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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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