The e¡ectofquorum-sensing blockers on the formationofmarinemicrobial communities and larval attachmentSergey Dobretsov1,2, Hans-Uwe Dahms1, Huang YiLi1, Martin Wahl2 & Pei-Yuan Qian1

1Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, PR China; and2Benthic Ecology, Leibniz-Institut fur Meereswissenschaften, IFM-GEOMAR, Kiel University, Kiel, Germany

Correspondence: Sergey Dobretsov,

Benthic Ecology, Leibniz-Institut fur

Meereswissenschaften, IFM-GEOMAR,

Kiel University, Dusternbrooker Weg 20,

24105 Kiel, Germany. Tel.: 149 431 600

4527; fax: 149 431 600 1671;

e-mail: sdobretsov@ifm-geomar.de

Received 15 June 2006; revised 13 October

2006; accepted 17 November 2006.

First published online 16 March 2007.

DOI:10.1111/j.1574-6941.2007.00285.x

Editor: Riks Laanbroek

Keywords

microbial communities; quorum-sensing

inhibitors; larval attachment; furanones;

antifouling.

Abstract

We studied the effect of the quorum-sensing (QS) blockers 5-hydroxy-3[(1R)-1-

hydroxypropyl]-4-methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-[(1S)-

1,2-dihydroxyethyl]furan-2(5H)-one (FUR2) and triclosan (TRI) on the forma-

tion of bacterial biofilms, and the effect of these biofilms on the larval attachment

of the polychaete Hydroides elegans and the bryozoan Bugula neritina. 14-day-old

subtidal biofilms were harvested from artificial substrata and were allowed to

develop in the laboratory with and without QS blockers. QS blockers inhibited the

production of violacein by the QS reporter strain Chromobacterium violaceum

CV026 and did not affect the metabolic activity of bacteria in multispecies

biofilms. At a concentration of 10�3 M all three tested compounds inhibited the

establishment of microbial communities, but at one of 10�4 M only FUR2

inhibited establishment. The tested QS blockers caused changes in bacterial density

and bacterial community structure, as revealed by terminal restriction fragment

length polymorphism and FISH. The groups most affected by QS blockers were

Alphaproteobacteria, Gammaproteobacteria and the Cytophagales. Larvae of H.

elegans and B. neritina avoided settling on biofilms that had developed in the

presence of QS blockers. Our results suggest that QS blockers directly control the

formation of multi-species biofilms, and indirectly – by means of biofilm proper-

ties � affect larval attachment on these modified biofilms.

Introduction

Natural and artificial substrata in the marine environment

are quickly colonized by marine micro- and macro-organ-

isms in a process known as ‘biofouling’. Biofouling causes

serious problems for marine industries and navies around

the world (Yebra et al., 2004). At present, the most effective

methods of biofouling control are based on the application

of substances such as tributyltin (TBT), copper or organic

compounds (e.g. Sea-Nine, Isothiazolone) (Thomas, 2001;

Yebra et al., 2004). All these chemicals are toxic and pollute

the aquatic environment (see the reviews of Evans, 1999;

Thomas, 2001). Accordingly, there is a need for the devel-

opment of ‘environmentally friendly’ nontoxic antifoulants.

Biofilms can enhance (Kirchman et al., 1982; Lau & Qian,

1997, 2001; Qian et al., 2003) or inhibit the larval settlement

of marine invertebrates and the attachment of algal spores

(Rodriguez et al., 1993; Egan et al., 2000, 2001; Dobretsov &

Qian, 2002; Huang & Hadfield, 2003). Moreover, chemical

compounds produced by bacteria can lead to the disruption

of biofilm formation and the prevention of biofouling

(Dobretsov et al., 2006). Hence, bacterial compounds may

be useful in the biotechnological development of an envir-

onmentally friendly protection against marine biofouling

(Clare et al., 1992; Holmstr�m & Kjelleberg, 1999; Arm-

strong et al., 2000).

Although bacteria are unicellular organisms, they can

control their growth and population densities (Xavier &

Bassler, 2003; March & Bentley, 2004). In order to achieve

this, bacteria have evolved a regulatory mechanism named

quorum sensing (QS) that consists of exuded info-chemicals

that activate or de-activate target bacterial genes responsible

for cell division and adhesion and, thus, control biofilm

formation and composition (Davies et al., 1998). Biofilm

formation, in turn, can control many processes at surfaces,

for example the uptake or release of compounds by host

organisms, bacterial virulence for the host, and biocorrosion

(Parsek & Greenberg, 2000; Sauer et al., 2002). The process of

QS is based on the production and release of low-molecular-

weight signal molecules (often called autoinducers). The

FEMS Microbiol Ecol 60 (2007) 177–188 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

extracellular concentration of QS molecules reflects the

population density of the producing organism. Bacteria can

perceive and react to such signal molecules, allowing the

whole cell population to initiate a concerted action once a

critical population density has been reached (de Nys et al.,

2000).

The intercellular signalling compounds that have received

the most attention so far are the N-acetyl homoserine

lactones (AHLs) that are restricted to Gram-negative bacter-

ia (Whitehead et al., 2001). The effects of AHLs are based on

gene products of the luxR gene analogues (Gray & Garey,

1999). Each Bacterial species may produce different AHL

analogues that differ in the length of the N-acetyl chains,

ranging from 4 to 14 carbons, and in the substitution at the

3-position of the side chain (McLean et al., 1997).

A second communication molecule, a furanosyl borate

diester found in Vibrio harveyi, has been suggested to

be active in interspecific signalling (Chen et al., 2002).

A genomic database analysis has indicated that such inter-

species communication possibly occurs throughout the

Eubacteria (Sperandio et al., 2001). The QS-producing

bacteria are highly diverse and fall within a large number

of species among Alpha-, Beta- and Gammaproteobacteria

(Manefield & Turner, 2002) which are dominant in tropical

waters (Webster et al., 2004; Huang et al., in press).

In contrast to Gram-negative bacteria, Gram-positive bac-

teria exude peptides as signal molecules (Miller & Bassler,

2001). QS signals produced by bacteria may also show

transphyletic effects and induce algal spore attachment

(Joint et al., 2002).

Because QS signals are important for bacterial growth,

adhesion and biofilm formation, the inhibition of QS signals

leads to the disruption of biofilm formation (Ren et al.,

2001a) and the inhibition of bacterial pathogenicity (Ren

et al., 2001b). Many marine organisms, such as the red alga

Delisea pulchra and the bacterium Aeromonas veronii, use

QS blockers to control epibiotic biofilm formation (McLean

et al., 1997, 2004; Manefield et al., 1999; Rice et al., 1999;

Bauer & Robinson, 2002; Zhang & Dong, 2004). Delisea

pulchra produces furanones that interfere with bacterial

AHLs and inhibit the growth of Gram-negative bacteria as

well as the settlement of invertebrate larvae (Steinberg et al.,

1997; Rasmussen et al., 2000; Kjelleberg et al., 2001). At the

same time, it is possible to propose that QS blockers can

control larval settlement indirectly by regulating the micro-

bial community structure of biofilms and the density of

bacteria, which in turn affects larval behaviour.

The aims of this study were to investigate (1) the effect on

the community structure and density of bacteria in multi-

species microbial communities of QS blockers at various

concentrations, and (2) the effect on larval attachment of

microbial communities that were treated or untreated by QS

blockers.

Materials and methods

Inhibition of QS by chemical compounds

The bacterium Chromobacterium violaceum CV026 (a mini-

Tn5 mutant) was used as an indicator organism for QS by

quantifying violacein synthesis. Prior to the bioassay, the

strain was grown overnight on 0.5% yeast extract and 1%

tryptone in filtered (0.22-mm) seawater. The inhibitive

effects of QS blockers on the production of violacein by

C. violaceum cultures were tested according to Martinelli

et al. (2004). Briefly, the experiments were conducted in 96-

well microplates that contained N-hexanoylhomoserine

lactone (HHL) at 3.7� 10�8 M, 100 mL of C. violaceum, and

QS blockers. Tested concentrations of QS blockers were

10�3–10�5 M. After exposure for 16 h at 27 1C, the plates

were dried and the absorbance of each cell well was

measured with an automatic plate reader at 590 nm. Cells

without QS blockers but with HHL and without HHL were

used as the positive and negative controls. In addition, the

effect of QS blockers on bacterial growth was investigated by

a comparison of bacterial culture turbidity at 660 nm.

Microbial film development

Biofilms were developed on polystyrene Petri dishes (Falcon

no. 1006) exposed to biofouling in the subtidal zone

(maximal depth = 3 m at high tide) during February and

March 2005. Additional experiments with a QS reporter

strain were carried out in September 2006. All dishes were

attached vertically to nylon ropes, and the upper end of each

rope was tied to the pier. A weight was attached to the lower

end of the rope to keep the rope straight. After 14 days, the

dishes were retrieved and microbial films were washed from

the dishes by a sterile brush into a sterile Petri dish filled

with filtered (0.22-mm) seawater (FSW). The biofilm sus-

pension obtained was filtered through a 45-mm mesh and

subsequently through ash filters (Whatman, N1) in order to

eliminate most of the diatoms. This microbial suspension

was used in further experiments (see below).

Biofilm treatment

The QS blockers 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-

methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-

[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one (FUR2, Sigma)

and triclosan (TRI, Sigma) were dissolved in FSW at

concentrations of 10�2, 10�3 and 10�4 M. Experiments were

performed in large Petri dishes (diameter 90 mm). Before

the commencement of each experiment, sterile polystyrene

slides (washed in 70% ethanol and rinsed three times in

sterile distilled water) cut from large Petri dishes were placed

on the bottom of each Petri dish. Thereafter, 9 mL of

microbial suspension and 1 mL of one type of the QS

FEMS Microbiol Ecol 60 (2007) 177–188c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

178 S. Dobretsov et al.

blockers were added in order to obtain a final concentration

of 10�3, 10�4 and 10�5 M. Each concentration was replicated

48-fold. Petri dishes without any chemicals added were used

as the controls. Biofilms were developed under laboratory

conditions (20 1C, no light) within 48 h according to Railkin

(1998). After the experiment, solutions were discarded and

the slides were rinsed several times with FSW. For each

concentration and compound, 10 replicates were used for

larval bioassay experiments, three replicates were used for

terminal restriction fragment length polymorphism (T-

RFLP) analysis, and the remaining 35 replicates were used

for bacterial counting and FISH, as well as for quantification

of respiratory active (alive) and nonactive (dead) cells. The

numbers of metabolically active cells in biofilms treated with

QS blockers and untreated (control) cells in biofilms were

determined by staining with 6 mM 5-cyano-2, 3-ditolyl

tetrazoliumchloride (CTC, Polysciences) in FSW according

to Haglund et al. (2002).

Larval cultivation and experiments with larvae

Adult broodstocks of the polychaete Hydroides elegans

(Haswell) and the bryozoan Bugula neritina (Linnaeus) were

collected from pilings and floating rafts in Port Shelter Bay,

Hong Kong (221190N, 1141160E). Batch cultures of poly-

chaete larvae were reared for 5 days to competent stage

according to Harder & Qian (1999). Bryozoan larvae were

obtained as described by Bryan et al. (1997); newly hatched

larvae were included in the bioassays. Still-water laboratory

bioassays were performed with five replicates in sterile

polystyrene Petri dishes (no. 1006, Falcon) that contained

20 larvae and the slide with biofilm developed under various

QS treatments in 4 mL of sterile-filtered (0.22-mm) seawater

(FSW). Prior to the larval bioassays, the biofilms were

washed with several portions of sterile FSW in order to

remove remaining QS blockers. Larval attachment assays

were run at 28 1C under continuous illumination for 1 h.

After this time, attached juveniles were counted and attach-

ment was expressed as percentages.

Biofilm analysis

Terminal restriction length polymorphism

The bacterial community compositions of biofilms on

polystyrene slides were compared using T-RFLP analysis

(Liu et al., 1997). For this purpose, the entire surface area of

each slide (120 mm2) was swabbed with sterile cotton balls.

Three swabs from each treatment were individually sus-

pended in 1 mL of extraction buffer (100 mM Tris-HCl,

100 mM EDTA, 100 mM sodium phosphate, 1.5 M sodium

chloride, 1% CTAB; at pH = 8) in 2-mL microcentrifuge

tubes. For lysing, the samples were subjected to three

cycles of freezing and thawing followed by incubation

for 2 h in 20% sodium dodecylsulfate (SDS) at 65 1C.

The cotton swabs were removed, and, after centrifugation

(8832 g� 5 min�1), the total DNA in the supernatant was

extracted and purified twice in a volume of 24 : 1 chlorofor-

m:isoamyl-alcohol, followed by precipitation in isopropanol

at room temperature for 15 min. The precipitated DNA was

washed with cold 70% ethanol and resuspended in 50 mL of

autoclaved double-distilled water and then frozen at

� 80 1C until use.

PCR of 16S rRNA genes of bacterial communities was

performed in a total volume of 25mL containing 1 mL of

DNA template, 250mM of each deoxyribonucleotide tripho-

sphate (dATP, dCTP, dGTP, dTTP; Pharmacia Biotechnol-

ogy), 1 U of DNA Taq polymerase (Amersham Biosciences)

and 0.8 mM of each universal primer: 341F forward (50-

CCTACGGGAGGCAGCAG-30) and 926R reversal (50-

CCGTCAATTCCTTTRAGTTT-30). The 926R primer was

labelled at the 50 end with 6-carboxy fluorescein (FAM) dye.

The thermocycling conditions were as follows: 95 1C for

2 min (1 cycle); 15 cycles of 95 1C for 30 s, 60 1C for 3 min,

and 72 1C for 3 min (the annealing temperature started at

60 1C and was reduced to 45 1C in increments of 1 1C

cycle�1); 10 cycles of 95 1C for 30 s, 45 1C for 3 min, and

72 1C for 3 min; and 72 1C for 10 min. Amplified DNA (4 mL

of PCR mixtures) was visualized by gel electrophoresis on a

1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer.

Fluorescently labelled PCR products from triplicate PCR

amplifications were combined and purified with the

Wizards PCR preps DNA purification system (Promega,

WI) according to the manufacturer’s protocol. Purified

amplicons were digested with 20 U MspI (Boehringer Man-

nheim Biochemicals, IN) at 37 1C for 6 h. Aliquots of

digested products (10 mL) were mixed with 0.5mL of internal

size standard (ET550-R, Amersham Biosciences, UK). This

mixture was denatured for 2 min at 95 1C and immediately

chilled on ice prior to capillary electrophoresis on a Mega-

BACETM genetic analyser (Amersham Biosciences) operated

in the genotyping mode. After electrophoresis, the lengths of

the fluorescently labelled terminal restriction fragments

(T-RFs) were determined by comparison with internal size

standards using the FRAGMENT PROFILER software (Amersham

Biosciences, UK). The lengths of T-RFs obtained by the

analyser were rounded up to the nearest integral values.

Peaks that were o 1.5 bp from a larger peak were classified

as its ‘shoulders’ and eliminated (Dunbar et al., 2001).

For each sample, peaks over a threshold of 50 fluorescence

units (Blackwood et al., 2003) and whose peak heights

contributed for at least 1% to the integrated height (Buchan

et al., 2003) were used for analysis. Terminal fragments

o 35 and 4 500 bp were excluded from the analysis in

order to avoid detection of primers and uncertainties of size

determination.

FEMS Microbiol Ecol 60 (2007) 177–188 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

179Antifouling activity of QS blockers

FISH

Biofilm slides were fixed in 4% paraformaldehyde sus-

pended in phosphate-buffered saline for 8 h at 4 1C and

transferred to ethanol–phosphate-buffered saline (1 : 1) at

20 1C until further processing. In order to perform FISH,

slides were dehydrated with 96% ethanol and air-dried. For

each treatment we had five replicated slides. For each slide,

only one fluorescent probe (Table 1) labelled with Cy3 was

used. Hybridization solution was mixed in a ratio of 1 : 1

with the appropriate fluorescently labelled oligonucleotide

and applied to each slide. Slides were incubated in 50-mL

Falcons tubes at 46 1C in the dark for 1.5 h. After hybridiza-

tion, slides were carefully removed and rinsed immediately

in warmed wash buffer (46 1C). Slides were rinsed in fresh

water to remove excess salt, air-dried, mounted, and stained

with the DNA-binding fluorochrome 4,6-diamidino-2-phe-

nylindole (DAPI, Vectashield, Vector Lab; concentration

0.5mg mL�1) for 15 min. The total number of bacteria and

the number of particular groups of bacteria in five randomly

selected fields of view were estimated using an epifluores-

cence microscope (Axiophot, Zeiss, Germany; magnifica-

tion � 1000 with excitation peaks at 359 and 647 nm).

Statistical analysis

The normality assumption was verified with Shapiro–Wilk’s

test (Shapiro & Wilk, 1965), and homogeneity of the data

with Barlett’s or Levene’s test (Zar, 1999). In order to

improve the normality of the data they were arcsine-

transformed. In the case of zero attached larvae, a value of

4n�1 (where n is the number of larvae in a single replicate)

was assigned to improve the arcsine transformation (Zar,

1999). The effect of concentration and chemical treatment

on larval attachment was determined by two-way ANOVA. The

differences between the treatments and the control were

determined by one-way ANOVA followed by a Dunnet posthoc

test (Quinn & Keough 2002). In all cases, the threshold for

significance was 5%. The data presented in all figures were

not transformed.

The T-RFLP patterns of different bacterial community

DNA samples were used for the construction of the Bray–

Curtis similarity matrix based on the total number of T-RFs

observed in all samples compared with the presence or

absence of these T-RFs in individual samples. The construc-

tion of a multi-dimensional scaling (MDS) plot to demar-

cate the similarity of microbial communities was performed

using the PRIMER program (Plymouth Marine Laboratory,

UK). In all cases, the threshold level for significance was 5%.

Results

Inhibition of QS by chemical compounds

All tested compounds significantly inhibited the production

of violacein by C. violaceum in the presence of HHL (Fig. 1;

ANOVA: F = 169.2, Po 0.0001). The most effective substances

were FUR1 and FUR2, which significantly (ANOVA, Dunnet

test, Po 0.05) inhibited bacterial QS at concentrations of

10�3–10�5 M. Violacein formation was reduced by up to

90% in comparison with the control, while there was no

inhibition of bacterial growth. The results of this experiment

suggest that the tested compounds do indeed interfere with

the signalling system of QS.

The effect of QS blockers on the development ofbiofilms

The densities of bacteria in biofilms were affected by

chemical compounds (Fig. 2). TRI and FUR1 were effective

at the highest concentration (10�3 M), while FUR2 signifi-

cantly (Po 0.05) decreased bacterial densities relative to the

control at all concentrations equal or above 10�4 M. None of

the compounds was active at 10�5 M. The percentage of

metabolically active cells (81� 3%) in biofilms treated by

chemical compounds was similar to that in the nontreated

biofilms (79� 5%), suggesting that QS blockers did not

have any toxic effects on bacteria from biofilms.

T-RFLP analysis (Table 2) revealed that the tested com-

pounds changed bacterial community profiles at some

concentrations (Fig. 3). Bacterial communities formed in

the presence of QS blockers were characterized by the

absence of certain terminal restriction fragments (T-RFs or

ribotypes) and the presence of others. The highest number

Table 1. Oligonucleotide probes used for FISH analysis

Probe Sequence Specificity

Percentage

formamide Reference

EUB338 GCTGCCTCCCGTAGGAGT Most common bacteria 20 Amann et al. (1990)

ALF968 GGTAAGGTTCTGCGCGTT Alphaproteobacteria 20 Manz et al. (1992)

BET42a GCCTTCCCACTTCGTTT Betaproteobacteria 35 Manz et al. (1992)

GAM42a GCCTTCCCACATCGTTT Gammaproteobacteria 35 Manz et al. (1992)

CF319a TGGTCCGTGTCTCAGTAC Cytophaga–Flavobacteria 35 Manz et al. (1992)

LGC354a YSGAAGATTCCCTACTGC Some Firmicutes spp. 35 Meier et al. (1999)

NonEUB338 ACTCCTACGGGAGGCAGC Negative control probe 20 Amann et al. (1990)

FEMS Microbiol Ecol 60 (2007) 177–188c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

180 S. Dobretsov et al.

of bacterial ribotypes was found in the control biofilms,

while the lowest was found in the presence of QS blockers,

such as 10�3 and 10�4 M FUR2 (Table 2). Most similar to

the controls were the biofilms developing in the presence of

10�5 and 10�4 M TRI and 10�5 M FUR2 (Fig. 3). The

biofilms treated with the other QS compounds and concen-

trations differed both from each other and from the control

(Fig. 3, Table 2). Some bacterial strains occurred only in the

presence of certain treatments. For example, the presence of

T-RFs at 158, 218, 264, 267 and 313 bp were specific to the

control treatments, while T-RFs at 66 and 178 bp were

present in biofilms developed with FUR1 treatment, and a

T-RF at 375 bp was present in biofilms developed with FUR2

treatment. All QS blockers showed the presence of unique

bacterial ribotypes (TRFs at 254, 285, 334–335 and 361bp)

that were absent in the control. In contrast, some T-RFs at

68, 126 and 176 bp were present in all treatments (Table 2).

The community composition of microbial films varied

among the treatments, as shown by FISH (Fig. 4). In all

treatments, FISH detected some bacteria that were stained

by the DAPI probe, but did not hybridize to the probes

designed to target Alphaproteobacteria, Betaproteobacteria,

Gammaproteobacteria, the Cytophaga–Flavobacterium group

of Bacteroides and Firmicutes. The probe for the most

common Eubacteria stained 98% of bacterial cells that were

also stained by DAPI. The amount of nonspecific binding

was low and did not exceed 3%. In all microbial films,

Alphaproteobacteria, Gammaproteobacteria and the Cyto-

phaga–Flavobacterium group of Bacteroides dominated

(Fig. 4).

All tested QS blockers at 10�3 M changed the relative

densities of specific bacterial groups in microbial biofilms

(Fig 4). At the concentration 10�4 M, only TRI significantly

(ANOVA: Dunnet, Po 0.05) decreased the relative densities of

Alphaproteobacteria, Betaproteobacteria and Gammaproteo-

bacteria. At the concentration 10�5 M, both TRI and FUR1

decreased the relative densities of Alphaproteobacteria and

Gammaproteobacteria. The effect of QS blockers was group-

FUR1

Rel

ativ

e ac

tivity

(%

)R

elat

ive

activ

ity (

%)

0

20

40

60

80

100

FUR2

Concentration (M)10-3 10-4 10-5 control

Concentration (M)10-3 10-4 10-5 control

Concentration (M)10-3 10-4 10-5 control

Rel

ativ

e ac

tivity

(%

)

0

20

40

60

80

100

TRI

0

20

40

60

80

100

**

*

**

*

*

*

Fig. 1. The effect of QS blockers (TRI, FUR1 and FUR2) at 10�3, 10�4 and

10�5 M on the production of violacein by the bacterium C. violaceum

CV026. Inhibition of the violacein production was observed by adding

QS blockers and HHL to C. violaceum CV026 cultures. A value of 100% is

equal to the violacein production induced by HHL at 3.7� 10�8 M. Data

that are significantly different from the control (no QS blockers) accord-

ing to a Dunnet test (ANOVA: Po 0.05) are indicated by asterisks. Data are

means (n = 3)� SD.

TRI FUR1 FUR2 Control

Bac

teria

l den

sity

x 1

03 ce

ll m

m–

2

0

5

10

15

20

2510–3M

10–4M10–5M

*

*

**

Fig. 2. The density of bacteria in biofilms treated with 10�3 M (black),

10�4 M (dark grey) or 10�5 M (light grey) of TRI, FUR1 or FUR2. Densities

of bacteria in biofilms treated by QS blockers that are significantly lower

than densities of the control ones (biofilms formed without QS blockers)

according to a Dunnet test (ANOVA: P o 0.05) are indicated by asterisks.

Data are means of five replicates� SD.

FEMS Microbiol Ecol 60 (2007) 177–188 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

181Antifouling activity of QS blockers

Table 2. T-RFLP profiles of bacterial communities formed in the pre-

sence of 10�3 M, 10�4 M and 10�5 M of TRI, FUR1 and FUR2 or without

quorum-sensing blockers (CON)

TRFs

(bp)

TRI FUR1 FUR2

CON10�3 10�4 10�5 10�3 10�4 10�5 10�3 10�4 10�5

57 Xa

59 Xb

62 Xb Xa Xb

63 Xa Xa Xb

64 Xa Xb

66 Xa Xa Xa Xa

68 Xa Xa Xa Xa Xa Xb Xa Xa Xa Xa

72 Xb

75 Xb

77 Xb Xa Xa Xb Xb Xa

79 Xa Xa Xa Xa Xa Xa

80 Xa Xa Xa Xa Xa Xa

88 Xb Xa

89 Xa Xa Xa Xa Xa Xa

90 Xa Xa Xa Xa Xa Xb Xa Xa Xa

91 Xa Xa Xa Xa Xa Xa Xb Xa Xa

92 Xa Xb

93 Xa

102 Xa Xa Xa Xb Xa Xa Xa

103 Xb

116 Xb

123 Xb Xa Xa Xa Xb

124 Xa Xa Xa Xa Xa Xa Xa Xa

125 Xa

126 Xa Xa Xa Xa Xa Xa Xa Xa Xa Xa

127 Xb Xa Xa Xb

128 Xa Xa Xb

129 Xa Xa Xb Xb Xa

130 Xb Xb Xa Xb

131 Xa Xb

142 Xb

145 Xa Xa Xa Xa

149 Xa

150 Xa

153 Xa Xa Xa Xa Xb Xa Xa Xa

154 Xa

158 Xa

163 Xa

172 Xb Xb

173 Xa

175 Xa Xa

176 Xa Xa Xa Xa Xa Xa Xb Xb Xb Xa

177 Xa Xa Xb

178 Xb Xb Xa

179 Xb

181 Xa

182 Xb

183 Xb

189 Xb

193 Xb Xb

194 Xa Xb Xa Xa

195 Xa

203 Xa

204 Xa Xa

Table 2. Continued.

TRFs

(bp)

TRI FUR1 FUR2

CON10�3 10�4 10�5 10�3 10�4 10�5 10�3 10�4 10�5

206 Xa Xa Xa Xa Xa Xa Xa Xa

207 Xa Xa Xb Xa

208 Xb Xa Xa

209 Xa

218 Xa

219 Xa Xa

220 Xb

246 Xa Xa Xa

247 Xa Xa Xa

248 Xa

253 Xb Xb Xb Xb

254 Xa Xb Xa

264 Xa

267 Xb

279 Xa Xb Xb

286 Xb

292 Xa

293 Xa Xb Xb Xb

294 Xb Xa

295 Xa Xa Xa Xb

298 Xa Xb

299 Xa Xa Xb Xb

300 Xa Xa Xb Xa Xa

302 Xa Xa

303 Xb Xa Xa Xa Xa Xb Xa Xa

310 Xa Xa Xa Xb Xa Xa

313 Xb

314 Xa Xb Xa Xa

315 Xa

329 Xa

333 Xa Xa Xa Xa

334 Xa Xa Xa Xa

335 Xa Xb Xa Xa

336 Xb Xb Xa

338 Xb Xa Xa Xb Xb

359 Xa

361 Xb Xa Xa Xa Xa

363 Xa

365 Xb Xb Xa

367 Xa Xa Xa

368 Xa Xa

370 Xa Xa Xa Xa Xa

371 Xa

372 Xa

374 Xa Xa Xa Xa Xa

375 Xa Xa Xa

397 Xa

419 Xa

420 Xa Xa Xa Xa

421 Xa Xa Xb Xa

422 Xa Xa

432 Xb

433 Xa

435 Xa Xb

436 Xb

441 Xa

FEMS Microbiol Ecol 60 (2007) 177–188c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

182 S. Dobretsov et al.

specific (Fig. 4). For example, at the concentration 10�3 M,

TRI significantly (ANOVA: Dunnet, Po 0.05) decreased the

relative density of Betaproteobacteria, while FUR1 at this

concentration decreased the relative density of Alphaproteo-

bacteria, and FUR2 at a similar concentration decreased the

density of Gammaproteobacteria as well as of the Cyto-

phaga–Flavobacterium of Bacteroides.

The effect of treated biofilms on larvalattachment

The effect of larval attachment of B. neritina varied accord-

ing to the compound tested (ANOVA: F = 5.5, Po 0.008), its

concentration (ANOVA: F = 12.9, Po 0.0001) and the combi-

nations of these factors (ANOVA: F = 2.9, Po 0.04, Fig. 5).

The percentage of larval attachment of B. neritina larvae on

the biofilms developed without QS-inhibiting compounds

(control) was 94� 5%. Larval attachment was similar to the

control on the biofilms treated with low concentrations

(10�4–10�5 M) of FUR2 and TRI and all tested concentra-

tions of FUR1 (Fig. 5). The biofilms developed in the

Table 2. Continued.

TRFs

(bp)

TRI FUR1 FUR2

CON10�3 10�4 10�5 10�3 10�4 10�5 10�3 10�4 10�5

445 Xa

451 Xa

483 Xb

Total

number TRFs

31 32 28 29 37 38 27 27 33 52

The presence and the fragment sizes (bps) of individual TRFs are denoted

as follows: Xa, present in three out of three replicates; Xb, present in two

out of three replicates; the presence in one out of three replicates is

omitted. Blank cells indicate the absence of particular TRFs in a given

biofilm.

TRI, triclosan; FUR1, 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-

2(5H)-one; FUR2, (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-

2(5H)-one.

FUR1

FUR2

TRI

CON

Stress: 0.1310

1010

1010

10

10

1010

10 1010

1010

101010

10

1010

10

1010

1010

1010

ConCon

Con

Fig. 3. Multi-dimensional scaling (MDS) showing similarities in bacterial

communities formed in the presence of TRI (grey square), FUR1 (white

triangle) and FUR2 (black triangle) or without quorum-sensing blockers

(CON, white rhombus). The MDS plot is based on the Bray–Curtis

similarity matrix of the presence and absence of T-RFLP fragments in

various biofilm samples. Analysis of this matrix was based on two

dimensions. Numbers on the graph correspond to the concentrations of

the compounds used for biofilm treatment. Three replicated samples

were used for T-RFLP analysis. The stress value in the right upper corner

determines the fit of the reproduced distance matrix to the observed

distance matrix.

10–3 M

10–4 M

10–5 M

% D

AP

I cou

nt

0

20

40

60

80

100 Alpha BettaGamma CFLG

*

**

*

% D

AP

I cou

nt0

20

40

60

80

100

*

*

*

Treatment

TRI FUR1 FUR2 Control

% D

AP

I cou

nt

0

20

40

60

80

100

*

**

Fig. 4. Quantitative estimates of biofilm community composition in the

presence of TRI, FUR1 and FUR2 using group-specific FISH probes (see

Table 1) for Alphaproteobacteria (black), Betaproteobacteria (light grey),

Gammaproteobacteria (dark grey), Cytophaga–Flavobacteria (oblique

stroke) and some Firmicutes spp. (dashed). In control dishes (CON) no

QS blockers were added. All densities are expressed as a percentage

of total bacterial numbers obtained using dual hybridization by DAPI

staining. Densities of bacteria that are significantly lower than the control

ones according to a Dunnet test (ANOVA: Po 0.05) are indicated by

asterisks. Data are means (n = 5)� SD.

FEMS Microbiol Ecol 60 (2007) 177–188 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

183Antifouling activity of QS blockers

presence of high concentrations (10�3 M) of TRI and FUR2

significantly reduced (ANOVA: Dunnet test, Po 0.05) larval

attachment.

Both compounds (ANOVA: F = 15.3, Po 0.0001) and the

concentration (ANOVA: F = 32.1, Po 0.0001) significantly

affected biofilm formation, which in turn affected larval

attachment of Hydroides elegans (Fig. 6). The biofilms

formed at high concentrations (10�3 M) of all tested chemi-

cals significantly inhibited (ANOVA: F = 7.3, Dunnet test,

Po 0.05) the attachment of larvae compared with the

nontreated biofilms. Larval attachment on such biofilms

was reduced 1.3–4 times (Fig. 6). FUR2 reduced the attrac-

tiveness of biofilms more strongly than the other com-

pounds did at concentrations of 10�3–10�4 M.

Discussion

In earlier experiments, furanones produced by the red alga

Delisea pulchra were shown to inhibit larval attachment

(Steinberg et al., 1997), grazing (de Nys et al., 1996), and

microbial attachment (Manefield et al., 1999). In this study

we investigated for the first time an indirect control of larval

attachment by quorum-sensing (QS) blockers via the mod-

ification of marine bacterial communities.

Our experiments demonstrated that some chemical

compounds not only significantly decreased production of

violacein by C. violaceum induced by AHLs (quorum-

sensing signals) but also decreased bacterial densities and

modified the composition of microbial communities, as

revealed by both FISH and T-RFLP analysis. Triclosan

(TRI), 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-

2(5H)-one (FUR1) and (5R)-3,4-dihydroxy-5-[(1S)-1,2-di-

hydroxyethyl]furan-2(5H)-one (FUR2) at 10�3 M had the

most striking effect on the density and composition of

bacterial communities. The effects of tested compounds

decreased with decreasing concentration of QS blockers.

FISH analysis showed that bacteria affected by the QS

blockers predominantly belonged to the Alphaproteobacter-

ia, Gammaproteobacteria or the Cytophaga–Flavobacterium

of the Bacteroides group. T-RFLP analysis demonstrated that

some bacterial ribotypes were absent after QS blocker

treatment, while other bacterial species were not affected by

QS blockers. This result suggests that the effect of QS

blockers was selective in our experiment. Analogously, the

AHL communication system (affected by the compounds

tested in this study) is highly species-specific (Whitehead

et al., 2001). For example, AHLs with side chains different

from the native ones interfere with signalling in the bacter-

ium Vibrio fischeri (Schaefer et al., 1996). Moreover, im-

plication of different AHLs leads to a different composition

of bacterial communities (McLean et al., 2005).

How did QS blockers affect the composition of microbial

communities in our experiments? We used two types of QS

blockers: furanones (FUR1 and FUR2), which inhibit quor-

um-sensing signals mediated by N-acylhomoserine lactones

(AHLs) (Rasmussen et al., 2000; Martinelli et al., 2004), and

triclosan (TRI), which inhibits the fatty acid biosynthesis

(Zhang & Dong, 2004). It has been shown that furanones

and triclosan interfere with AHL-mediated QS systems of

Gram-negative bacteria, in which a LuxR-type receptor

protein and a LuxI-type AHL synthase are the central

components (Fuqua & Greenberg, 2002; Zhang & Dong,

2004). Taking into account that in our experiments not all

Treatments

TRI FUR1 FUR2 Control

Atta

chm

ent %

0

20

40

60

80

100

10–3 M10–4 M10–5 M

*

*

Fig. 5. The effect of biofilms formed in the presence of TRI, FUR1 and

FUR2 at 10�3 M (black), 10�4 M (dark grey) and 10�5 M (light grey) on the

larval attachment of Bugula neritina. Attachment of larvae was ex-

pressed as a percentage. Levels of attachment of B. neritina on biofilms

treated by QS blockers that are significantly lower than levels of

attachment on the control (biofilms formed in FSW) according to a

Dunnet test (ANOVA: Po 0.05) are indicated by asterisks. Data are means

(n = 5)� SD.

TRI FUR1 FUR2 Control

Atta

chm

ent %

0

20

40

60

80

100 10–3 M10–4 M10–5 M

**

*

*

Fig. 6. The effect of biofilms formed in the presence of triclosan TRI,

FUR1 and FUR2 at 10�3 M (black), 10�4 M (dark grey) and 10�5 M (light

grey) on the larval attachment of Hydroides elegans. Attachment of

larvae was expressed as a percentage. Levels of attachment on biofilms

treated by QS blockers that are significantly lower than levels of

attachment on the control (biofilm formed with FSW) according to a

Dunnet test (ANOVA: Po 0.05) are indicated by asterisks. Data are means

(n = 5)� SD.

FEMS Microbiol Ecol 60 (2007) 177–188c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

184 S. Dobretsov et al.

tested concentrations of QS blockers were toxic to bacteria

or inhibited production of violacein by C. violaceum, we

conclude that the chemicals at concentrations of

10�3–10�5 M interact only with the quorum-sensing signal-

ling system of Gram-negative bacteria, thus inhibiting their

attachment, growth, and the formation of biofilms. Some of

bacterial strains that do not use AHL-mediated QS systems

may take advantage of the low density of competitors and

increase their growth. In our experiments, QS blockers did

not increase the density of low G1C Gram-positive bacteria

(mostly Firmicutes), as visualized by a LGC354a probe

(Meier et al., 1999). This suggests that some unique ribo-

types appearing in biofilms treated with QS blockers belong

to other bacterial groups.

A variety of QS blockers have been isolated from plants,

bacteria and fungi (reviewed by Zhang & Dong, 2004). For

example, halogenated furanones from the marine alga Deli-

sea pulchra inhibit the growth, swarming and biofilm

formation of Pseudoalteromonas aeruginosa and Escherichia

coli by interfering with bacterial AHLs (Rasmussen et al.,

2000; Hentzer et al., 2003; Ren et al., 2004). Furanones are

also produced by marine bacteria, green, red and brown

algae, sponges, fungi, and ascidians (Kjelleberg et al., 2001),

as well as by higher plants (Slaughter 1999). For example,

butenolides [2(5H)-furanones] have been isolated from

Streptomyces and from Hortonia spp. (Cho et al., 2001).

Gram-positive Bacillus spp. and Gram-negative Agrobacter-

ium tumefaciens and Arthrobacter spp. (Zhang et al., 2002,

2004) produced AHL-lactonase, which degrades bacterial

AHL signals. It is important to note that most of the QS

signals and their inhibitors were tested only in biomedical

experiments and against monospecies biofilms (for reviews

see Bauer & Robinson, 2002; Smith et al., 2003; Zhang &

Dong, 2004). Information about QS signals and their

inhibitors in the marine environment is fairly limited.

Examples include the production of AHLs by Roseobacter

spp. isolated from ‘marine snow’ (Gram et al., 2002), by

Roseobacter sp. and Vibrio sp. associated with sponges

(Taylor et al., 2004), and by marine free-living Alphaproteo-

bacteria and bacteria associated with eukaryotic algae

(Wagner-Dobler et al., 2005). It is possible to conclude that

QS signals and their inhibitors may play an important role

in aquatic microbial communities and should be a main

target of future studies.

In our experiments, the tested compounds modified

biofilms, which, in turn, affected the larval attachment of

the bryozoan B. neritina and the polychaete H. elegans. This

effect is concentration-dependent: all QS blockers at the

concentration 10�3 M affected the formation of biofilms,

which in turn inhibited larval attachment, but at the

concentration of 10�4 M only biofilms formed in the pre-

sence of FUR2 decreased the attachment of H. elegans

compared with the control. Were the concentrations of QS

blockers in our investigation realistic? Unfortunately, actual

concentrations of AHLs and QS blockers in marine biofilms

have not yet been determined. Taking into account that the

concentration of halogenated furanones in the alga Delisea

pulchra reaches 3–25 mg g�1 (Wright et al., 2000) it is

possible to propose that QS blockers in biofilms may exist

at similar concentrations. In the laboratory, halogenated

furanones from D. pulchra inhibit bacterial attachment at

10 mM (Maximilien et al., 1998), and QS signals in biofilms

of P. aeruginosa at 10 mM (Hentzer et al., 2003) and in

biofilms of C. violaceum at 100 mM (Martinelli et al., 2004).

These examples demonstrate that furanones can inhibit QS

and bacterial attachment at wide range of concentrations

that are similar to those used in this study.

Variations in the larval settlement response to the bio-

films treated with QS blockers can be explained in two ways.

First, compounds may decrease bacterial density, which in

turn causes low levels of larval attachment (Lau & Qian,

1997; Huang & Hadfield, 2003). Second, compounds may

modify the diversity of microbial communities. Bacterial

species composition and abundance in a microbial biofilm

can determine the biofilm inductiveness towards larval

settlement (Lau & Qian, 1997; Huang & Hadfield, 2003;

Qian et al., 2003; Dobretsov & Qian, 2006).

In conclusion, the experiments described here demon-

strate that QS blockers may affect larval attachment indir-

ectly by the modification of bacterial films. This finding may

inspire the search for novel antifouling technologies that

could be used in the control of both micro- and macrofoul-

ing. Overall, the indirect effects of QS blockers on larval

settlement deserve further investigation.

Acknowledgements

We thank Dr Urs Sequin (Switzerland) for providing us with

a synthetic furanone 5-hydroxy-3[(1R)-1-hydroxypropyl]-

4-methylfuran-2(5H)-one, and two anonymous reviewers

for their comments. This work was supported by RGC

grants (HKUST6402/05 M and CA04/05.SC01) to P.Y.Q.

and partially by a grant from the Alexander von Humboldt

Foundation to S.D.

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