Aquaculture 311 (2011) 258–260

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

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N-acylhomoserine lactone-degrading Bacillus strains isolated from

aquaculture animals
Tom Defoirdt a,b,⁎, Loan Doan Thanh a, Bart Van Delsen a, Peter De Schryver b, Patrick Sorgeloos a,
Nico Boon b, Peter Bossier a
a
Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Gent, Belgium
b
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Infections caused by antibiotic-resistant pathogenic bacteria can cause considerable losses in aquaculture.
Received 27 September 2010 Many aquaculture pathogens regulate the expression of virulence genes through quorum sensing, bacterial
Received in revised form 25 November 2010 cell-to-cell communication with small signal molecules. N-acyl homoserine lactones (AHLs) are the most
Accepted 25 November 2010
extensively studied class of quorum sensing signals. AHL-degrading enrichment cultures were previously
Available online 2 December 2010
isolated from the intestinal tract of healthy aquaculture animals and found to increase the survival of fish and
Keywords:
shrimp larvae. In this study, we isolated Gram-positive spore-forming strains from AHL-degrading enrichment
Quorum sensing cultures originating from whiteleg shrimp and European sea bass. Five selected isolates showed good AHL
Quorum quenching degradation abilities in a nutrient-rich background, simulating the presence of high levels of other nutrients as
Bacillus is the case in a gastrointestinal environment. Indeed, degradation rates between 0.7 and 0.9 mg l− 1 h− 1 were
Sea bass observed in Luria–Bertani medium supplemented with 5 mg l− 1 N-hexanoyl-L-homoserine lactone. The
Whiteleg shrimp selected isolates were confirmed to belong to the genus Bacillus by 16S rDNA sequencing and might be
interesting novel biocontrol strains for use in aquaculture.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction types of quorum sensing molecules is N-acylhomoserine lactones

FAO reports consider disease outbreaks as a significant constraint
to the development of the aquaculture sector worldwide (Subasinghe
et al., 2001). Traditionally, bacterial infections are treated by applying
antimicrobials. Many farmers also use antibiotics in a prophylactic
way, even when pathogens are not evident (Holmström et al., 2003).
This practice has resulted in the development of (multiple) antibiotic
resistance (Cabello, 2006), which makes antibiotic treatments
ineffective in controlling diseases (Karunasagar et al., 1994). There-
fore, there is an urgent need for alternative control techniques.
Many bacterial pathogens (inculding different Vibrio and Aero-
monas species) regulate the expression of virulence genes through
quorum sensing, bacterial cell-to-cell communication with small
signal molecules, and consequently, quorum sensing disruption has
been suggested as an alternative strategy to control bacterial
infections in aquaculture (Defoirdt et al., 2004). One of the major
⁎ Corresponding author. Laboratory of Aquaculture & Artemia Reference Center,
Rozier 44, B-9000 Gent, Belgium. Tel.: + 32 9 264 37 54; fax: + 32 9 264 41 93.
E-mail address: Tom.Defoirdt@UGent.be (T. Defoirdt).
URLs: http://www.aquaculture.ugent.be, http://LabMET.UGent.be (T. Defoirdt).
(AHLs), produced by many different Gram-negative bacteria (Fuqua
et al., 2001). AHLs are typically produced by a homolog of Vibrio
fischeri LuxI and detected by a homolog of Vibrio fischeri LuxR.
The ability to interfere with AHL quorum sensing by degrading the
signal molecules seems to be widely distributed in the bacterial
kingdom (Dong et al., 2007). Bacillus species were amongst the first
bacteria reported to degrade AHLs by producing lactonase enzymes,
which inactivate AHLs by opening the lactone ring (Dong et al., 2000;
Lee et al., 2002). We previously isolated AHL-degrading enrichment
cultures (mixed cultures obtained by subsequent culturing intestinal
microbiota in a medium containing AHLs as the sole carbon source)
from the intestinal tract of healthy shrimp and fish (Tinh et al., 2007;
Cam et al., 2009). The addition of these mixed cultures increased
survival of different aquaculture species, including turbot (Scophthal-
mus maximus) larvae (Tinh et al., 2008) and larvae of the giant
freshwater prawn (Macrobrachium rosenbergii) (Nhan et al., 2010). In
this study, we aimed at isolating AHL-degrading Bacillus strains from
the AHL-degrading enrichment cultures obtained previously from the
gastrointestinal tract of aquatic animals. As Bacillus strains are
increasingly being used as probionts in aquaculture (Decamp et al.,
2008; Hong et al., 2005) and because AHL degradation protects
aquatic animals from infection, AHL-degrading Bacillus spp. might be
interesting novel biocontrol strains for use in aquaculture.

0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.11.046
 T. Defoirdt et al. / Aquaculture 311 (2011) 258–260 259

8
P3/pME6000
2.3. Detection of N-hexanoyl-L-homoserine lactone (HHL)
P3/pME6863
LT3 A stock solution of HHL was prepared by dissolving HHL (Fluka) in
6 LT6
LT8 200 μl of ethanol (95%) and then further diluted to a final
LT12 concentration of 1000 mg l− 1 by adding sterile distilled water. A
LCDR16
[HHL] (mg l-1)

plate diffusion method was used for quantitative detection of HHL

4
using Chromobacterium violaceum CV026 as a reporter (Tinh et al.,
2007). This strain does not produce AHLs and produces the purple
pigment violacein in the presence of exogenous AHLs (McClean et al.,
2
1997). CV026 was grown to an optical density of around 2 at 550 nm
in a buffered (pH 6.5) normal Luria-Bertani (LB) medium containing
20 mg l− 1 kanamycin and spread over buffered (pH 6.5) LB plates
0
Subsequently, 10 μl of the sample solution was applied to the center of
the plates and the plates were incubated at 28 °C for 48 h. After the
incubation, the zone of purple-pigmented CV026 was measured and
0 2 4 6 8 10 12 14 the concentration of HHL in the sample was calculated based on a
Time (h) standard curve correlating the diameter of purple-pigmented
C. violaceum CV026 to HHL concentration.
Fig. 1. N-hexanoyl-L-homoserine lactone (HHL) degradation by the selected isolates. At
the different time points, 10 μl of filter sterilized supernatants of cultures grown in LB20
medium supplemented with 5 mg l− 1 HHL was spotted on LB agar covered with a lawn
2.4. AHL degradation assay
of the AHL reporter strain Chromobacterium violaceum CV026. The remaining HHL
concentration was calculated based on a regression line of the purple zone of CV026 as Each isolate was inoculated at 108 CFU ml− 1 in a buffered LB20
function of the concentration of HHL (based on a dilution series of a HHL stock medium (pH 6.5) supplemented with 5 mg l− 1 HHL. At regular time
solution). Strains P3/pME6000 and P3/pME6863 were used as negative and positive
intervals, 1 ml samples from each culture were taken and filtered over
controls, respectively.
a 0.2 μm filter. The HHL concentration in the cell-free supernatants
was determined as described above. Pseudomonas P3/pME6000 and
2. Materials and methods Pseudomonas P3/pME6863 (=pME6000 + the Bacillus AHL lactonase
gene aiiA; Molina et al., 2003), grown under the same culture
2.1. AHL-degrading enrichment cultures conditions, were used as negative and positive controls, respectively.

Two AHL-degrading enrichment cultures were used to isolate
Bacillus strains: EC5-Tinh, previously enriched from whiteleg shrimp
(Penaeus vannamei; Tinh et al., 2007), and EC5-Cam, previously
enriched from European sea bass (Dicentrarchus labrax; Cam et al.,
2009). The enrichment cultures were stored at −80 °C.
2.2. Isolation of Bacillus strains from the cultures
The AHL-degrading enrichment cultures were inoculated in Nine
Salt Solution (NSS) containing 5 mg l− 1 N-hexanoyl-L-homoserine
lactone (HHL) as the sole carbon and nitrogen source. The composi-
tion of the NSS was as follows: NaCl (17.6 g l− 1), Na2SO4 (1.47 g l− 1),
NaHCO3 (0.08 g l− 1), KCl (0.25 g l− 1), KBr (0.04 g l− 1), MgCl26.H2O
(1.84 g l− 1), CaCl22.H2O (0.41 g l− 1), SrCl26.H2O (0.008 g l− 1), H3BO3
(0.008 g l− 1). The solution was buffered to pH 6.5 with 200 mg l− 1 3-
(N-morpholino)-propanesulfonic acid). The suspensions were incu-
bated at 28 °C for 48 h on a shaker (120 rpm). After the incubation, the
cultures were pasteurised (30 min 70 °C). This procedure was
repeated once, after which the pasteurised cultures were plated on
Luria-Bertani agar containing 20 g l− 1 synthetic sea salt (LB20).
Colonies were picked and tested for AHL degradation (see below).
The 5 best AHL degraders (out of 27 isolates) were selected for further
testing.
2.5. Identification of the isolates by 16S rRNA gene sequencing
PCR targeting a 1500 base pair fragment of the 16S rRNA gene of
the isolates was performed according to Boon et al. (2002) using the
primer pair GM3f and GM4r (Biolegio, Nijmegen, The Netherlands).
PCR was performed with a GeneAmps PCR system 2700 thermal
cycler (PE Applied Biosystems, Nieuwerkerken a/d Ijssel, The
Netherlands) using the program: 95 °C for 5 min, 32 cycles of 94 °C
for 1 min, 42 °C for 1 min, 72 °C for 3 min and finally an extension
period of 72 °C for 10 min. DNA sequencing of the obtained PCR
products was carried out at IIT Biotech (Bielefield, Germany). The
nucleotide sequences of the isolates were deposited in the Genbank
database (http://www.ncbi.nlm.nih.gov/Genbank). Homology
searches were completed with the BLAST server of the National
Center for Biotechnology Information for the comparison of the
nucleotide query sequence against a nucleotide sequence database
(blastn).
3. Results and discussion
Bacillus species are frequently used as probiotics, both for humans
and animals. In aquaculture, the use of Bacillus species as probiotics is
expanding rapidly and a number of commercial biocontrol products
are composed of mixtures of Bacillus spores (Hong et al., 2005). In this

Table 1
N-hexanoyl-L-homoserine lactone (HHL) degradation rate of selected AHL-degrading Bacillus isolates in LB20 medium supplemented with 5 mg l− 1 HHL, GenBank sequence
accession numbers of partial 16S rDNA sequences of the isolates and related strains sharing 100% sequence identity with the sequences of the isolates (based on NCBI BLAST).

Isolate Source HHL degradation rate (mg l− 1 h− 1) Accession no. Related strainsa (GenBank accession no.)

LT3 EC5-Tinh 0.7 HQ235052 Ba JMC-UBL 06 (HM451437.1), Bt JMC-UBL 03 (HM451439.1)

LT6 EC5-Tinh 0.7 HQ235053 Ba B (HQ200405.1), Bc HMT6(HQ156459.1),
Bs ov2004-03268-01 (GU585579.1), Bt ODPY (HM770098.1)
LT8 EC5-Tinh 0.7 HQ235054 Ba JMC-UBL 06 (HM451437.1), Bc HMT6(HQ156459.1), Bt JMC-UBL 03 (HM451439.1)
LT12 EC5-Tinh 0.9 HQ235055 Ba JMC-UBL 06 (HM451437.1), Bt JMC-UBL 03 (HM451439.1)
LCDR16 EC5-Cam 0.8 HQ235056 Ba JMC-UBL 06 (HM451437.1), Bt JMC-UBL 03 (HM451439.1)
a
Ba: Bacillus anthracis; Bc: Bacillus cereus; Bs: Bacillus subtilis, and Bt: Bacillus thuringiensis.
260 T. Defoirdt et al. / Aquaculture 311 (2011) 258–260
study, we isolated Bacillus strains based on their capability to degrade
N-acylhomoserine lactones (AHLs). As a starting point, we used AHL-
degrading enrichment cultures (containing Bacillus spp.) which have
been shown before to enhance survival of aquaculture animals (Tinh
et al., 2008; Cam et al., 2009). Bacillus strains were isolated from the
enrichment cultures by sequentially culturing in a medium containing
N-hexanoyl-L-homoserine lactone (HHL) as the sole carbon and
nitrogen source and pasteurising the cultures, followed by plating on
LB20 agar. In this way, 27 different isolates were obtained. Based on
preliminary HHL degradation tests, 5 isolates able to degrade HHL
down to below detection limit within 12 h were selected for further
experiments. HHL was used as a test compound because it is produced
by aquaculture pathogens such as Aeromonas hydrophila, Aeromonas
salmonicida, Edwardsiella tarda and Vibrio salmonicida (Swift et al.,
1997; Morohoshi et al., 2004; Bruhn et al., 2005).
The isolates were inoculated at 108 CFU ml− 1 in buffered LB20
medium supplemented with 5 mg l− 1 HHL in order to determine
whether they are able to degrade AHLs in a nutrient-rich background,
simulating the presence of high levels of other nutrients as is the case in
a gastrointestinal environment. All isolates degraded HHL down to
below detection limit within 6–9 h (Fig. 1). The HHL concentration also
decreased in the negative control during the first 6 h, which was
probably due to adsorption to the recipients or bacterial cells because
the concentration remained constant afterwards. Based on the fate of
HHL during the experiment, HHL degradation rates were calculated. The
isolates showed degradation rates between 0.7 and 0.9 mg HHL l− 1 h− 1
(Table 1), which is more than double the HHL degradation rate of
positive control strain P3/pME6863 (0.3 mg HHL l− 1 h− 1). The slower
degradation by the positive control strain might be due to lower activity
at relatively high salt concentrations.
The selected isolates were further characterised phenotypically
and all were found to be Gram-positive and spore-forming rods. All
isolates were able to grow both at low (5 g l− 1) and high (35 g l− 1)
salt concentrations, indicating that they could be used over a wide
range of salinities. The isolates were confirmed to belong to the genus
Bacillus by sequencing of the 16S rDNA. An NCBI BLAST search
revealed that the isolates were most closely related to Bacillus
anthracis, Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis
(Table 1). Bacillus species including Bacillus cereus and Bacillus
thuringiensis have been reported before to contain the AHL-degrading
lactonase enzyme AiiA, confering biocontrol activity against quorum
sensing plant pathogens (Dong et al., 2002; Lee et al., 2002). However,
to the best of our knowledge, AHL-degrading Bacillus spp. have never
been isolated from aquaculture settings before.
In conclusion, in this study, we isolated five Bacillus strains from
AHL-degrading enrichment cultures originating from whiteleg shrimp
and European sea bass, that are able to quickly degrade AHLs in a
nutrient-rich background. Because different studies have shown
before that AHL-degrading bacteria can decrease mortality caused
by aquaculture pathogens, these isolates might be interesting novel
biocontrol strains for use in aquaculture. In further research, we plan
to test the efficacy of the isolates in different host–pathogen settings.
Acknowledgements
The authors thank the “Fonds voor Wetenschappelijk Onderzoek”
(FWO-Vlaanderen) for financial support. T.D. is a postdoctoral fellow
of FWO-Vlaanderen. This work is supported by project no. G.A.064.10N
granted by FWO-Vlaanderen.
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