the inhibition of
signal molecule biosynthesis, (2) the application of quorum sensing antagonists (including natural
occurring as well as synthetic halogenated furanones, antagonistic quorum sensing molecules and
undefined exudates of higher plants and algae), (3) the chemical inactivation of quorum sensing
signals by oxidised halogen antimicrobials, (4) signal molecule biodegradation by bacterial
lactonases and by bacterial and eukaryotic acylases and (5) the application of quorum sensing
agonists. The few reports that deal with disruption of quorum sensing of aquatic pathogens, together
with the results obtained with human and plant pathogens, indicate that this new approach has

Abbreviations: AHL, acylated homoserine lactone; AI, autoinducer.

* Corresponding author. Tel.: +32 9 264 59 76; fax: +32 9 264 62 48.
E-mail address: Willy.Verstraete@UGent.be (W. Verstraete).

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2004.06.031
70 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

potential in fighting infections in aquaculture. However, before this new strategy can be applied in
aquaculture, the impact of quorum sensing disruption on the virulence of aquatic pathogens and the
impact of the proposed quorum sensing disrupting techniques on the aquaculture system of interest
should be studied in more depth.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Pathogen; Infection; Quorum sensing; Quorum quenching; Biodegradation; Antagonism

1. Introduction

According to FAO statistics, aquaculture is one of the fastest-growing food-

producing industries. Worldwide, the sector has increased at an average rate of 9.2%
per year since 1970. As a comparison, capture fisheries and terrestrial farmed meat
production increased at a rate of 1.4% and 2.8%, respectively (FAO, 2002). FAO
reports consider disease outbreaks as a significant constraint to development of the
sector worldwide, with annual losses of billions of dollars (Subasinghe, 1997). Diseases
caused by pathogenic or opportunistic bacteria such as Vibrio spp. and Aeromonas spp.
can be problematic in the intensive rearing of molluscs, fish and shrimp as they can
cause losses up to 100% (Moriarty, 1999; Zhang and Austin, 2000; Muroga, 2001;
Soto-Rodriguez et al., 2003).
So far, conventional approaches such as the use of antibiotics and disinfectants
have only had limited success in the prevention or cure of aquatic disease
(Subasinghe, 1997). Moreover, their frequent use is leading to the rapid development
of (multiple) resistance (Dias et al., 1995; Molina-Aja et al., 2002; Vivekanandhan et
al., 2002; Vattanaviboon et al., 2003). Therefore, according to FAO, there is an urgent
need for alternative control techniques in aquaculture, with the emphasis on
prevention, which is likely to be more cost-effective than cure (Subasinghe, 1997).
Disease prevention can be achieved by improved management including the
prevention of the transmission of pathogens between farms (e.g. by quarantine;
Subasinghe, 1997), the improvement of the water quality (e.g. by bioaugmentation;
Grommen and Verstraete, 2002; Moriarty, 1998), the avoidance of stress (e.g. due to
high stocking densities, handling and temperature and salinity changes; Brock and
Bullis, 2001) and a good hygiene (e.g. the disinfection of culture tanks, culture water
and eggs; Brock and Bullis, 2001). Alternative strategies to the use of antimicrobials
that have been applied successfully in aquaculture include the so-called microbial
matured water (Skjermo and Vadstein, 1999), the greenwater system (Tendencia and
dela Peña, 2003), bacteriophage therapy (Nakai and Park, 2002), the application of
probiotics (for reviews see Gatesoupe, 1999; Verschuere et al., 2000) and the
application of immunostimulants (for reviews see Sakai, 1999; Smith et al., 2003b)
and vaccines (for reviews see Newman, 1993; Gudding et al., 1999; Heppell and
Davis, 2000). More alternatives are very welcome since no anti-infective technique
seems to be able to solve every problem alone. This paper deals with an alternative
strategy that has not received much attention in aquaculture so far: disruption of
bacterial quorum sensing.
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 71

2. The new target: bacterial cell-to-cell communication

Quorum sensing is a mechanism in which bacteria coordinate the expression of certain

genes in response to their population density by producing, releasing and detecting small
signal molecules. This mechanism was first discovered in the marine bacterium Vibrio
fischeri (Nealson et al., 1970) and was thought to be restricted to only a limited series of
species. Later on, similar systems were found to be present in many other Gram-negative
bacteria. These Gram-negative bacteria use acylated homoserine lactones (AHLs) as signal
molecules (Fig. 1A, for reviews see Fuqua et al., 2001; Miller and Bassler, 2001;

Fig. 1. Three major quorum sensing systems. (A) AHL-mediated quorum sensing. The I protein is the AHL
synthase enzyme. The AHL molecules diffuse freely through the plasma membrane. As population density
increases, the AHL concentration increases as well and once a critical concentration has been reached, AHL binds
to the R protein, a response regulator. The AHL-R protein complex activates or inactivates transcription of the
target genes. (B) Peptide-mediated quorum sensing in Gram-positive bacteria. A peptide signal (PS) precursor
protein is cleaved, releasing the actual signal molecule. The peptide signal is transported out of the cell by an ATP
binding cassette (ABC) transporter. Once a critical extracellular peptide signal concentration is reached, a sensor
kinase (SK) protein is activated to phosphorylate the response regulator (RR). The phosphorylated response
regulator activates transcription of the target genes. (C) Quorum sensing in V. harveyi. In this system, there are
two types of signal molecules. AI-1 is an AHL and its biosynthesis is catalysed by the luxLM enzyme. AI-2 is a
furanosyl borate diester; its biosynthesis is catalysed by the LuxS enzyme. AI-1 and AI-2 are detected at the cell
surface by the LuxN and LuxP–LuxQ receptor proteins, respectively. At low cell density, LuxN and LuxQ
autophosphorylate and transfer phosphate to LuxO via LuxU. The phosphorylated LuxO is an active repressor for
the target genes. At high cell density, LuxN and LuxQ interact with their autoinducers and change from kinases to
phosphatases that drain phosphate away from LuxO via LuxU. The dephosphorylated LuxO is inactive.
Subsequently, transcription of the target genes is activated by LuxR. Redrawn after Miller and Bassler (2001).
72 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

Whitehead et al., 2001). AHLs of different species differ in the acyl side chain, which
usually contains between 4 and 14 carbons and which can have an oxo or a hydroxyl
substitution at the third position. In addition to the AHL-mediated systems in Gram-
negative bacteria, some Gram-positive bacteria were also found to regulate a variety of
processes in response to their population density. The quorum sensing systems of
Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus aureus, for instance,
have been extensively studied (for reviews see Dunny and Leonard, 1997; Miller and
Bassler, 2001). In contrast to the Gram-negative bacteria, these bacteria use secreted
peptides as signal molecules (Fig. 1B). A third major quorum sensing system was recently
unravelled in the luminescent bacterium Vibrio harveyi (Chen et al., 2002). The V. harveyi
system has two components: autoinducer 1 (AI-1) and autoinducer 2 (AI-2). Both can
activate bioluminescence by a phosphorylation and dephosphorylation cascade (Fig. 1C,
for reviews see Miller and Bassler, 2001; McNab and Lamont, 2003; Xavier and Bassler,
2003). AI-1 is an AHL and because of its specificity, it is probably used for intraspecies
communication. AI-2, on the other hand, is a furanosyl borate diester. Because AI-2
activity has been detected in many other species (Gram-negative as well as Gram-positive)
and because AI-2 biosynthesis is closely linked to the methyl cycle, this type of quorum
sensing is believed to be used for interspecies communication and might be involved in the
detection of the growth phase and the growth potential of the bacterial community (Xavier
and Bassler, 2003).

Table 1
The quorum sensing systems of different aquatic pathogens and the link between quorum sensing and virulence
factor expression and/or virulence as such
Species Signal Quorum sensing-regulated References
virulence (factors)
Aeromonas BHLa, HHLb biofilm formation, Swift et al. (1997),
hydrophila exoprotease production Swift et al. (1999),
Lynch et al. (2002)
Aeromonas BHLa, HHLb serine protease production Swift et al. (1997)
salmonicida
Vibrio ODHLc unknown Milton et al. (1997)
anguillarum
Vibrio harveyi OHBHLd, siderophore production, Bassler et al. (1993),
AI-2 production of type III Lilley and Bassler (2000),
secretion system components, Manefield et al. (2000),
extracellular toxin production Mok et al. (2003)
Vibrio unknown opacity McCarter (1998)
parahaemolyticus
Vibrio vulnificus AI-2 protease and haemolysin McDougald et al. (2000),
production, lethality to mice Kim et al. (2003)
Yersinia ruckeri unidentified unknown Temperano et al. (2001)
AHL
a
BHL: N-butanoyl-l-homoserine lactone.
b
HHL: N-hexanoyl-l-homoserine lactone.
c
ODHL: N-(3-oxodecanoyl)-l-homoserine lactone.
d
OHBHL: N-(3-hydroxybutanoyl)-l-homoserine lactone.
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 73

Phenotypes that are controlled by a quorum sensing system include luminescence,

conjugation, nodulation, swarming, sporulation, biocorrosion, antibiotic production and
most importantly biofilm formation and the expression of virulence factors such as lytic
enzymes, toxins, siderophores and adhesion molecules (De Kievit and Iglewski, 2000;
Miller and Bassler, 2001; Whitehead et al., 2001; De Windt et al., 2003). Quorum sensing
systems are found in a still growing list of bacteria that are pathogenic to plants, animals
and humans (for reviews see De Kievit and Iglewski, 2000; Williams et al., 2000). The list
includes (but is not restricted to) the aquatic pathogens Aeromonas hydrophila, Aeromonas
salmonicida, Vibrio anguillarum, V. harveyi, Vibrio parahaemolyticus, Vibrio vulnificus
and Yersinia ruckeri (Table 1). For most of them, a link between quorum sensing and
virulence factor expression has been demonstrated (Table 1). These quorum sensing
pathogens probably increase their chances to infect their host successfully by delaying
virulence factor production until the population density is large enough to overwhelm the
host’s immune system (Donabedian, 2003). It has been shown that inactivating the quorum
sensing system of quorum sensing pathogens can result in a significant decrease in
virulence factor expression (Jones et al., 1993; Swift et al., 1999) and in a decrease of
virulence as such (Wu et al., 2001). As the importance of quorum sensing in virulence
development of pathogenic bacteria became clear, disruption of quorum sensing was
suggested as a new anti-infective strategy (Finch et al., 1998). Subsequently, several
research groups started to investigate different techniques that could disrupt the quorum
sensing systems of pathogens. The techniques that have been developed so far will be
discussed throughout the following paragraphs.

Fig. 2. Schematic overview of different strategies that have been developed to disrupt bacterial quorum sensing.
(A) Inhibition of signal molecule biosynthesis by the application of substrate analogues. (B) Blocking signal
transduction by the application of quorum sensing antagonists. (C) Chemical inactivation and biodegradation of
signal molecules. (D) Application of quorum sensing agonists to evoke virulence factor expression at low
population density. See text for details.
74 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

3. Inhibition of signal molecule biosynthesis

A first quorum sensing disrupting technique aims at inhibiting signal molecule

biosynthesis (Fig. 2A). In many cases, homologues of the V. fischeri LuxI protein catalyse
the biosynthesis of Gram-negative AHL signal molecules, using acyl–acyl carrier proteins
(for the acyl chain) and S-adenosylmethionine (for the homoserine lactone moiety) as
substrates (Whitehead et al., 2001). In an attempt to block AHL biosynthesis, Parsek et al.
(1999) found that analogues of S-adenosylmethionine (such as S-adenosylcysteine) could
inhibit activity of the Pseudomonas aeruginosa LuxI homologue RhlI by up to 97%.
Database research revealed that no AHL synthase sequence motifs were present in other
enzymes with S-adenosylmethionine binding sites. Therefore, it might be possible to use
the S-adenosylmethionine analogues as specific quorum sensing inhibitors, without
affecting other vital processes in prokaryotic and eukaryotic organisms. However, further
research is necessary to elucidate whether AHL biosynthesis inhibitors would be useful to
combat infections in aquaculture since the paper of Parsek et al. (1999) is the only one
dealing with this type of compounds so far.

4. Application of quorum sensing antagonists

4.1. Antagonists for AHL-mediated quorum sensing

4.1.1. Delisea pulchra halogenated furanones

As quorum sensing-mediated processes are often involved in the interaction with plant
and animal hosts, it might not be surprising that these higher organisms have developed
mechanisms to disrupt quorum sensing. One of these mechanisms is the production of
quorum sensing antagonists: molecules that can bind to quorum sensing response
regulators, but fail to activate them (Fig. 2B). The red marine alga D. pulchra has
developed such a defense mechanism to protect itself from extensive bacterial colonisation
(Givskov et al., 1996). The alga produces halogenated furanones as antagonists for AHL-
mediated quorum sensing. Because of their structural similarity with AHLs (Fig. 3), the

Fig. 3. Structural similarity between AHL and the D. pulchra halogenated furanone (5Z)-4-bromo-5-
(bromomethylene)-3-butyl-2(5H)-furanone (Compound 2). The R in the AHL molecule is usually an alkyl
group consisting of between 3 and 13 carbons, which can have an oxo or hydroxyl substitution at the second
carbon.
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 75

halogenated furanones most probably bind to LuxR type proteins without activating them
(Manefield et al., 1999; Rasmussen et al., 2000). Givskov et al. (1996) showed that
swarming of the pathogen Serratia liquefaciens on agar plates could be inhibited
completely by adding 100 mg/l of (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-
furanone (Compound 2). This furanone could also suppress the expression of bio-
luminescence genes, located on a reporter plasmid in S. liquefaciens, without affecting the
growth rate of the bacterium. Interestingly, Manefield et al. (2000) found that the furanone
inhibited extracellular toxin production in a pathogenic V. harveyi strain. Mortality in the
shrimp Penaeus monodon was reduced to 50% after intramuscular injection with diluted
cell supernatant extracts from V. harveyi cultures grown in the presence of the halogenated
furanone, compared to extracts from untreated cultures.

4.1.2. Antagonistic AHL molecules

Apart from the D. pulchra furanones, it was shown that some naturally occurring
AHL molecules could stimulate quorum sensing-regulated pigment production in the
Gram-negative bacterium Chromobacterium violaceum, while other AHLs completely
inhibited this phenotype (McClean et al., 1997). The stimulatory or inhibitory effect
was linked to the structure of the acyl side chain of the molecules: AHLs with an acyl
side chain containing up to eight carbons were stimulatory, acting as quorum sensing
agonists. AHLs with an acyl chain containing 10 carbons or more, on the other hand,
were inhibitory and acted as quorum sensing antagonists. Apart from that, research by
Zhu et al. (1998) indicated that heterologous AHLs are potent antagonists for AHL-
mediated quorum sensing in bacteria that express the LuxR-type proteins at native
levels. Overexpression of these proteins abolished the repression. Interestingly, research
by Swift et al. (1997) and Swift et al. (1999) indicated that heterologous AHLs are
able to reduce virulence factor production by the aquatic pathogens A. hydrophila and
A. salmonicida. In the first report, Swift et al. (1997) showed that serine protease
production by A. salmonicida was delayed and that the final concentration was
reduced to 50% by applying N-(3-oxododecanoyl)-l-homoserine lactone at a
concentration of 10 AM. In a second research, Swift et al. (1999) demonstrated that
N-(3-oxodecanoyl)-l-homoserine lactone, at a concentration of 10 AM, inhibited
quorum sensing-regulated exoprotease production by the Gram-negative pathogen A.
hydrophila as well. Two other 3-oxo substituted long-acyl AHLs, N-(3-oxododeca-
noyl)-l-homoserine lactone and N-(3-oxotetradecanoyl)-l-homoserine lactone, had a
similar effect.

4.1.3. Synthetic AHL-mediated quorum sensing antagonists

Based on the findings mentioned above, several research groups started to investigate
the activities of different synthetic AHL and furanone analogues with respect to quorum
sensing (Reverchon et al., 2002; Smith et al., 2003a; Hentzer et al., 2002). As far as we
know, a synthetic derivative of the D. pulchra halogenated furanones, (5Z)-4-bromo-5-
(bromomethylene)-2(5H)-furanone, is the most active AHL antagonist mentioned in
literature thus far. This furanone, dosed in a concentration of 10 AM, could almost
completely reduce virulence factor expression in pure cultures of P. aeruginosa PAO1
(Hentzer et al., 2003). Interestingly, the furanone was equally active on biofilm bacteria
76 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

compared to planktonic cells, making them susceptible to sodium dodecyl sulphate and
antibiotics. In the absence of the furanone, on the contrary, 100- to 1000-fold higher doses
of antibiotics are required to eradicate biofilm bacteria compared to their planktonic
counterparts (Anwar and Costerton, 1990). Furthermore, Hentzer et al. (2003) showed that
the furanone was also active in vivo in mouse lungs after infection with a P. aeruginosa
strain harboring a fluorescent AHL reporter plasmid. The fluorescence signal from the P.
aeruginosa strain was significantly reduced after subcutaneous injection of the furanone.
After 8 h, however, the signal reappeared, indicating that the furanone had cleared from
the mouse blood.

4.1.4. Antagonists produced by higher plants and micro-algae

In an attempt to learn whether eukaryotes can interact with bacterial quorum sensing,
Teplitski et al. (2000) found that exudates from higher plants, such as pea, rice, soybean,
tomato, crown vetch and Medicago truncatula also influence AHL-mediated quorum
sensing. Reverse phase high-performance liquid chromatography revealed that there are
several different AHL mimicking substances present in extracts from pea and M.
truncatula seedlings (Teplitski et al., 2000; Gao et al., 2003). In contrast to D. pulchra,
these plants secrete substances that stimulate AHL-dependent quorum sensing as well as
substances that inhibit such responses. Interestingly, similar results were recently obtained
for micro-algae (Teplitski et al., 2004). The algae Chlamydomonas reinhardtii,
Chlamydomonas mutablis, Chlorella vulgaris and Chlorella fusca all stimulated quorum
sensing-regulated luminescence in wildtype V. harveyi. In contrast, colonies of C.
reinhardtii inhibited AHL-mediated luminescence in several different Escherichia coli
AHL reporter strains. The inhibition of luminescence was shown not to be due to toxicity
or to an inhibition of luminescence as such since the luminescence of a constitutively
luminescent derivative of E. coli was not inhibited. Apart from that, culture age as well as
growth conditions were shown to be important factors in the production of AHL
mimicking substances by C. reinhardtii. The levels of these substances were considerably
higher if the alga was cultured phototrophically compared to culturing on a medium
containing acetate as a carbon source. Unfortunately, the chemical nature of the quorum
sensing mimic compounds secreted by higher plants and micro-algae still has to be
elucidated.

4.2. Antagonists for AI-2-mediated quorum sensing

The AI-2 structure has only recently been elucidated (Chen et al., 2002). Therefore, not
much effort has been done to disrupt AI-2-mediated quorum sensing so far. In one report,
however, Ren et al. (2001) found that the halogenated D. pulchra furanone Compound 2,
previously described as an AHL antagonistic analogue, could completely inhibit AI-2-
regulated swarming of E. coli. Moreover, the furanone decreased thickness of E. coli
biofilms by 55% and the percentage of live cells in the biofilms by 87%. Finally, the
furanone also inhibited AHL-mediated as well as AI-2-mediated luminescence in V.
harveyi. The furanone might also attenuate virulence of this aquatic pathogen since the
expression of some virulence factors was also found to be regulated by its quorum sensing
system (Table 1).
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 77

4.3. Significance for aquaculture

Altogether, the results mentioned above indicate that the application of quorum sensing
antagonists in aquaculture might constitute an alternative approach for preventing or
combatting infections caused by pathogens that regulate virulence factor expression by an
AHL-mediated quorum sensing system (such as A. hydrophila, A. salmonicida and V.
harveyi) as well as by an AI-2-mediated system (such as V. harveyi and V. vulnificus).
Apart from that, they also indicate that the positive effect of the so-called microbial
matured water might partly be due to the presence of antagonistic AHLs. Indeed, the
production of microbial matured water takes place on a biofilter with a previously
established biofilm (Skjermo and Vadstein, 1999) and very high AHL concentrations were
reported in biofilms before (Charlton et al., 2000). Finally, the results obtained in the
researches with the macro-alga D. pulchra and with the unicellular algae Chlamydomonas
and Chlorella indicate that algae might be useful to control infections in aquaculture as
they disrupt the quorum sensing systems of pathogenic bacteria.

5. Chemical inactivation of quorum sensing molecules

It has been established for a long time that AHLs are chemically inactivated via alkaline
hydrolysis, yielding the cognate acyl-homoserine (Voelkert and Grant, 1970). To our
knowledge, the only other chemical inactivation that has been studied so far is the reaction
with oxidised halogen antimicrobials (Fig. 2C). These antimicrobials, at a concentration of
approximately 0.14 mM, were found to decrease the concentration of 3-oxo-substituted
AHLs to about one-fourth after 1 min incubation, but had no effect on unsubstituted ones
(Borchardt et al., 2001). Moreover, the inactivation of 3-oxo AHLs was shown to proceed
in the presence of polysaccharide biofilm compounds despite the much higher
concentration of the latter compared to the AHL concentration. In a further study,
Michels et al. (2003) unravelled the inactivation mechanism by liquid chromatography-
mass spectrometry. Apparently, the reaction kinetics are largely influenced by the pH of
the reaction mixture. At pH 6, a 3-oxo AHL molecule reacts quickly with two molecules
of hypobromous or hypochlorous acid, yielding a 2,2-dihalo-3-oxo AHL molecule.
Subsequently, the acyl chain is hydrolysed, yielding a fatty acid and 2,2-dihalo-N-
ethanoyl-l-homoserine lactone (Fig. 4). At pH 3, the reaction stops after the halogenation
steps, whereas at pH 8, the lactone ring of 2,2-dihalo-N-ethanoyl-l-homoserine lactone is
hydrolysed, yielding 2,2-dihalo-N-ethanoyl-l-homoserine. These data indicate that
treating culture water with low concentrations of strong oxidising agents, such as ozone
(Summerfelt, 2003), might be useful as an anti-infective therapy in aquaculture by
removing the quorum sensing molecules of pathogens.

6. Enzymatic inactivation and biodegradation of quorum sensing molecules

The ability to degrade AHLs seems to be widely distributed in the bacterial kingdom
(Fig. 2C). Enzymes that are able to inactivate AHLs have been discovered in species
78 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

Fig. 4. Reaction between a 3-oxo AHL and halogen antimicrobials (HOX; hypobromous or hypochlorous acid) at
pH 6. First, two a-halogenation reactions occur, yielding 2,2-dihalo-3-oxo AHL. Subsequently, the acyl chain is
hydrolised, yielding a fatty acid and 2,2-dihalo-N-ethanoyl-l-homoserine lactone. The R is an alkyl group
consisting of between 3 and 13 carbons, which can have an oxo or hydroxyl substitution at the second carbon.
Redrawn after Michels et al. (2000).

belonging to the h-Proteobacteria (Zhang et al., 2002), the â-Proteobacteria (Leadbetter

and Greenberg, 2000; Lin et al., 2003; Uroz et al., 2003) and the g-Proteobacteria (Uroz et
al., 2003) as well as in some Gram-positive species (Dong et al., 2002; Lee et al., 2002;
Uroz et al., 2003). These bacteria might block the quorum sensing systems of their
bacterial competitors to obtain a selective advantage over them. This could be the case, for
instance, for those microbes living in proximity of bacteria that regulate the production of
antibiotics via quorum sensing (Pierson et al., 1998). The actual inactivation of the signal
compound can be mediated by two types of enzymes: AHL lactonases and AHL acylases
(Fig. 5). Moreover, eukaryotic acylases might be able to inactivate AHLs as well (Xu et
al., 2003).

6.1. Bacterial AHL lactonases

Dong et al. (2000) screened more than 500 field and laboratory bacterial isolates for
AHL-inactivating activity. Of these, 24 isolates showed different levels of enzymatic
activity in eliminating AHLs. The enzyme responsible for the AHL-inactivating activity
(AiiA) was isolated from the strain that showed the strongest activity, Bacillus sp. strain
240B1. The purified enzyme, at a concentration of 50 mg/l, reduced the concentration of
N-(3-oxohexanoyl)-l-homoserine lactone from 20 AM to about 5 AM after 10 min.
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 79

Fig. 5. Enzymatic inactivation of AHLs. Cleavage of the amide bond by an AHL acylase enzyme yields a fatty
acid and homoserine lactone. Cleavage of the lactone ring by an AHL lactonase enzyme yields the corresponding
acylated homoserine. The R is an alkyl group consisting of between 3 and 13 carbons, which can have an oxo or
hydroxyl substitution at the second carbon.

Electrospray ionisation-mass spectrometry of the hydrolysis product revealed that the AiiA
enzyme opens the lactone ring to produce N-(3-oxohexanoyl)-l-homoserine (Dong et al.,
2001). Further research demonstrated that genes encoding AHL-degrading lactonases are
widespread in many Bacillus species (Dong et al., 2002; Lee et al., 2002; Dong et al.,
2004). These AiiA homologues showed about 90% sequence homology at the amino acid
level.
The first evidence indicating that enzymatic AHL inactivation could be used as a
biocontrol strategy was provided in the study of Dong et al. (2000). In this study,
expression of the AiiA enzyme in transformed Erwinia carotovora decreased the
production of cell wall degrading enzymes by the pathogen to about 10% and inhibited
soft rot disease symptoms in susceptible plants almost completely. In a further in vivo
study, Molina et al. (2003) tested the efficacy of using an AHL-degrading Bacillus sp.
strain for the biocontrol of plant diseases. The Bacillus sp. strain could reduce potato tuber
soft rot caused by E. carotovora to about 15% and crown gall in tomato caused by
Agrobacterium tumefaciens to about 10%. AHL degradation by the Bacillus sp. strain
offered a protection as effective as or better than antibiotic production by a Pseudomonas
chlororaphis biocontrol strain. Moreover, degradation of AHLs had not only a preventive,
but also a curative biocontrol activity. Recently, similar results were obtained with Bacillus
thuringiensis (Dong et al., 2004).

6.2. Bacterial AHL acylases

6.2.1. AHL acylases in Variovorax paradoxus and Ralstonia

At the same time as Dong et al. (2000) discovered an AHL-degrading Bacillus sp.
strain, Leadbetter and Greenberg (2000) isolated a strain that could use AHLs as the sole
source of carbon and nitrogen. This strain, V. paradoxus VAI-C, was enriched on a
medium with 500 mg/l N-(3-oxohexanoyl)-l-homoserine lactone as the sole source of
carbon and nitrogen. The researchers deduced from experiments with radiolabeled AHL
80 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

that V. paradoxus cleaves the AHL by an AHL acylase enzyme, releasing homoserine
lactone and a fatty acid. Subsequently, the fatty acid is used as carbon source via the h-
oxidation pathway. Further research is needed to elucidate how the bacterium obtains the
nitrogen from the homoserine lactone moiety. More recently, Flagan et al. (2003) isolated a
bacterium, Arthrobacter sp. strain VAI-A, capable of degrading and utilising the
nitrogenous breakdown products of AHL signal molecules. Interestingly, the AHL-
dependent growth rate and yield of a coculture of the Arthrobacter sp. strain and V.
paradoxus VAI-C were superior to those of monocultures of these bacteria, indicating that
consortia may have a synergistic effect in quorum sensing signal turnover and
mineralisation.
Recently, Lin et al. (2003) isolated an AHL-inactivating bacterium, Ralstonia sp. strain
XJ12B, from a mixed-species biofilm. The enzyme responsible for the AHL-inactivating
activity (AiiD) was purified and subsequently, N-(3-oxodecanoyl)-l-homoserine lactone
was incubated with the purified enzyme. Electrospray ionisation-mass spectrometry of the
hydrolysis product demonstrated that the AiiD enzyme hydrolyses the amide bond of
AHLs. Expression of the AiiD enzyme in transformed P. aeruginosa PAO1 inhibited
swarming of the pathogen and reduced mortality of the nematode Caenorhabditis elegans
to about 15%, compared to wildtype PAO1.

6.2.2. Specific AHL inactivation by AHL acylases produced by pseudomonads

Another recent research, by Huang et al. (2003), indicated that some substrate
specificity of AHL-inactivating enzymes can exist. The researchers isolated a soil
pseudomonad, strain PAI-A, that inactivated AHLs by means of an AHL acylase
enzyme. In contrast to V. paradoxus and Ralstonia sp. strain XJ12B, the pseudomonad
could only utilise AHLs with acyl side chains longer than eight carbons. Since the 16S
rRNA gene from strain PAI-A showed high similarity to the one from the pathogen P.
aeruginosa PAO1, the ability of the pathogen to degrade AHLs was investigated as
well. In accordance with the results obtained for the strain PAI-A, the pathogen started
to grow on long-acyl AHLs but not on short-acyl AHLs. The investigators presumed
that degradation of its own long-acyl AHL, N-(3-oxododecanoyl)-l-homoserine lactone,
may play a role in the regulation of the quorum sensing system of P. aeruginosa. Such
a signal turnover control mechanism had already been found in A. tumefaciens (Zhang
et al., 2002).

6.3. Eukaryotic acylases

Since several bacterial AHL acylases had been found to inactivate AHLs by
deacylation, Xu et al. (2003) investigated the ability of an eukaryotic counterpart of
these bacterial enzymes to inactivate AHL molecules. Different AHLs were shown to be
inactivated by the porcine kidney acylase I enzyme. Since the inactivation was best at high
pH, it could have been due to simple alkaline hydrolysis of the lactone ring. However, the
acylase could reduce a model biofilm, made mainly by a Pseudomonas and a
Microbacterium species, indicating that the enzyme might be able to inactivate AHLs at
environmentally relevant concentrations. Nevertheless, further research is needed to
investigate to what extent eukaryotic acylases can indeed inactivate AHLs and whether
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 81

these enzymes have any significance in the in vivo inhibition of quorum sensing-mediated
gene expression.

6.4. Enzyme activity and substrate threshold levels

Several bacteria were shown to be able to degrade AHLs if these molecules were
present at AM to mM concentrations (Dong et al., 2000; Leadbetter and Greenberg,
2000). Unfortunately, no enzyme activity studies have been conducted so far. Moreover,
previous research indicated that the biodegradation abilities of micro-organisms can be
limited to above certain threshold substrate levels (Janssens et al., 1997; Kovar et al.,
2002). As transcription of quorum sensing-regulated genes can be activated at AHL
concentrations as low as 1 nM (Andersen et al., 2001), it still has to be established
whether substrate specificity of the AHL-degrading enzymes is sufficiently high to
decrease the AHL concentration to below this threshold. However, mixed substrate
growth experiments indicate that threshold concentrations might be lower or even non-
existent if other carbon sources are present (as in most natural ecosystems) than if there
is only one single carbon source (Egli, 2004). Indeed, several in vivo studies showed
that the degradation of AHLs might continue until below the level needed for activation
of quorum sensing-regulated virulence genes (Dong et al., 2000; Molina et al., 2003;
Uroz et al., 2003; Dong et al., 2004).

6.5. Significance for aquaculture

The data mentioned above indicate that bacteria that are able to degrade quorum
sensing molecules might be useful as biocontrol agents in aquaculture. Hence, it is of
interest to investigate whether quorum sensing molecule degraders would be good
probionts. Apart from that, the reports dealing with AHL-degrading Bacillus spp. (Dong et
al., 2000; Molina et al., 2003; Dong et al., 2004) suggest that the positive effect of Bacillus
spp., used as probionts in aquaculture (Moriarty, 1998; Rengpipat et al., 2003), might
partly be due to inactivation of quorum sensing molecules—apart from the production of
growth-inhibiting substances.

7. The opposite way: application of quorum sensing agonistic analogues

All techniques discussed so far aim to inactivate quorum sensing-regulated virulence

factor expression. Mäe et al. (2001), however, tested an opposite strategy: they activated
quorum sensing-regulated virulence factor expression by using quorum sensing agonists.
The idea behind this strategy was that by adding the signal molecule of a pathogen,
virulence factor expression would be activated at low population density (Fig. 2D).
Subsequently, the virulence factors could trigger the activation of the host’s defense
system allowing resistance to develop. In the research of Mäe et al. (2001), disease in
tobacco plants caused by E. carotovora was reduced to 10% by applying a 5 mM solution
of the pathogen’s own AHL. Furthermore, the ability of E. carotovora to cause disease
after local inoculation with 106 pathogens per plant was decreased to about half in
82 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88

transgenic tobacco plants producing the pathogen’s AHL compared to wildtype lines. If
the infection was already established or if the inoculum size was increased by a factor 4 or
more, however, there was no significant difference between AHL-producing and control
plants. Apparently, the pathogen was present in sufficiently large numbers to overwhelm
the defense system of the plant in these cases. Taken together, these results are in
accordance with the original hypothesis that quorum sensing is used to avoid premature
virulence factor production and subsequent activation of plant defense responses. As this
research was conducted with plants, it would be very interesting to conduct similar tests
with (aquatic) animals to investigate whether their immune systems—specific or
nonspecific—could be activated successfully if a quorum sensing pathogen is present
by the application of the microbe’s signal molecule.

8. Limitations to disruption of bacterial quorum sensing

8.1. Resistance development

A first limitation to the use of quorum sensing disrupting techniques as a new anti-
infective therapy might be resistance development. Indeed, research by Zhu et al. (1998)
indicates that bacteria could simply circumvent quorum sensing blockade by over-
expressing quorum sensing genes. These researchers found that many synthetic AHL
analogues were potent inhibitors of quorum sensing responses in wildtype A. tumefaciens,
whereas in a transformed strain that overexpressed the A. tumefaciens LuxR homologue
TraR, inhibition was not detected for any of the analogues. However, as disruption of
quorum sensing is less likely to pose a selective pressure for development of resistance
than conventional antibacterial compounds do (Hentzer et al., 2003), the chance that a
quorum sensing mutant will arise might be rather small.

8.2. Lack of specificity

Apart from resistance development, the lack of specificity could also confine the use of
quorum sensing disrupting techniques to control pathogens. Most quorum sensing
disrupting techniques developed so far are not specifically blocking the quorum sensing
system of one or more pathogens. Most AHL degrading bacteria, for instance, inactivate a
wide range of AHL molecules. However, not all bacteria found to contain a quorum
sensing system are pathogens. Quorum sensing was shown, for example, to be involved in
nodule formation by Rhizobium species and in antibiotic production by fluorescent
pseudomonads (Pierson et al., 1997; Pierson et al., 1998). As could be the case for these
growth promoting and biocontrol activities of plant-associated bacteria, gross disruption of
quorum sensing might adversely affect yet unknown favourable quorum sensing-regulated
processes in aquatic ecosystems. On the other hand, it will probably be difficult to develop
techniques that only disrupt the quorum sensing system of a single pathogenic species. It is
clear that much more knowledge about the occurrence and function of quorum sensing in
aquaculture systems will be necessary in order to be able to develop a new anti-infective
strategy.
 T. Defoirdt et al. / Aquaculture 240 (2004) 69–88 83

9. Conclusions and further perspectives

The results obtained by using techniques that disrupt quorum sensing systems of
pathogenic bacteria indicate that it is a promising alternative for antibiotics in fighting
bacterial infections. This new approach might also have value in aquaculture since a link
between quorum sensing and virulence factor expression in several aquatic pathogens has
been demonstrated. Unfortunately, data about the impact of quorum sensing on virulence
(i.e. the net result of all virulence factors) of aquatic pathogens are still lacking.
Fundamental research in the quorum sensing domain will undoubtedly provide more
precise insights into the mechanism by which the expression of quorum sensing-regulated
genes is activated or inhibited. This research will make it possible, for example, to conduct
a more directed search for antagonists. So far, most of the research has been done on the
AHL-mediated quorum sensing systems of Gram-negative human and plant pathogens.
However, it can be expected that more techniques to disrupt quorum sensing systems of
other pathogens will be developed in the future.
Before this new strategy can be applied in aquaculture, there are some important topics
that should be faced. First of all, the impact of disrupting the quorum sensing system of
several aquatic pathogens on their virulence should be studied in full depth in order to
elucidate whether it is a valid anti-infective strategy in aquaculture. Secondly, the different
quorum sensing disrupting techniques should be investigated further in order to determine
which technique(s) suits best for the aquaculture system of interest. In this view, one should
try to get an idea about the impact of the techniques on the health of the final consumer of the
aquaculture product and also on the aquaculture system itself since gross disruption of
quorum sensing might adversely affect yet unknown favourable quorum sensing-regulated
processes. Moreover, some practical problems—such as the cost of a treatment and how to
deliver the quorum sensing disrupting compound or organism to the site of action—should
be considered. Finally, the problem of eventual resistance development should not be
neglected although disruption of quorum sensing is less likely to pose a selective pressure for
development of resistance than conventional antibacterial compounds do.

Acknowledgements

The authors wish to acknowledge financial support from the bInstituut voor de
aanmoediging van Innovatie door Wetenschap en Technologie in VlaanderenQ (IWT grant
no. 31205) and the bFonds voor Wetenschappelijk OnderzoekQ (project no. 3GO230.02).
Special thanks go to Wim De Windt, Roeland Grommen, Fernando Morgan (Posdoctoral
Fellow supported by the Francqui Foundation of Belgium) and Klara Van Driessche for
critically reading the manuscript.

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