ORIGINAL ARTICLE
Caenorhabditis elegans-based in vivo screening ofbioactives from marine sponge-associated bacteria againstVibrio alginolyticusS. Durai, L. Vigneshwari and K. Balamurugan
Department of Biotechnology, Alagappa University, Karaikudi, India
Keywords
Caenorhabditis elegans, in vivo screening,
quorum quenching, Sponge-associated
bacteria, Vibrio alginolyticus.
Correspondence
Krishnaswamy Balamurugan, Department of
Biotechnology, Alagappa University, Karaikudi
630 003, India.
E-mail: [email protected]
2013/1233: received 20 June 2013, revised
10 August 2013 and accepted 21 August
2013
doi:10.1111/jam.12335
Abstract
Aim: To establish Caenorhabditis elegans based in vivo method for screening
bioactives from marine sponge associated bacteria (SAB) against Vibrio species.
Methods and Results: About 256 SAB isolates were screened for their ability
to rescue C. elegans infected with Vibrio species. The chloroform extract of the
positive isolate was subjected to column fractionation and purity of the active
fraction was analysed using HPLC. Further, the components were elucidated
using GC/MS. The active fraction was tested for its in vivo rescue activity,
antibacterial and anti-QS activity. In vivo colonization reduction and biofilm
inhibition efficiency were assessed using GFP-tagged V. alginolyticus using
confocal laser scanning microscopy (CLSM). The ability of the active fraction
in modulating expression of V. alginolyticus quorum sensing (QS) regulators
luxT and lafK was measured using real-time PCR. The results indicated that
the chloroform extract of SAB4.2 displayed significant rescue activity against
V. alginolyticus by inhibiting the QS pathway. HPLC analysis of the active
fraction revealed a single major peak and GC/MS analysis suggested Pyrrolo
[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) as the major
constituent. The potent bacterial isolate was identified as Alcaligenes faecalis.
Conclusions: In vivo screening using C. elegans identified a marine isolate that
inhibits the virulence of V. alginolyticus by interrupting the QS pathway.
Significance and Impact of the Study: The study provides a C. elegans based
in vivo screening method for identifying bioactives from natural resources by
overcoming the disadvantages of traditional in vitro plate assays.
Introduction
Since the discovery of antibiotics, micro-organisms have
been proven to be the source of various important life sav-
ing drugs. Innumerable terrestrial microbiomes have been
explored for their bioactive metabolites, and several com-
pounds have been identified against Gram-positive and
Gram-negative pathogens. As infectious microbes develop
resistance against several drugs, new sources for drug
discovery have become necessary to overcome multidrug-
resistance-related issues. Oceans are the source of structur-
ally diverse and novel anti-infectives. Sponges of the
phylum Porifera are the major biomass occupants of the
marine environment and only 1% of the phylum accounts
to fresh water origin (Radjasa et al. 2007). Besides being
the known source of several bioactive principles, marine
sponges also serve as the host for innumerable marine
micro-organisms (Wang 2006). In the recent years, symbi-
otic bacteria associated with sponges have been proven to
be the source of several lead molecules previously believed
as a metabolite of sponges itself (Zhang et al. 2005;
Newman and Hill 2006). Bioactive compounds isolated
from these sponges gains utmost importance due to their
similarity with compounds isolated from terrestrial
microbes of highly varied phylogeny (Perry et al. 1988).
Vibrio alginolyticus is a mesophilic marine bacterium
responsible for vibriosis, wound infections and gastroenter-
itis in humans. Infection and mortality caused by
Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology 1329
Journal of Applied Microbiology ISSN 1364-5072
V. alginolyticus in aquaculture results in significant eco-
nomic losses every year (Lee et al. 1996; Liu et al. 2011).
The increase in antibiotic resistance of V. alginolyticus iso-
lates at clinical and aquaculture environments and the
insufficient information about the mechanism of develop-
ment of antibiotic resistance have made it difficult to
control V. alginolyticus infections. Although the pathogen-
associated infections can be treated successfully with the
appropriate antibiotics, they can often lead to serious med-
ical conditions especially in immune-compromised
patients (Reilly et al. 2011). Hence, it becomes necessary to
find novel antivirulent compounds to treat V. alginolyticus
infections. The adhesive property of V. alginolyticus is
another important reason for its successive and efficient
invasion of the host system (Snoussi et al. 2008). Many
studies in recent years have proven the ability of Vibrio
spp. in developing biofilm during infection (Yildiz and
Visick 2009). The bacterium develops resistance to antibi-
otics by initial attachment and development of multilay-
ered, smeared colonies. Biofilm development in the host
and the production of extracellular products are attributes
of its virulence and pathogenicity. V. alginolyticus QS path-
ways like LuxO and LuxR were elucidated in recent reports
(Rui et al. 2008). luxT and lafK in V. alginolyticus are
found to play important roles in regulation of V. alginolyti-
cus virulence (Ye et al. 2008; Liu et al. 2012). Traditional
methods of antibiotic discovery primarily screen for com-
pounds that affect the growth and survival of bacterial
pathogens and identify compounds with almost similar
mechanism of action. The development of resistance
against these structurally similar antibiotics demands the
need for a new strategy of antimicrobial discovery (Moy
et al. 2006). Hence, identifying a potent QS-inhibitor
against V. alginolyticus will be an efficient method to
restrict the virulence by avoiding development of any
subsequent antibacterial resistance.
Marine invertebrates have developed highly specific
relationships with numerous micro-organisms, and these
associations have been recognized for ecological and bio-
logical importance (Armstrong and Van Baalen 1979;
Sponga et al. 1999; Strahl et al. 2002). It has been
reported that the ratio of micro-organisms with antimi-
crobial activity from invertebrates is higher than from
other sources (Ivanova et al. 1998; Burgess et al. 1999),
suggesting that invertebrate-associated micro-organisms
might play a chemical defence role for their hosts. Several
recent studies have proven the efficiency of marine bacte-
rial extracts to inhibit quorum sensing in Gram-positive
and Gram-negative bacteria (Bakkiyaraj and Pandian
2010; Nithya et al. 2010; Nithyanand et al. 2010).
Using live animal models as a platform for screening is
the most accurate alternative for in vitro solid assay
plates, mediating antimicrobial discovery. The main
advantage of using live models is that both efficacy and
toxicity of the screened candidate are validated simulta-
neously (Matthews and Kopczynski 2001). Current drug
discovery processes rely on popular model systems such
as yeast, worms and flies due to their high similarity with
the human system in their biological mechanisms and
protein functions (Zon and Peterson 2005; Kaletta and
Hengartner 2006). The main benefit of using these mod-
els in the drug discovery process for in vivo selection and
validation is the development of a potent technology for
the discovery of tomorrow’s small molecular medicines
(Powell and Ausubel 2008).
Caenorhabditis elegans is a well-established and popu-
larly accepted model for studying bacterial pathogenicity
and screening for antimicrobials (Bhavsar and Brown 2006;
Kurz and Ewbank 2007). Recently, we have established
C. elegans as a model system for V. alginolyticus (Durai
et al. 2011b) and studied the changes in C. elegans at both
physiological and molecular levels against Vibrio parahae-
molyticus infection (Durai et al. 2011a). With this knowl-
edge on the interaction of C. elegans with Vibrio spp., the
present study has screened and characterized several
sponge-associated bacteria (SAB) collected from the Palk
bay region of India, for their anti-infective activity against
pathogenic Vibrio spp. infection under in vivo conditions.
Materials and methods
Bacterial strains and C. elegans
Reference bacterial strains obtained from the American
Type Culture Collection (ATCC) included V. alginolyticus
(ATCC 17749), V. parahaemolyticus (ATCC 17802) and
Vibrio vulnificus (ATCC 29307). All the cultures were
maintained in Zobell marine medium 2216 (HiMedia,
Mumbai, India). Escherichia coli OP50 was maintained in
LB medium. V. alginolyticus was GFP tagged and main-
tained according to a standardized protocol (Sawabe et al.
2006). C. elegans WT Bristol N2 obtained from Caenor-
habditis Genetics Center (CGC) was routinely maintained
at 20°C on nematode growth medium (NGM) seeded with
E. coli OP50 as per the standard methods (Brenner 1974).
In all assays, the size of bacterial inoculum was kept
constant at 6�7 9 108 cells ml�1 (0�3 OD at 600 nm).
Collection, isolation and identification of sponge-
associated bacteria
The marine sponge samples of Haliclona spp. were col-
lected at the depth of 5 m from the costal waters of
Mandapam, Palk Bay North (longitude 79° 8″ East; lati-
tude 9°17″ North). Sponge samples were washed gently
with sterile seawater to remove debris and loosely bound
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In vivo screening of anti-infectives S. Durai et al.
bacteria and then transported to the laboratory in sterile
condition. The sponge samples were squeezed and
swabbed using sterile cotton swabs (HiMedia, India) in
different regions. The cotton swabs were directly used to
inoculate autoclaved marine agar medium (HiMedia).
Colonies of different morphology were selected and
maintained as pure culture in the marine agar medium.
Sterile environment was maintained throughout to avoid
external bacterial contamination in pure cultures. The
positive isolate SAB4.2 was identified by 16S rRNA gene
sequence analysis according to Nithyanand et al. 2011.
The identified sequence was then submitted to the
GenBank database.
C. elegans rescue assay
For the preliminary screening, a single colony from the
pure culture of each SAB was inoculated into 2 ml marine
broth and incubated at 37°C overnight. Each culture was
centrifuged at 9391 g, and the cell-free culture supernatant
was filtered using 0�2-lm filters and extracted with an
equal volume of chloroform 1 : 1 (v/v). The solvent frac-
tion was collected and evaporated to remove solvent under
reduced pressure. The resultant crude extract was dissolved
in 100 ll of molecular grade water and tested for the ability
to rescue C. elegans from V. alginolyticus infection. Rescue
assay was performed in liquid medium with c. 50 worms
transferred into each well of a 24-well plate, and 100 ll ofthe crude extract was used to analyse the rescue efficacy
against pathogen infection. The crude extracts of SAB
isolate which showed significant rescue activity against
V. alginolyticus was also tested against other Vibrio strains.
C. elegans exposed to Vibrio spp. alone served as a negative
control and uninfected C. elegans exposed to SAB4.2 alone
served as the positive control.
Purification and identification of active compound
Silica gel column and thin-layer chromatography
To identify the component of the crude extract responsible
for the rescue activity, the SAB4.2 extract was purified
using silica column chromatography with a solvent system
that was standardized using thin-layer chromatography
(TLC). The crude extract was purified using 20 9 2 cm sil-
ica gel columns (SRL, Mumbai, India), with a mesh size of
60–120 lm packed into a total column volume of 40 ml. A
methanol/chloroform/hexane solvent system in the ratio of
0�5 : 2�5 : 7 was used as a mobile phase and the absorbed
compound was eluted using methanol/water (75 : 25)
solution at a flow rate of 2 ml min�1. The purity of the
elutant was confirmed by TLC. Individual fractions were
analysed for their in vivo rescue activity. Active fractions
were collected many times and pooled together and
evaporated. Survival assay was performed using the
column-purified fraction at different concentrations
(150–600 lg ml�1). The toxicity of the SAB extract was
tested by exposing C. elegans to the active fraction in all
test concentrations (150–600 lg ml�1). C. elegans exposed
to V. alginolyticus alone served as a negative control and
uninfected C. elegans exposed to SAB4.2 alone served as
the positive control.
HPLC and GC/MS analysis
The column fraction displaying rescue activity was analy-
sed using high-pressure liquid chromatography (HPLC),
to check the purity. Column-purified active fraction
(5 mg) was redissolved in 0�5 ml of HPLC grade metha-
nol and introduced into a SHIMADZU system (Japan)
with C18 silica gel column. Fractions were eluted using
binary gradient of methanol with water from 0�1% to
100% (v/v). The major peaks were collected and checked
for rescue activity, and the active fraction was analysed
using gas chromatography/mass spectrometry. GC/MS
analysis of the column purified active fraction was
conducted using a Shimadzu GCMS-QP 2010 plus sys-
tem comprising a manual injector. The gas chromato-
graph is interfaced to a mass spectrometer equipped with
RXi-5MS (5% diphenyl, 95% dimethyl polysiloxane)
fused to a capillary column (30 9 0�25 9 0�25 lm). For
GC/MS detection, an electron ionization system was
operated in electron impact mode with ionization energy
of 70 eV. Helium gas (99�9%) was used as a carrier gas
at a constant flow rate of 1�0 ml min�1 and an injection
volume of 2 ll (splitless). The injector temperature was
maintained at 260°C, the ion-source temperature was
250°C and the oven temperature was programmed from
140°C (for 2 min) with an increase of 10°C min�1 to
250°C as the final temperature. Mass spectra were taken
at 70 eV at a scan interval of 0�5 s and from 45
to 450 Da. The solvent delay was 4 min, and the total
GC/MS running time was 30 min. The relative percentage
amount of each component was calculated by comparing
its average peak area to the total area. Real-time analyser
(Agilent Technologies, Palo Alto, CA, USA) was used to
handle the mass detector, mass spectra and chromato-
grams. Metabolites were identified by comparison with
the NIST database and standards.
Antibacterial and QSI assay
The effect of SAB4.2 column-purified active fraction
(600 lg ml�1) on cell proliferation of V. alginolyticus was
determined by a growth curve assay. The growth curve of
V. alginolyticus was determined for up to 24 h, along with
a quorum-sensing inhibition assay. The quorum-sensing
inhibition assay was performed using Chromobacterium
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S. Durai et al. In vivo screening of anti-infectives
violaceum ATCC 12472 in a 24-well plate liquid medium
as mentioned previously (Nithya et al. 2010). Cinnamal-
dehyde (75 mmol l�1) was the positive standard control
as at lower concentration and it has been proven to inhi-
bit quorum sensing without inhibiting bacterial growth
(Niu et al. 2006; Brackman et al. 2008).
Bacterial colonization assay
The effect of the column-purified SAB4.2 extract on in vivo
colonization in C. elegans intestine was analysed using
GFP-tagged V. alginolyticus. C. elegans exposed to GFP-
tagged V. alginolyticus in the presence and the absence of
SAB extract were examined under confocal laser scanning
microscope (CLSM) for their intensity of GFP fluorescence
which is directly proportional to the intestinal colonization
by V. alginolyticus. Worms exposed to GFP-tagged E. coli
served as negative controls. In addition, any GFP-like
pseudo-fluorescence activity of SAB4.2 against V. parahae-
molyticus was used as another control to avoid any false-
positive results. Also, the colony forming unit (CFU) count
of the sample isolated from the infected C. elegans was per-
formed on TCBS agar plates according to standardized
procedures (Durai et al. 2011b).
Pharyngeal pumping assay
To determine the pharyngeal pumping rate, infected
worms and column-purified SAB4.2 extract treated
worms were placed on NGM plates seeded with E. coli
OP50 and V. alginolyticus respectively. Pharyngeal pump-
ing was observed manually using a stereomicroscope
continuously for a minute. Animals infected with
V. alginolyticus served as controls against treated animals.
The pumping rate in the presence of E. coli OP50 was
also monitored and compared.
In vitro biofilm inhibition and microscopic observation
For visualization by light microscopy, biofilms were
allowed to grow on glass pieces (c. 1 9 1 cm) placed in
24-well polystyrene plates supplemented with the column-
purified bacterial extract (50–300 lg ml�1) and incubated
for 24 h at 37°C. The glass pieces were removed from the
plate and stained with crystal violet. The stained glass
pieces were placed on slides with the biofilm pointing up
and inspected by light microscopy. Visible biofilms were
documented with an attached digital camera (Nikon,
Tokyo, Japan). The biofilms were also monitored under
CLSM (Carl Zeiss, Goettingen, Germany) after washing
with PBS and staining with 0�01% acridine orange. A
488 nm Ar laser and 500–640 nm band pass emission fil-
ters were used to excite and detect the stained cells. CLSM
images were obtained from 24 h control and treated bio-
films and processed using Zen 2009 image software.
Quantitative analysis of biofilm inhibition was performed
using liquid assay (Nithya et al. 2010).
In vivo biofilm inhibition and CLSM analysis
Approximately, 25 worms per well were taken in 24-well
polystyrene plates containing Zobell Marine Broth 2216
(ZMB) and the column purified SAB4.2 extract. The wells
without SAB4.2 extract acted as controls. Each well was
inoculated with 1% (0�3 OD at 600 nm) of GFP-tagged
V. alginolyticus and incubated at 25°C. After 24 h, a
worm from each well was taken washed gently with M9
buffer to remove planktonic cells and observed for
adhered bacterial cells using CLSM. The density of the
surface biofilm was measured by intensity profile analysis
using Zen software 2009. The intensity of the GFP
recorded was directly proportional to the amount of
bacterial biofilm.
RNA isolation and qPCR
To analyse the regulation of quorum-sensing genes in
V. alginolyticus, total RNA was isolated from both V. alg-
inolyticus and V. alginolyticus treated with SAB4.2
column-purified active fraction for 12 h. Total RNA was
isolated using a guanidine thiocyanate/phenol extraction
method. RT-PCR was performed using Superscript III
Kit (Invitrogen) according to the manufacturer’s instruc-
tions. RT-PCR was followed by a standard real-time PCR
method in a single-well format in which the V. alginolyti-
cus QS-specific primers and the primers for the house-
keeping gene (rpoB) (Table 1) with their PCR mix
(SYBR Green kit, Applied Biosystems) were combined
separately at a predefined ratio. The PCR cycle numbers
were always titrated according to the manufacturer’s and
previously established protocols to ensure that the reac-
tion was within the linear range of amplifications. The
steady-state levels of QS-specific gene mRNA were
assessed from the cycle threshold (Cq) values during the
real-time PCR of the candidate QS-specific gene product
relative to the Cq values of the rpoB using a relative rela-
tionship method supplied by the manufacturer (Applied
Biosystems).
Statistical analysis
All the experiments were conducted thrice, and one-way
ANOVA (using SPSS Ver. 17.00) was used to compare the
mean values of each treatment. The significant difference
between the means of the parameter was calculated by
using Dunnett’s test (P < 0�05).
1332 Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology
In vivo screening of anti-infectives S. Durai et al.
Results
Alcaligenes faecalis (SAB4.2) extract rescued C. elegans
from V. alginolyticus infection
Among the screened isolates, crude chloroform extracts
(100 ll) of eight SAB isolates had rescued C. elegans
from V. alginolyticus infection during the preliminary
screening. Crude extract from the isolate SAB4.2 signifi-
cantly (P < 0�05) promoted the survival rate of C. elegans
by 60% against V. alginolyticus infection in 48 h postin-
fection. In the assay, C. elegans exposed to Vibrio spp.
displayed complete death (negative control) and
C. elegans exposed to SAB4.2 extract alone (without
Vibrio spp. infection) displayed 100% survival (positive
control). The ability of the SAB4.2 crude extract to rescue
C. elegans against other Vibrio spp. was also tested. The
activity of SAB4.2 extract against the other Vibrio strains
was not as significant as against V. alginolyticus. Hence,
bioactivity of the SAB4.2 isolate against V. alginolyticus
was explored in further studies. A full-length 16S rRNA
gene sequence of the SAB4.2 isolate was PCR amplified,
sequenced and aligned to the closest match using BLAST.
The strain SAB4.2 was identified as A. faecalis. The entire
16S rRNA gene sequence was submitted to the GenBank
database with the accession number JQ040510.
Purification and characterization of SAB4.2 extract
In total, 20 fractions (2 ml each) collected from silica gel
column were tested for their rescue activity. The active
fraction (fraction number 8) was collected repeatedly,
pooled and evaporated for further assays. The survival
assay, with varying concentrations of the column purified
extract indicated 300 lg ml�1 as the minimum dose
required to display a significant rescue activity of >50%(P < 0�05) (Fig. 1). We observed that the SAB4.2 active
fraction displayed no toxic effect on C. elegans. Further,
no reduction in life span was observed in C. elegans
exposed to the active fraction alone in all the tested con-
centrations (P < 0�05). Before proceeding with further
assays, the purity of the active fraction was analysed using
HPLC. The HPLC chromatogram displayed a single
major peak at the RT of 52�52. The fraction correspond-
ing to the major peak was collected and tested for the
rescue activity. Other minor peaks with no significant
intensity were also tested for rescue activity. The results
of the rescue assay indicated activity only in the fraction
corresponding to the major peak and not in other frac-
tions. The column-purified active fraction was character-
ized using GC/MS. The results of the mass spectrometry
indicated Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-
(2-methylpropyl) as major constituent (29�29%) (Fig. 2).
The other compounds identified include L-proline
(7�72%) and uric acid (4�52%). Considering the HPLC
chromatogram and the GC/MS analysis, it is clear that
the major peak corresponds to Pyrrolo[1,2-a]pyrazine-
1,4-dione,hexahydro-3-(2-methylpropyl) which is the
only component to display rescue activity.
Growth curve and QS inhibition
The column-purified active fraction of SAB4.2 did not
inhibit cell proliferation of V. alginolyticus even at a
high test concentration of 600 lg ml�1 (Fig. 3a). This
result suggested that the SAB4.2 active fraction inhibited
only the virulence of V. alginolyticus without affecting
the bacterial cell proliferation. QS Inhibition assay
performed with the marker strain C. violaceum further
confirmed the ability of SAB4.2 to prohibit bacterial
communication by inhibiting colour formation (Viola-
cein) in vitro equal to cinnamaldehyde the positive
control (Fig. 3b).
Table 1 Gene-specific primers for qPCR
S. No Gene Primer sequence (5′–3′)
1 luxT-FP GCGTAGTAAAGAAGATACCGA
luxT-RP GGAAGTGATGGCTAATACCGG
2 lafK-FP GAATCGGGAACGGGTAAAGAA
lafK-RP GGTGAACGCGCCTTTTACAT
3 rpoB-FP TCCGTATTCCCGATTCAGAG
rpoB-RP CAGCGGTGCAGAATAAGTCA
60 7040 5020 3010
Nem
atod
e su
rviv
al in
%
800
Time in hours
120
100
80
60
40
20
0
Figure 1 Survival curve for Caenorhabditis elegans exposed to Vibrio
alginolyticus, in the presence of various concentrations (150–
600 lg ml�1) of column-purified SAB4.2 extract. C. elegans exposed
to V. alginolyticus alone displayed complete death (negative control)
and C. elegans exposed to SAB4.2 without V. alginolyticus displayed
100% survival (positive control). Data are expressed in percentage of
live C. elegans in each group (P < 0�05). (□) V. alginolyticus alone;
(M) V. alginolyticus SAB4.2 150 lg; ( ) V. alginolyticus SAB4.2
300 lg; ( ) V. alginolyticus SAB4.2 450 lg; ( ) V. alginolyticus
SAB4.2 6050 lg and ( ) SAB4.2 alone 600 lg.
Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology 1333
S. Durai et al. In vivo screening of anti-infectives
Active fraction reduced in vivo colonization of
V. alginolyticus
The intensity of GFP fluorescence observed in the gut of
C. elegans was directly proportional to the density of
pathogen load in C. elegans. CLSM analysis of C. elegans
indicated reduced colonization of V. alginolyticus in the
presence of SAB4.2 extract at a significantly lower con-
centration (250 lg ml�1) (Fig. 4a). This in turn indicated
the in vivo efficacy of the SAB4.2 active fraction to pre-
vent bacterial adherence, thereby inhibiting colonization
in C. elegans intestine. Assays performed using higher
concentrations of SAB4.2 active fraction (300–500 lg ml�1) displayed similar results. It should be noted
that the in vivo colonization reduction concentration of
the SAB4.2 active fraction was lower than the optimal
concentration observed in rescue assay (300 lg ml�1)
proving the efficiency of the extract in inhibiting the
adhesion property of V. alginolyticus. The in vitro time
course CFU count of infected C. elegans calculated in the
presence of the active fraction (250 lg ml�1) further sup-
ported the CLSM data on the reduced intestinal coloniza-
tion by V. alginolyticus (Fig. 4b).
The presence of the SAB extract improved nematode
activity
In C. elegans, food intake by pharyngeal pumping is
directly correlated with the activity of animals. It is
important to note that the decrease in pharyngeal pump-
ing is a symptom of disease, illness or infection. The pha-
ryngeal pumping of C. elegans was visually scored
between control worms infected with V. alginolyticus and
worms infected in the presence of the SAB4.2 active
extract (250 lg ml�1). The results indicated a gradual
decrease in pharyngeal pumping during infection, in the
absence of the SAB4.2 extract. In contrast, worms
infected in the presence of the active extract displayed
pumping rate equal to that of worms treated with the
food source E. coli OP50 (Fig. 5).
In vitro and In vivo antibiofilm activity of SAB4.2
The effect of the SAB4.2 extract on V. alginolyticus bio-
film formation was studied on glass slides using crystal
violent staining, followed by light microscopic analysis.
The inhibition of biofilm was also studied using CLSM,
wherein the untreated biofilm always showed higher sur-
face coverage in contrast to the treated samples that dis-
played disrupted biofilm (Fig. 6a). Quantification of
in vitro biofilm inhibition indicated a gradual increase
with increase in extract concentration. At a concentration
of 250 lg ml�1, the SAB4.2 extract displayed significant
(75%) biofilm inhibition activity (Fig. 6b).
In vivo biofilm analysis from C. elegans CLSM micro-
graphs displayed a significant difference in the surface
biofilm between the control and the treated samples
(Fig. 6c). The density of adhering bacteria on C. elegans
(a)
(b)
250
200
150
100
50
0
0
100
30 50 70 90 110 130 150 170 190 210 230 250
244
201173154
153
125
126103
9170
6541
270 290 310
O
N
NH
O
330
10 20 30Retention time (RT)
PDA Multi 1
mA
U
40 50min
52·5
20
Figure 2 (a) HPLC analysis of column-purified active fraction of SAB4.2 (b) GC/MS spectra of the major compound Pyrrolo[1,2-a]pyrazine-1,4-
dione,hexahydro-3-(2-methylpropyl) in column purified active fraction of SAB4.2.
1334 Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology
In vivo screening of anti-infectives S. Durai et al.
surface was imaged and analysed using the software pro-
vided with the CLSM instrument. The intensity of fluo-
rescence in the control animal without the SAB extract
treatment was much higher (250 units) than the SAB4.2
treated samples (50 units). The above results confirmed
the ability of the SAB4.2 to reduce V. alginolyticus adher-
ence both in vitro and in vivo.
Regulation of QS responsible genes
The luxT gene, a strong regulator of the V. alginolyticus
QS system and lafK gene (responsible for a swarming
phenotype which is under control of luxT) were reported
for their significant role in V. alginolyticus infection (Liu
et al. 2012). As the SAB4.2 active fraction displayed
significant reduction in the QS-mediated violacein
production in the C. violaceum strain and inhibited
QS-controlled biofilm formation in V. alginolyticus, the
effect of the SAB extract on the transcription of V. algi-
nolyticus QS genes was analysed using qPCR. The results
indicated a significant down regulation of luxT and lafK
genes in V. alginolyticus exposed to the SAB4.2 active
fraction (Fig. 7). Taken together, all these results ascer-
tain the quorum-sensing inhibition mechanism of the
SAB4.2 extract against V. alginolyticus. Further studies on
understanding the complete QS pathway regulation in
the presence of SAB4.2 active fraction will provide clear
information on the mechanism of action.
Discussion
Marine sponges are an untapped source of anti-infectives
and act as shelter for diverse microbial communities
(Garson 1993). The present study screened secondary
metabolites from marine bacterial isolates associated with
the sponge Haliclona simulans for their ability to rescue
C. elegans against Vibrio spp. infection. C. elegans provide
a strong platform for screening bioactives from medicinal
plants, natural product libraries and synthetic chemical
libraries against bacterial pathogens (Bhavsar and Brown
2006; Moy et al. 2006, 2009; Adonizio et al. 2008).
Hence, the primary objective of the study was to exploit
the amenability of the model organism for in vivo screen-
ing of anti-infectives from sponge-associated bacteria. In
contrast to traditional plate assay methods, the activity
and toxicity of the extracts under examination were vali-
dated in a single step while using in vivo screening (Moy
et al. 2006). The simplicity of C. elegans system allowed
2·5
1·5
0·5
2
2 4 6 8 10 12 14 16 18 20
1
00
Time (h)
O.D
@ 6
00 n
m
CV control Positive standard control Test Medium control
(a)
(b)
Figure 3 (a) Growth curve analysis: Influence of column-purified active fraction (600 lg ml�1) on growth of Vibrio alginolyticus. The data repre-
sent the mean value of experiments performed in triplicates. (b) QS Inhibition: Liquid assay in a multiwell plate for QS inhibition displaying CV
control; Cinnamaldehyde (75 mmol l�1) as a positive standard control; CV treated with SAB4.2 active fraction (250 lg ml�1) as test and sterile
medium with active fraction alone (250 lg ml�1) as medium control. The SAB4.2-treated sample showed no colour formation in comparison with
untreated CV control. No contamination from SAB4.2 active fraction is seen in medium control. ( ) V. alginolyticus control and ( ) V. algino-
lyticus + SAB4.2.
Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology 1335
S. Durai et al. In vivo screening of anti-infectives
for the screening of a large number of samples in a short
time period (Bhavsar and Brown 2006). Although C. ele-
gans have been previously used as a screening model in
the identification of anti-infectives, this is the first study
to validate the bioactivity of marine bacterial extract
using C. elegans based screening against V. alginolyticus.
Previously, C. elegans has been established as a model for
Vibrio spp. infection and the infection related phenotypic
0·90
0·80
0·70
0·60
0·50
0·40
0·30
0·20
0·10
0·004 8 12 24 36
Time in hours
Log
CF
U p
er w
orm
50 µm
50 µm 50 µm
50 µm
(B)
(A)a b
c d
Figure 4 (a) In vivo colonization reduction: Confocal laser scanning microscopic images of Caenorhabditis elegans infected with GFP tagged
Vibrio alginolyticus displaying reduced in vivo colonization upon treatment with the SAB4.2 chloroform extract; (a,b) Infection in the absence of
SAB extract; (c,d) Infection in the presence of the SAB4.2 extract (250 lg ml�1). (b) CFU assay: Kinetic analysis of bacterial colonization in
C. elegans in untreated and SAB4.2 extract (250 lg ml�1) treated samples. ( ) Untreated and ( ) treated.
1336 Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology
In vivo screening of anti-infectives S. Durai et al.
changes, including bacterial colonization, pharyngeal
activity and transcriptional regulation of immune respon-
sible genes, have been reported (Durai et al. 2011a,b).
With the knowledge about infection from our previous
studies, preliminary screening using chloroform extract of
SAB isolates was performed against V. alginolyticus to
observe various phenotypic parameters. The observed
parameters included (i) rescue of the nematode against
V. alginolyticus infection; (ii) negligent toxicity of the
extract to C. elegans and (iii) developmental changes in
C. elegans due to extract treatment (egg laying). Results
of the preliminary screening identified 8 SAB isolates that
promoted C. elegans survival against V. alginolyticus
infection without displaying toxicity and developmental
changes. Toxicity assays in C. elegans are well established
and have been reported to be comparable with higher
animal models (Williams and Dusenbery 1990) and
mammalian cell lines (Moy et al. 2006). Hence, the active
compound identified by the current C. elegans-based
screening assay can be easily taken to drug trials. The
results suggested that the SAB4.2 extract displayed signifi-
cant rescue activity (60%) only against V. alginolyticus
infection.
HPLC analysis of the column-purified active fraction
showed a single major peak that exhibited the rescue
activity. GC/MS analysis of the major fraction revealed the
presence of Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-
3-(2-methylpropyl) as the major constituent (29�29%).
The compound identified in the present study was
previously isolated from sponge-associated bacteria and
reported for its bioactivity including antibiofilm and anti-
larval settlement activity against Vibrio halioticoli (Dash
et al. 2009).
Identification of natural anti-infectives that target the
virulence of bacteria rather than the survival is considered
to be highly important (Clatworthy et al. 2007). To
understand the mechanism of action of SAB4.2 extract in
promoting survival of C. elegans, in vitro assays were per-
formed. From comparing the in vitro growth assay and
the in vivo rescue assay, it became clear that SAB4.2
rescues C. elegans from V. alginolyticus infection by a dif-
ferent mechanism without affecting the growth of the
bacteria. Also, it is observed that low concentration of
SAB4.2 is efficient under in vivo conditions compared
with in vitro. The reason behind this could be SAB4.2
mainly inhibits the bacterial cell to cell communications
that could reduce the quorum-sensing-mediated virulence
phenotypes like adhesion. Due to reduced adhesive prop-
erty of the pathogen the level of colonization in C. ele-
gans intestine might be reduced, and hence, there was a
reduction in pathogenesis. The other possible reason for
this could be the efficiency of SAB4.2 to support the
innate immune system of C. elegans and act efficiently to
digest the pathogen and cure the infection paving way for
survival in the presence of SAB4.2. Similar results regard-
ing efficient in vivo activity of screening compound was
also observed by Moy et al. 2006. The efficiency of high
throughput screen to identify compounds targeting the
virulence of V. cholera has been previously reported
(Hung et al. 2005). The ability of cinnamaldehyde deriva-
tives to interfere with the AI-2-dependent QS system of
Vibrio spp. and its ability to rescue Artimia shrimp
against Vibrio harveyi infection has been documented in
earlier studies (Brackman et al. 2009; Musthafa et al.
2011). Antipathogenic activity of marine bacterial isolates
in inhibiting the virulence of Gram-negative bacterium
200
180
160
140
120
100
80
60
40
20
04 8 12 24
Time in hours
Num
ber
of fl
ings
/min
ute
Figure 5 Pharyngeal pumping: Treatment with SAB4.2 extract (250 lg ml�1) promotes the activity of Caenorhabditis elegans against Vibrio
alginolyticus infection. (&) Control; (h) untreated and ( ) treated
Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology 1337
S. Durai et al. In vivo screening of anti-infectives
(A)
(B)
a
b
c
240180120
600
0
100
100
200
200
300
300
400400
500
500
600
600
700
700
800
800Intensity
Y (µm) x (µm)
240180120
600
0
100
100
200
200
300
300400
400
500
500
600
600
700
700
800
800
Intensity
Y (µm)
x (µm)
240180120
600
0
100
100
200
200
300
300400
400
500
500
600
600
700
700
800
800Intensity
Y (µm)x (µm)
Bright field Confocal
99·70 µm
99·70 µm
99·70 µm
100
90
80
70
60
50
40
30
20
10
050 100 150 200 250 300
Concentration of SAB 4·2 extract in µg ml–1
% o
f bio
film
inhi
bitio
n
*
*
*
Figure 6 (A) In vitro biofilm inhibition: Effects of SAB4.2 chloroform extract at different concentrations on biofilm formation by Vibrio alginolyti-
cus on glass slides observed under light and Confocal microscopes, (a) Control untreated; (b) SAB4.2 100 lg ml�1and (c) SAB4.2 250 lg ml�1.
(B) Dose-dependent biofilm inhibition by SAB4.2 active fraction (100–250 lg ml�1). Statistically significant (P < 0�05) values are indicated by *.
(C) In vivo biofilm inhibition: Representative confocal laser scanning microscopic images of biofilm disruption with SAB4.2 chloroform extract at
different concentrations on glass slides and on Caenorhabditis elegans outer surface after 24 h, (a) Control untreated; (b) SAB4.2 treated
(100 lg ml�1); (c) SAB4.2 treated (250 lg ml�1).
1338 Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology
In vivo screening of anti-infectives S. Durai et al.
(C)a
b
c
DistanceIntensity Ch1
Intensity Ch1
Intensity Ch1
Intensity Ch1
Intensity
Marker 1143·335 µm
99
Marker 2286·239 µm
68
Difference142·904 µm
–31
Distance
Distance (µm)
Intensity Ch1
Marker 1308·148 µm
65
Marker 2615·371 µm
15
Difference307·223 µm
–50
DistanceIntensity Ch1
Marker 1173·114 µm
10
Marker 2345·708 µm
23
Difference172·594 µm
13
250
200
150
100
50
0
Intensity
250
200
150
100
50
0
Intensity
250
200
150
100
50
0
0 50 100 150 200 250 300 350 400
Distance (µm)
0 100 200 300 400 500 600 700 800 900
Distance (µm)
0 50 100 150 200 250 300 350 400 450 500
Figure 6 (Continued)
Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology 1339
S. Durai et al. In vivo screening of anti-infectives
Pseudomonas aeruginosa by targeting the AHL-mediated
virulence factor has also been reported (Musthafa et al.
2011). In many cases, the marker strain C. violaceum was
used to identify the QS inhibition activity of marine
metabolites (Adonizio et al. 2008; McLean et al. 2008;
Bakkiyaraj and Pandian 2010; Nithya et al. 2010;
Annapoorani et al. 2012). Hence, the ability of SAB4.2 to
inhibit QS was studied initially using the marker strain
C. violaceum. The assay suggested that SAB4.2 signifi-
cantly quenched the QS-mediated pigment production in
C. violaceum.
Intestinal colonization and persistence are claimed as
the major routes by which V. alginolyticus establishes
infection in C. elegans (Durai et al. 2011b). The persis-
tence and integrity of the bacterium in C. elegans intes-
tine has a crucial role in infection manifestation. In vivo
integrity of bacteria leads to the enhanced accumulation
of bacterial cells resulting in the formation of an extracel-
lular matrix and provides the environment necessary for
biofilm formation (Sousa et al. 2009). It was observed
that adhesive property and biofilm formation play major
roles in V. alginolyticus infection in human and marine
hosts (Snoussi et al. 2008). In the current study, CLSM
analysis of SAB4.2 treated C. elegans revealed reduced
pathogen colonization in the intestine. In vitro bacterial
colonization assay proved the ability of SAB4.2 extract in
reducing pathogen CFU in the infected worm intestine.
Biofilm assay performed in vitro proved the ability of
SAB4.2 to reduce the adherence of V. alginolyticus on a
glass slide. The in vivo biofilm assay on C. elegans also
confirmed reduced adhesion of V. alginolyticus in the
presence of SAB4.2 extract. Previously, many marine and
nonmarine micro-organisms have been proven to reduce
virulence by inhibiting biofilm formation against patho-
genic bacteria (Valle et al. 2006; Jiang et al. 2011).
Recently, SAB belonging to Bacillus spp. was reported to
inhibit biofilm formation in E. coli (Sayem et al. 2011).
Extracts from coral associated bacteria and coral associ-
ated actinomycetes were also reported to have antibiofilm
activity against Streptococcus pyogenes (Nithyanand et al.
2010) and clinical isolates of S. aureus (Bakkiyaraj and
Pandian 2010). Inhibiting virulence without affecting the
growth of bacteria is a better way of screening to avoid
emergence of antibacterial resistance behaviour against
antibiotic compounds. Hence, natural compounds inhib-
iting the conserved virulence pathway of many patho-
genic bacteria can become a potent lead to combat future
bacterial infections. The quorum-sensing system among
the bacterial community is a primary target of recent
screening studies to reduce virulence without cell lethal-
ity. The V. alginolyticus QS system was identified to be
similar to that of V. harveyi, involving the LuxO and
LuxR system controlling the major virulent factors (Liu
et al. 2012; Rui et al. 2008; Ye et al. 2008). Recently, a
new regulator, LuxT, was identified to control LuxO at
the transcriptional level and LuxR at post-transcriptional
level (Liu et al. 2012). The effect of SAB4.2 extract on
V. alginolyticus luxT regulation was proven for the first
time using real-time PCR. Down regulation of luxT and
lafK upon SAB4.2 treatment might have prevented
V. alginolyticus from adherence and in vivo colonization,
thereby preventing infection in the nematode. The effi-
ciency of C. elegans-based screening for the validation of
QS regulators from South Florida medicinal plant has
been previously reported against P. aeruginosa (Adonizio
et al. 2008). With the results of the present study and
previous validation, it is evident that C. elegans-based
screening can identify not only antimicrobials and
immune modulators (Moy et al. 2006) but also new leads
attenuating bacterial virulence.
To our knowledge, this is the first study employing an
in vivo screening system to identify sponge associated
bacteria which produce bioactive compounds with rescue
activity against V. alginolyticus infection in C. elegans. The
present study confirmed that using such efficient screening
systems with stringent criteria for identification of novel
anti-infective producers will lead to new perspectives of
screening methods.
Acknowledgements
This study was supported in part by the Grants from
University Grants Commission (UGC), Department of
Biotechnology (DBT), Council of Scientific & Industrial
Research (CSIR), and Department of Science and Tech-
nology (DST), Ministry of Science and Technology, India
to Dr. K. Balamurugan. Financial support to S. Durai
provided by Lady Tata Memorial Trust in the form of
10
9
8
7
6
5
4
3
2
1
0luxT lafK
Fol
d ch
ange
in m
RN
A le
vel
Figure 7 Regulation of Vibrio alginolyticus QS regulator genes, luxT
and lafK in control and SAB4.2 extract (250 lg ml�1) treated
samples. (&) Control and (h) treated.
1340 Journal of Applied Microbiology 115, 1329--1342 © 2013 The Society for Applied Microbiology
In vivo screening of anti-infectives S. Durai et al.
Senior Scholarship is thankfully acknowledged. The
authors acknowledge the computational and Bioinformat-
ics facility provided by Alagappa University Bioinformat-
ics Infrastructure Facility (funded by DBT, GOI; Grant
No. BT/BI/25/001/2006). Also, the authors thankfully
acknowledge Miss. S. Krishnaveni for her timely help in
manuscript preparation.
Conflict of Interest
No conflict of interest declared.
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