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Influence of Glucose Concentrations on Biofilm Formation, Motility, Exoprotease
Production, and Quorum Sensing in Aeromonas hydrophila
Article in Journal of food protection · February 2013
DOI: 10.4315/0362-028X.JFP-12-321 · Source: PubMed
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Influence of Glucose Concentrations on Biofilm Formation,Motility, Exoprotease Production, and Quorum Sensing in
Aeromonas hydrophila
IQBAL KABIR JAHID,1,2 NA-YOUNG LEE,1 ANNA KIM,1 AND SANG-DO HA1*
1School of Food Science and Technology, Chung-Ang University, 72–1 Nae-Ri, Daedeok-Myun, Ansung, Kyunggido 456–756, South Korea; and
and 2Department of Microbiology, Jessore Science and Technology University, Jessore 7408, Bangladesh
Influence of Glucose Concentrations on Biofilm Formation,Motility, Exoprotease Production, and Quorum Sensing in
Aeromonas hydrophila
IQBAL KABIR JAHID,1,2 NA-YOUNG LEE,1 ANNA KIM,1 AND SANG-DO HA1*
1School of Food Science and Technology, Chung-Ang University, 72–1 Nae-Ri, Daedeok-Myun, Ansung, Kyunggido 456–756, South Korea; and
and 2Department of Microbiology, Jessore Science and Technology University, Jessore 7408, Bangladesh
MS 12-321: Received 18 July 2012/Accepted 16 October 2012
ABSTRACT
Aeromonas hydrophila recently has received increased attention because it is opportunistic and a primary human pathogen.
A. hydrophila biofilm formation and its control are a major concern for food safety because biofilms are related to virulence.
Therefore, we investigated biofilm formation, motility inhibition, quorum sensing, and exoprotease production of this
opportunistic pathogen in response to various glucose concentrations from 0.05 to 2.5% (wt/vol). More than 0.05% glucose
significantly impaired (P , 0.05) quorum sensing, biofilm formation, protease production, and swarming and swimming
motility, whereas bacteria treated with 0.05% glucose had activity similar to that of the control (0% glucose). A stage shift
biofilm assay revealed that the addition of glucose (2.5%) inhibited initial biofilm formation but not later stages. However,
addition of quorum sensing molecules N-3-butanoyl-DL-homoserine lactone and N-3-hexanoyl homoserine lactone partially
restored protease production, indicating that quorum sensing is controlled by glucose concentrations. Thus, glucose present in
food or added as a preservative could regulate acyl-homoserine lactone quorum sensing molecules, which mediate biofilm
formation and virulence in A. hydrophila.
Aeromonas hydrophila is opportunistic and a primary
pathogen of amphibians, reptiles, fish, and humans (1). A.hydrophila causes various human infections such as
gastrointestinal tract syndromes, wound and soft tissue
infections, and bloodborne dyscrasias (29). A. hydrophilarecently has been categorized as an emerging foodborne
pathogen (17), with food and/or water as possible sources of
human infection (14).Biofilms are architecturally complex assemblies of
microorganisms that form on biotic or abiotic surfaces
or at interfaces. Biofilms are characterized by interactions
between microorganisms embedded in a matrix of extra-
cellular polymeric substances created by the microbial
populations and exhibit altered phenotypes with respect to
growth rate and gene transcription (7). Like many other
microorganisms, A. hydrophila can form biofilms in
laboratory settings on stainless steel (23), glass (40), and
vegetables (8). A. hydrophila possesses two distinct types of
flagella, polar and lateral, for swimming and swarming,
respectively. Both flagellar systems play vital roles in
pathogenesis (34) and optimal biofilm formation (18). A.hydrophila secretes metalloprotease, zinc protease, and
serine protease, which play a vital role in invasiveness
and generation of infection (5).
Bacterial genes and their respective phenotypes are
regulated in a population-dependent manner with the aid of
small diffusible signal molecules through a phenomenon
referred to as quorum sensing (QS) (36). Gram-negative
bacteria produce acyl-homoserine lactone (AHL) QS
systems to regulate a variety of physiological processes,
including virulence. A. hydrophila produces N-3-butanoyl-
DL-homoserine lactone (C4-HSL) synthase, encoded by
ahyI, and N-3-hexanoyl homoserine lactone (C6-HSL)
synthase, encoded by ahyR, at a ratio of 70:1 (35).Modulation of virulence, exoprotease production, T3SS and
T6SS protein expression, and biofilm development by QS has
been previously reported (15, 23, 36). Khajanchi et al. (14)also reported that AHLs and cytotoxic activity levels were
higher in clinical strains than in strains isolated from water,
establishing the importance of QS in human infection.
Because biofilms are directly related to virulence (31)and sessile cells are resistant to antibiotics and various
disinfectants (24), a better approach is to inhibit biofilm
formation rather than attempt to kill bacteria already in
established biofilms (32). Blockage of QS is a new
phenomenon, and many antibacterial agents have been
identified recently to combat disease in this manner (2).Furukawa et al. (10) reported that sugar fatty acid esters
inhibited biofilm formation by foodborne pathogens. Simi-
larly, EDTA (3), bacteriocins, and competition for nutrients
(31) inhibited biofilm formation by Listeria monocytogenes.* Author for correspondence. Tel: 82-031-670-4831; Fax: 82-031-675-4853;
E-mail: sangdoha@cau.ac.kr.
239
Journal of Food Protection, Vol. 76, No. 2, 2013, Pages 239–247doi:10.4315/0362-028X.JFP-12-321Copyright G, International Association for Food Protection
Glucose also has been reported as having antibiofilm effects
on Escherichia coli (13) and Bacillus cereus (33).Chestnut honey (38) and vanillin (30) also have been
reported as having antibiofilm and anti-QS effects on A.hydrophila. The present study was conducted to examine
the effect of various glucose concentrations on biofilm
formation, motility, exoprotease production, and QS in A.hydrophila.
MATERIALS AND METHODS
Bacterial strains, culture media, and growth conditions.
The strains used for the study were A. hydrophila strains KCTC
2358 (isolated from a can of milk with a fishy odor), KCTC 11533
(isolated from surface water), and KCCM 32586 (a clinical
isolate). The bioreporter strain Chromobacterium violaceumCV026 (Animal, Plant and Fisheries Quarantine and Inspection
Agency, Seoul, Korea) also was included. A 20% (wt/vol) glucose
(Merck, Darmstadt, Germany) solution was prepared by filter
sterilization with 0.22-mm-pore-size filters (Millipore Corporation,
Billerica, MA). Nutrient broth (NB; Difco, BD, Sparks, MD) was
used for all the experiments except the violacein assay, for which
we used Luria-Bertani (LB) medium (Difco, BD). Before each
experiment, the cultures were activated by transferring from the
280uC freezer on nutrient agar plates to 30uC for overnight
incubation. A single colony from the plate was inoculated in 5 ml
of NB and incubated overnight at 30uC for 48 h without shaking
and then diluted 1:50 in NB or LB containing 0, 0.05, 0.25, 0.5,
1.0, and 2.5% (wt/vol) glucose. Bacteria were grown at 30uCunless otherwise indicated.
Quantitative biofilm formation assay. The quantitative
biofilm assay was performed according to the procedure described
by O’Toole (28) with minor modifications. The cultures were
grown in NB for 48 h without shaking and then diluted (1:50)
in NB containing various concentrations of glucose, and 100-ml
aliquots were placed in each well of a 96-well polystyrene
microtiter plate (BD, Franklin Lakes, NJ). The microtiter plate was
incubated at 30uC for 72 h without shaking. After incubation, the
absorbance was determined as an indicator of cell growth at 600 nm
with a microplate reader (Spectra Max 190, Molecular Devices,
Sunnyvale, CA). The plate was washed in a small tub of water after
the bacterial cultures were discarded, air dried overnight, stained
with 125 ml of 0.1% (wt/vol) crystal violet (CV) by incubating for
30 min at room temperature, and dried again overnight. The CV
was solubilized with 125 ml of 95% (vol/vol) ethanol at room
temperature for 10 min, and the absorbance was read at 595 nm
with a microplate reader. Biofilm formation was determined using
equation 1, normalized to planktonic growth according to Teh et al.
(37):
BFI~(AB{CW)
(GB{GW)ð1Þ
where BFI is the biofilm formation index, AB is the optical density
at 595 nm (OD595) of the CV-stained attached microorganisms,
CW is the OD595 of the stained blank wells containing
microorganism-free medium only, GB is the OD600 of the cell
growth in suspended culture, and GW is the OD600 of the blank
well. The degree of biofilm production was classified into three
categories according to Martinez-Medina et al. (25): weak (0.1 .
BFI # 0.5), moderate (0.5 . BFI # 1), or strong (BFI . 1).
Motility assay (swimming and swarming). Swimming is
defined as flagellum-directed movement in an aqueous environment,
and swarming is defined as multiple, lateral flagellum-directed rapid
movement on solid surfaces. Both forms of motility were examined
in the present study using protocols described elsewhere (6, 12) with
slight modifications. For swimming motility, 1.5 ml of the same OD
cultures were spotted at the center of a plate containing 25 ml of NB
and 0.3% Bacto-agar (Difco, BD) and incubated face up for 24 h at
25uC. The assay was performed after the plates were allowed to dry
for 12 h. To measure swarming, the same culture volume was
inoculated on the surface of an agar plate containing LB and 0.5%
Bacto-agar (Difco, BD) and incubated at 25uC for 48 h. After
incubation, the diameter of the area of motility of the strains was
measured, and the plates were photographed.
Exoprotease assay. Exoprotease activity was assessed using
a Fluoro protease assay kit (G-bioscience, St. Louis, MO).
Overnight cultures were diluted (1:50) with cultures with the same
OD in different concentrations of glucose containing fresh NB and
incubated for 48 h with shaking at 200 rpm. After incubation,
supernatants were collected by centrifugation at 16,060 | g. The
supernatant (50 ml) from each glucose condition was added to
150 ml of fluorescein isothiocyanate–conjugated substrate and
incubated at room temperature for 1 h. Fluorescence was measured
at 485 nm excitation and 530 nm emission with a fluorescence
microplate reader (Spectra Max Gemini EM, Molecular Devices).
The data were interpreted using the trypsin standard supplied with
the kit. The medium with fluorescent substrate was used as the
negative control.
Production of AHLs. Violacein production was quantified
using procedures similar to those previously described (20) with
some modifications. The A. hydrophila strains were grown for 48 h,
and C. violaceum CV026 was grown in LB for 24 h at 28uC with
shaking at 200 rpm. The A. hydrophila strains were streaked
perpendicularly to the C. violaceum CV026 on LB agar plates with
different concentrations of glucose and incubated for 24 h at 28uC.
All of the culture grown on the C. violaceum CV026 plate was
collected by rubbing it off plate, and this culture was mixed with
500 ml of dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis,
MO), which resulted in release of the violacein pigment. After
centrifugation at 16,060 | g for 15 min, 200 ml of colored DMSO
from each condition was evaluated at 585 nm with a microplate
reader.
SEM of biofilm experiments. Biofilm formation of A.hydrophila strain KCTC 11533 was observed using scanning
electron microscopy (SEM) after incubation for 72 h on
polystyrene coupons (2 by 2 cm) prepared in house in 5 ml of
NB containing different concentrations of glucose. The selected
coupons were rinsed three times with phosphate-buffered saline
(PBS; pH 7.2). The adhered cells were fixed in 4% glutaraldehyde
in PBS for 2 h and then washed three times for 15 min with PBS.
The fixed cells were serially treated with ethanol (50% for 15 min,
60% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min,
and 100% two times for 15 min each) and successively dehydrated
by soaking in 33, 50, 66, and 100% hexamethyldisilazane in
ethanol for 15 min each. The dehydrated samples were examined
under a scanning electron microscope (S-3400, Hitachi High
Technologies, Tokyo, Japan) according to the procedure described
previously (20).
Biofilm inhibition determined with the stage shift assay.For the stage shift biofilm assay, we followed the procedure
described by Chang et al. (3). A. hydrophila KCTC 11533 was
inoculated without glucose into NB, 48-h cultures were diluted to
240 JAHID ET AL. J. Food Prot., Vol. 76, No. 2
1:50 in NB, and 100-ml aliquots were inoculated into a microtiter
plate and incubated at 30uC for 6 days. At various time intervals,
2.5% (wt/vol) glucose was added to the microtiter wells, and
biofilm inhibition was calculated using equation 2:
biofilm reduction (%)~(A2:5{B2:5)
(A0{B0)
� �100 ð2Þ
where A2.5 denotes the average BFI at specific times with the
addition of 2.5% glucose and bacteria, B2.5 denotes the BFI of the
specific time with the addition of 2.5% glucose in blank wells, A0
denotes the BFI after 6 days in the control (0% glucose), and B0
denotes the BFI of blank wells.
Induction of exoprotease inhibition using exogenousAHLs. N-Acyl homoserine lactones C4-HSL and C6-HSL were
purchased commercially (Cayman Chemical Company, Arbor, MI),
and 100 mmol stock solutions were prepared by dissolving the
AHLs in DMSO according to the manufacturer’s instructions and
stored at 220uC until further use. A. hydrophila KCTC 11533 was
inoculated into NB containing different concentrations of glucose,
and final concentrations of 20 mmol of C4-HSL and C6-HSL, both
singly and in combination, were used to examine protease activity
using the Fluoro protease assay kit according to the procedure
described for exoprotease activity. The percent induction by
exogenous AHLs with and without the addition of HSL and blank
values with or without HSL were calculated using equation 3:
induction protease (%)~P(HC{HB)
P(C{B)
� �100 ð3Þ
where HC denotes the average absorbance in the supernatant with
the addition of cells, HSL, and glucose; HB denotes the absorbance
value in a blank with glucose and HSL without the addition of cells;
C denotes the average absorbance concentration in control (0%)
glucose with cells without HSL; and B denotes the average of 0%
glucose blank absorbance values without bacterial cells and HSL.
The values were converted to protease concentrations at specific
glucose concentrations and HSL, which are denoted as P(HC 2 HB)
and control glucose as P(C 2 B).
Statistical analysis. To determine the inhibitory effect of
glucose on motility, biofilm formation, exoprotease activity, and
QS, all experiments were repeated three times. The data were
analyzed with an analysis of variance using SAS software (version
9.1, SAS Institute Inc., Cary, NC) for a completely randomized
design. When the effect was significant (P , 0.05), means were
separated with Duncan’s multiple range test.
RESULTS
Biofilm formation. The results of biofilm formation
after exposure to different glucose concentrations, based on
CV staining on the polystyrene solid surface of 96-well
microtiter plates, are shown in Figure 1. The representative
results from A. hydrophila KCTC 2358 showed a gradual
reduction of biofilm formation with the addition of glucose
in NB (Fig. 1a). Addition of 0.05% glucose did not
significantly reduce biofilm formation compared with the
control, whereas 0.25% glucose significantly (P , 0.05)
inhibited biofilm formation of all three strains (Fig. 1b).
Glucose at 2.5% completely inhibited biofilm formation
(Fig. 1b) without any significant effect (P . 0.05) on
planktonic growth; therefore, biofilm reduction by glucose
was not due to growth inhibition. The mean planktonic
growth values, as indicated by OD600 values, were 0.22, 0.75,
and 0.34 for strains KCTC 2358, KCTC 11533, and KCCM
32586, respectively, in control broth (0% glucose). The mean
growth (OD600) values were 0.21, 0.72, and 0.33, respec-
tively, with 0.25% glucose and 0.19, 0.71, and 0.29,
respectively, with 2.5% glucose. The average BFI scores
for biofilm formation were 1.3, 1.04, and 0.31 for strains
KCTC 2358, KCTC 11533, and KCCM 32586, respectively,
in control broth (0% glucose) and were reduced to 0.33, 0.39,
and 0.074, respectively, in 0.25% glucose. The results also
showed significant (P , 0.05) strain-specific BFI values;
strain KCCM 32586 had low biofilm formation in control
broth. In general, the results indicate that regardless of strain
concentrations of glucose higher than 0.05% had an
inhibitory effect on biofilm formation.
Swarming and swimming motility. Because motility
is one of the contributing factors for virulence and biofilm
formation and because A. hydrophila cells have both lateral
FIGURE 1. Biofilm formation by A. hydrophila. (a) Microtiter wells with strain KCTC 2358. (b) Biofilm formation index values for threeA. hydrophila strains growth with various glucose concentrations and stained with crystal violet. Values are the mean ¡ standard error ofthe mean for three independent experiments. Within each variable, values with the same letter are not significantly different according toDuncan’s multiple range test (P . 0.05).
J. Food Prot., Vol. 76, No. 2 INFLUENCE OF GLUCOSE CONCENTRATIONS ON BIOFILM OF A. HYDROPHILA 241
and polar flagella, motility was tested using semisolid agar
with incubation for 24 h for swarming motility and 48 h for
swimming motility. Figure 2 shows both types of motility
of all three A. hydrophila strains of different origins. A slow
reduction in bacterial motility was found in medium
supplemented with increasing concentrations of glucose
(Fig. 2a). No movement from the site of inoculation was
observed with 2.5% glucose. Motility of all strains was
significantly impaired (P , 0.05) by the addition of glucose
(Fig. 2b and 2c), following the same trend as observed for
the biofilm inhibition assay. Mean diameters of swarming
motility (Fig. 2b) were 39.7, 18.3, and 38.3 mm for strains
KCTC 2358, KCTC 11533, and KCCM 32586, respective-
ly, on control plates, which was significantly reduced to
10.7, 7.0, and 11.3 mm, respectively, with the addition of
0.25% glucose. Mean diameters of swimming motility
(Fig. 2c) were 61.6, 20.7, and 46.7 mm, respectively, on
control plates, with significant reductions to 17.7, 14.3, and
5.7 mm, respectively, with the addition of 0.25% (wt/vol)
glucose, indicating the same inhibition trend as seen for
swarming. Motility also was strain specific; strain KCTC
11533 was significantly less motile (P , 0.05).
Exoprotease production. The pathogenic and virulence
characteristics of A. hydrophila are related to exoprotease
production. In control and 0.05% added glucose treatments,
exoprotease production was induced, but higher glucose
concentrations ($0.25%) significantly inhibited (P , 0.05)
the exoprotease activity in the A. hydrophila supernatant
(Fig. 3). Strain KCTC 2358 had significantly lower protease
production in the control broth than did the other two strains.
Protease concentrations were 10.9, 23.2, and 38.9 ng/ml for
strains KCTC 2358, KCTC 11533, and KCCM 32586,
respectively, in control supernatants and were reduced to 0.2,
7.5, and 1.4 ng/ml, respectively, with 0.25% glucose. Glucose
concentrations of 1.0 and 2.5% inhibited the secretion of
exoprotease to below the detection limit of the assay (Fig. 3).
FIGURE 2. Motility assay for A. hydrophila. (a) Swarmingmotility in a representative experiment with strain KCTC 2358.(b) Swarming motility assay (25uC) for all A. hydrophila strains.Values are the mean ¡ standard error of the mean of threeindependent experiments. (c) Swimming motility assay. Values arethe mean ¡ standard error of the mean of three independentexperiments. Within each variable, values with the same letter arenot significantly different according to Duncan’s multiple rangetest (P . 0.05).
FIGURE 3. Exoprotease assay (30uC) in A. hydrophila strainsfor 48 h. Values are the mean ¡ standard error of the mean ofthree independent experiments. Within each variable, values withthe same letter are not significantly different according toDuncan’s multiple range test (P . 0.05).
242 JAHID ET AL. J. Food Prot., Vol. 76, No. 2
Violacein production. The QS regulation effect was
determined using the biosensor C. violaceum CV026 C6-
HSL transposon mutant strain. This mutant strain is negative
for violet color in the absence of AHLs but produces color
when AHLs are externally supplied. When A. hydrophilastrains are grown with CV026, the mutant produces color
because violacein is produced in response to AHLs secreted
by the test strains, and the intensity of the color is
proportional to the AHL concentration. Figure 4a shows
violacein production by CV026 cultures grown with strain
KCTC 11533 at 1.0 and 2.5% glucose in one experiment,
which did not result in color production. Figure 4b shows
violacein production for multiple experiments. Mean
violacein production was 0.922 and 0.720 with strains
KCTC 11533 and KCCM 32586, respectively, under
control conditions, and addition of 0.25% glucose signif-
icantly reduced (P , 0.05) the values to 0.679 and 0.223,
respectively. Violacein values were 0.075 and 0.046,
respectively, at 0.5% glucose, but addition of 1.0% glucose
did not result in color production. Culture with strain KCTC
2358 did not result in production of color by the biosensor
strain at any glucose concentration. These findings indicate
that in general, AHL production by A. hydrophila strains
was repressed by glucose.
Biofilm observation by SEM. Figure 5 shows images
of biofilm formation by strain KCTC 11533 with different
concentrations of glucose; this strain produced significantly
more biofilm and induced more violacein production than
did the other A. hydrophila strains. Three-dimensional,
thick, and firmly attached mushroom-like structures were
noted for the control culture and that with 0.05% glucose
(Fig. 5a and 5b), whereas addition of 0.25 and 0.5% glucose
resulted in only cell monolayers (Fig. 5c and 5d). Very few
bacteria were attached to the polystyrene surface in cultures
with 1.0 and 2.5% glucose (Fig. 5e and 5f). The bacterial
cell shapes also differed at noninhibiting concentrations
(#0.05%) and inhibiting concentrations ($0.25%) of
glucose (Fig. 5g through 5i). The cells in the control
biofilms (Fig. 5g) and the 0.05% glucose biofilms (Fig. 5h)
were oval, short rods, and those grown with 1.0% glucose
were elongated rods (Fig. 5i). These SEM results further
support biofilm inhibition by glucose at concentrations
$0.25%.
Stage shift biofilm assay. Figure 6 shows that the
addition of glucose (2.5%) at different stages of growth
of A. hydrophila KCTC 11533 significantly inhibited
(P , 0.001) biofilm formation within 48 h. The mean
decreases in the BFI compared with that at 6 days were
89.9, 83.3, 67.4, 50.1, and 18.4% at 0 h, 6 h, 12 h, 1 day,
and 2 days, respectively. The addition of glucose did not
significantly affect biofilm inhibition after 2 days.
Induction of protease inhibition with exogenousAHLs. The exogenous addition of 20 mmol C4-HSL and
20 mmol C6-HSL, both singly and in combination, resulted
in significantly higher (P , 0.05) protease production.
Figure 7 shows that in A. hydrophila KCTC 11533 the
combination of C4-HSL and C6-HSL resulted in higher
protease activity than obtained when each AHL was added
alone. The combination of both molecules restored protease
production to 83.4, 39.8, and 33.5% for media containing
0.25, 0.5, and 1.0% glucose, respectively. The external
supply of QS molecules did not restore this activity in
medium with 2.5% glucose.
DISCUSSION
Kirov (17) reported on the potential of A. hydrophila as
a human foodborne pathogen. Because biofilms may play a
vital role in virulence, we assessed the potential role of QS
and inhibition of QS in biofilm formation, motility, and
exoprotease activity in A. hydrophila. Other researchers
have noted inhibition of QS and biofilm formation by
compounds such as brominated furanones (11), D-amino
acids (19), cis-2-decenoic acid (4), and 7-hydroxyindole
(21). Although vanillin and honey have been reported to
inhibit biofilm formation in A. hydrophila (30, 38), we
FIGURE 4. Violacein production by reporter stain C. violaceum CV026 grown with A. hydrophila and different concentrationsof glucose. (a) One representative experiment of violacein production with A. hydrophila KCTC 11533. (b) Histogram values are the mean¡ standard error of the mean of three independent experiments. Within each variable, values with the same letter are not significantlydifferent according to Duncan’s multiple range test (P . 0.05).
J. Food Prot., Vol. 76, No. 2 INFLUENCE OF GLUCOSE CONCENTRATIONS ON BIOFILM OF A. HYDROPHILA 243
FIGURE 5. Scanning electron micrographs of biofilms formed on polystyrene coupons by A. hydrophila KCTC 11533 with differentconcentrations (wt/vol) of glucose: (a) 0%; (b) 0.05%; (c) 0.25%; (d) 0.5%; (e) 1.0%; (f) 2.5%; (g) 0%, higher magnification (10 mm);(h) 0.05%, higher magnification (10 mm); (i) 1.0%, higher magnification (10 mm).
FIGURE 6. Stage shift biofilm assay of A. hydrophila KCTC 11533grown with different concentrations of glucose. Values are the mean¡ standard error of the mean of three independent experiments.Within each variable, values with the same letter are not significantlydifferent according to Duncan’s multiple range test (P . 0.05).
FIGURE 7. Induction of protease inhibition in cultures of A.
hydrophila KCTC 11533 plus different concentrations of glucose byaddition of exogenous C4-HSL and C6-HSL. Values are the mean ¡
standard error of the mean of three independent experiments. Withineach variable, values with the same letter are not significantlydifferent according to Duncan’s multiple range test (P . 0.05).
244 JAHID ET AL. J. Food Prot., Vol. 76, No. 2
found that glucose at low concentrations (0.25%) also
inhibited biofilm formation (Fig. 1) without affecting
planktonic growth. In a recent study, 0.5% (wt/vol) honey
reduced E. coli O157:H7 biofilm formation by 98% through
inhibition of the expression of biofilm-related curli genes,
QS genes, and virulence genes (22). Kierek and Watnick
(16) found that biofilm formation was induced in Vibriocholerae by monosaccharides (glucose, galactose, and
mannose).
O’Reilly and Day (27) observed reduced production of
protease by A. hydrophila grown in 0.5% (wt/vol) glucose,
whereas fructose and sucrose did not influence protease
production. However, 0.25% (wt/vol) glucose significantly
reduced protease production by A. hydrophila in our study
(Fig. 3).
The findings in our study suggest that glucose may in-
hibit production of lateral and polar flagella under the
control of QS (Figs. 2 and 4). QS may moderate both
swarming and swimming motility, which were completely
inhibited by addition of 1.0 and 2.5% glucose; the strains
did not diffuse from the origin of inoculation (Fig. 2), and
no violacein was produced (Fig. 4) under these conditions
by C. violaceum. Polar and lateral flagellar systems for
swimming and swarming, respectively, in A. hydrophila are
associated with virulence (39, 41). Yu et al. (41) found an
interconnection between T3SS and T6SS proteins under the
control of temperature, RpoN, AxsA/ExsA, and QS. The QS
regulator (AhyR) is responsible for the upregulation of
T3SS and T6SS (15, 39).Although C. violaceum CV026 is a C6-HSL mutant, it
produces the violacein color when exposed to C8-AHL, C8-
3-oxo-AHL, and C4-AHL (9, 26). A. hydrophila secretes
C4-HSL and C6-HSL (35). The QS experiments (violacein
production) revealed that 0.5% glucose inhibited approxi-
mately 95% of QS activity, based on the response of the
CV026 biosensor (Fig. 4b). Culture of CV026 with A.hydrophila KCTC 2358 did not result in color production.
The activity of both serine protease and metalloprotease
in A. hydrophila is under the control of QS mechanisms
(36), which is also the case for protease production in
Aeromonas salmonicida (35). However, we found a
correlation between violacein production and exoprotease
production inhibition by glucose (Figs. 3 and 4). Scanning
electron micrographs of control cultures and those grown
with 0.05% glucose revealed three-dimensional mushroom-
like structures of sessile cells previously noted by other
researchers for wild types but not QS mutants (23). The
difference in cell shape as revealed by SEM (Fig. 5) also is
correlated with previous findings of the inhibitory effect of
honey on E. coli O157:H7 cells (22). The stage shift biofilm
assay results also revealed that glucose inhibited early stage
biofilm formation because QS is active at the initial stage of
biofilm formation (Fig. 6). A. hydrophila ahyI mutant strain
(this gene encodes a protein responsible for the synthesis of
C4-HSL) had substantially reduced serine protease and
metalloprotease activity, which could be restored by the
addition of exogenous C4-HSL (36). We found a significant
increase in protease production with the addition of exogenous
C4-HSL and C6-HSL (Fig. 7). These findings support our
hypothesis that glucose regulates QS, which in turn influences
motility, biofilm formation, and exoprotease activity.
In conclusion, glucose concentrations starting at 0.25%
(wt/vol) inhibited biofilm formation, motility, and protease
production by A. hydrophila via the signal cascade QS-
inhibiting pathway (Fig. 8). Glucose might bind to the
master PhoB/AxsA regulators of polar and lateral flagella,
which are also controlled by QS mechanisms. The regulation
of AHLs further modulates protease production, motility, and
biofilm formation. The four characteristics (biofilm forma-
tion, motility, protease production, and QS) of A. hydrophilastrains that were originally isolated from food, surface water,
and a clinical setting had different phenotypic patterns, and
regulation was strain specific. Strain KCTC 2358 had
significantly higher biofilm production and high motility
FIGURE 8. Conceptual model of the ef-fects of glucose on the inhibition of cellularphenotypes in A. hydrophila. Right arrowindicates induction of gene expression orstimulation of a phenotype; dashes indicaterepression of gene expression or repressionof a phenotype.
J. Food Prot., Vol. 76, No. 2 INFLUENCE OF GLUCOSE CONCENTRATIONS ON BIOFILM OF A. HYDROPHILA 245
with less evidence of QS and protease production. Strain
KCCM 32586 had less biofilm formation but higher protease
production and QS activity, and strain KCTC 11533 had
higher biofilm formation, QS, and exoprotease activity but
less motility. Thus, none of these strains had the same
phenotypic profile for these four characteristics. Inhibitory
activities were noted when glucose was added at concentra-
tions $0.25% (wt/vol), whereas no inhibitory effect was
observed with #0.05% (wt/vol) added glucose (Fig. 8).
The present findings indicate that glucose regulates
biofilm formation and virulence properties. Because food is
a complex matrix with many components, further studies are
required to elucidate the effects of glucose in various foods
on biofilm formation and virulence properties of A.hydrophila.
ACKNOWLEDGMENT
This research was supported by the Chung-Ang University Excellent
Freshman Scholarship grants.
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