Environmental Microbiology (2011) 13(1), 6273

doi:10.1111/j.1462-2920.2010.02308.x

3-Indolylacetonitrile Decreases Escherichia coli

O157:H7 Biofilm Formation and Pseudomonas
aeruginosa Virulence
emi_2308

62..73

Jin-Hyung Lee, Moo Hwan Cho and Jintae Lee*

School of Display and Chemical Engineering,
Yeungnam University, Gyeongsan-si,
Gyeongsangbuk-do 712749, Korea.
Summary
Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish
Pseudomonas aeruginosa virulence. However, indole
and bacterial indole derivatives are unstable in the
microbial community because they are quickly
degraded by diverse bacterial oxygenases. Hence,
this work sought to identify novel, non-toxic, stable
and potent indole derivatives from plant sources for
inhibiting the biofilm formation of E. coli O157:H7 and
P. aeruginosa. Here, plant auxin 3-indolylacetonitrile
(IAN) was found to inhibit the biofilm formation of
both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN more effectively inhibited biofilms
than indole for the two pathogenic bacteria. Additionally, IAN decreased the production of virulence
factors including 2-heptyl-3-hydroxy-4(1H)-quinolone
(PQS), pyocyanin and pyoverdine in P. aeruginosa.
DNA microarray analysis indicated that IAN repressed
genes involved in curli formation and glycerol
metabolism, whereas IAN induced indole-related
genes and prophage genes in E. coli O157:H7. It
appeared that IAN inhibited the biofilm formation of E.
coli by reducing curli formation and inducing indole
production. Also, corroborating phenotypic results
of P. aeruginosa, whole-transcriptomic data showed
that IAN repressed virulence-related genes and
motility-related genes, while IAN induced several
small molecule transport genes. Furthermore, unlike
bacterial indole derivatives, plant-originated IAN was
stable in the presence of either E. coli or P. aeruginosa. Additionally, indole-3-carboxyaldehyde was
another natural biofilm inhibitor for both E. coli and
P. aeruginosa.
Received 20 January, 2010; accepted 11 June, 2010. *For corresponding. E-mail jtlee@ynu.ac.kr; Tel. (+82) 53 810 2533; Fax (+82)
53 810 4631.

2010 Society for Applied Microbiology and Blackwell Publishing Ltd

Introduction
Anti-virulence compounds have been suggested as alternative ways to fight infectious diseases because unlike
antimicrobials, anti-virulence compounds do not affect
growth and so there is less chance of developing resistance (Hentzer et al., 2002; Lesic et al., 2007). For
example, natural brominated furanones (produced by the
red macroalga Delisea pulchra) and their synthetic derivatives inhibit the biofilm formation of Escherichia coli (Ren
et al., 2001) and P. aeruginosa (Hentzer et al., 2003) by
interrupting their quorum sensing (Hentzer et al., 2003;
Ren et al., 2004). A quorum-sensing kinase inhibitor
decreased the virulence of E. coli O157:H7 without affecting its growth (Rasko et al., 2008). Hence, inhibiting the
cell signalling is an important way to fight infectious diseases, and it is necessary to discover novel compounds
that inhibit the biofilm formation and the virulence factor
production of pathogenic bacteria.
Indole is an intercellular signal (Lee and Lee, 2010). A
variety of both Gram-positive and Gram-negative bacteria
(Lee and Lee, 2010) produce indole using tryptophanase
(TnaA; EC 4.1.99.1) that can reversibly convert tryptophan into indole, pyruvate and ammonia (Newton and
Snell, 1965). Indole plays diverse biological roles in E. coli
and other bacteria. For example, indole controls the
virulence of pathogenic E. coli (Anyanful et al., 2005;
Hirakawa et al., 2009) as well as P. aeruginosa, which
cannot produce indole (Lee et al., 2009), and indole controls biofilm formation in E. coli (Di Martino et al., 2003;
Lee et al., 2007a) and in V. cholerae (Mueller et al., 2009).
Additionally, humans maintain a symbiotic relationship
with indole-producing enteric bacteria over a long period
of time. Hence, recent studies have suggested that indole
and the derivatives that are derived from bacterial indole
play beneficial roles in the human immune system (Wikoff
et al., 2009; Bansal et al., 2010).
In contrast to indole-producing bacteria, many nonindole-producing bacteria, plants and animals produce
diverse oxygenases, which can oxidize indole and
produce indole derivatives (Ensley et al., 1983; Lee and
Lee, 2010). Hence, six hydroxyindoles and three indigoid
compounds were previously investigated as biofilm
inhibitors for E. coli O157:H7 (Lee et al., 2007b) and

3-Indolylacetonitrile inhibits E. coli O157:H7 biofilm 63

Results
The main goals of this research were to determine
whether the indole derivatives from plant sources influenced E. coli biofilm formation and P. aeruginosa virulence and to determine the genetic mechanism using
DNA microarrays and phenotypic assays.
3-Indolylacetonitrile and indole-3-carboxyaldehyde
are potent biofilm inhibitors of E. coli O157:H7
and P. aeruginosa
Five indole derivatives derived from plant sources and
indole-3-propioninc acid found in animal sources were

E.coli O157:H7
Biofilm formation (OD570/620)

A 3.0

OH
O

2.5

NH

N
H

N
H

2.0

OH

N
H

1.5

OH

NH

N
H

1.0

O
OH

0.5
0.0

N
H

N
H

None Indole 7HI

IAA

MI

I3PA

I3C

CN

N
H

I3CA IAN

E.coli O157:H7

B 3.0
Biofilm formation (OD570/600)

anti-virulence compounds for P. aeruginosa (Lee et al.,

2009). Although the previous results provided a starting
point for anti-virulence drugs, indole and the bacterial
indole derivatives are rapidly degraded by many pathogenic bacteria, such as P. aeruginosa, and increased the
biofilm formation of P. aeruginosa (Lee et al., 2009).
Therefore, the use of bacterial indole derivatives is limited
against some pathogenic bacteria that have developed
defence systems against these derivatives.
Plants have developed advanced defence mechanisms
in order to survive in their ecosystems, and hence, plant
secondary metabolites are major pharmaceutical sources
(Zhao et al., 2005; Li and Vederas, 2009). Among these
compounds, several indole derivatives have shown a
variety of biological functions with respect to bacteria
and humans. For example, indole-3-carbinol and 3,3diindolylmethane (Fig. 1A) that originate from cruciferous
vegetables, exhibit antimicrobial, antiviral and anticancer
activities (Higdon et al., 2007; Fan et al., 2009). Like
indole-3-acetic acid, 3-indolylacetonitrile (IAN, Fig. 1A) is
a naturally occurring plant growth hormone (auxin) (Jones
et al., 1952) that is synthesized from tryptophan in cruciferous vegetables (Brassica), such as broccoli, cauliflower
and cabbage (Kutcek et al., 1960). Additionally, indole3-propionic acid (Fig. 1A), which is found in animal blood
(Wikoff et al., 2009), is a powerful antioxidant against the
Alzheimer b-amyloid (Chyan et al., 1999).
The goal of this study was to identify novel, non-toxic,
stable and potent indole derivatives that inhibited the
biofilm formation of E. coli O157:H7 and P. aeruginosa.
Five indole derivatives from cruciferous vegetables and
indole-3-propionic acid were investigated as biofilm inhibitors for E. coli O157:H7 and P. aeruginosa and as antivirulence compounds for P. aeruginosa. In order to
understand the molecular basis of the biofilm inhibition of
IAN (the most effective E. coli biofilm inhibitor among the
tested chemicals), DNA microarrays, phenotypic assays
and scanning electron microscopy (SEM) were utilized.
This is the first report to use IAN in order to inhibit E. coli
O157:H7 biofilm formation and to reduce P. aeruginosa
virulence.

2.5
2.0
1.5
1.0
0.5
0.0
0

25

50

100

150

-1

IAN (mg ml )
Fig. 1. A. Effect of indole derivatives [indole, 7-hydroxyindole (7HI),
3-indoleacetic acid (IAA), 3,3-methylene bisindole (MI),
indole-3-propioninc acid (I3PA), indole-3-carbinol (I3C),
indole-3-carboxyaldehyde (I3CA) and 3-indolylacetonitrile (IAN)] at
100 mg ml-1 on normalized (OD570/OD620) biofilm formation of E. coli
O157:H7 in LB medium at 37C after 24 h in 96-well plates. All of
the indole derivatives were dissolved in DMSO. DMSO and indole
were used as the negative and positive controls respectively. The
structures of indole, 7HI, IAA, MI, I3PA, I3C, I3CA and IAN are
shown.
B. Dose-dependent effect of IAN (0, 25, 50, 100 and 150 mg ml-1)
on E. coli O157:H7 biofilm formation. At least two independent
experiments were conducted (total 12 wells), and the error bars
indicate one standard deviation.

screened for their ability to inhibit the biofilm formation of

E. coli O157:H7 in LuriaBertani (LB) medium in 96-well
plates for 24 h at 37C. Indole 3-acetic acid (IAA), 3,3methylene bisindole (MI), indole-3-propioninc acid (I3PA),
indole-3-carbinol (I3C), indole-3-carboxyaldehyde (I3CA)
and IAN were dissolved in dimethylsulfoxide (DMSO) and
screened at a concentration of 100 mg ml-1. Dimethylsulfoxide [0.1% (v/v)] and indole (100 mg ml-1) were used as
the negative and positive controls respectively. Dimethylsulfoxide [0.1% (v/v)] alone did not affect cell growth,
biofilm formation, and virulence production in both E. coli
O157:H7 and P. aeruginosa. In the absence of the indole
derivatives, E. coli O157:H7 formed robust biofilms
(Fig. 1). I3CA and IAN most effectively reduced E. coli

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

1.4

Toxicity

1.2

To test the toxicity of indole derivatives, the specific

growth rate was measured in LB medium. The growth rate
of E. coli O157:H7 was 1.32 0.03 h-1 in the absence
of the indole derivatives, whereas the growth rate
was 1.28 0.01 h-1 with 100 mg ml-1 IAN (Fig. 3B)
and
1.08 0.02 h-1
with
100 mg ml-1
indole-3carboxyaldehyde (I3CA). The growth rate of P. aeruginosa
was 1.45 0.12 h-1 in the absence of the indole derivatives, whereas the growth rate was 1.38 0.13 h-1 with
100 mg ml-1 IAN (Fig. 3C) and 1.42 0.10 h-1 with
100 mg ml-1 I3CA. For IAN, a concentration as high as
150 mg ml-1 did not affect the cell growth of E. coli
O157:H7 (1.29 0.08 h-1). Therefore, IAN was not toxic
to E. coli O157:H7 and P. aeruginosa.

0.6
0.4
0.2
0.0

None

Indole

7HI

IAA

I3CA

IAN

Fig. 2. Effect of indole derivatives [indole, 7-hydroxyindole (7HI),

3-indoleacetic acid (IAA), indole-3-carboxyaldehyde (I3CA) and
3-indolylacetonitrile (IAN)] at 100 mg ml-1 on normalized
(OD570/OD620) biofilm formation of P. aeruginosa PAO1 in LB
medium at 37C after 7 h in 96-well plates. Indole and the indole
derivatives were dissolved in DMSO. DMSO and indole were used
as the negative and positive controls respectively. At least two
independent experiments were conducted (total 12 wells), and the
error bars indicate one standard deviation.

O157:H7 biofilm formation (11-fold for I3CA and 24-fold

for IAN), whereas IAA did not greatly affect E. coli
O157:H7 biofilm formation (Fig. 1A). Under the same conditions, indole inhibited E. coli O157:H7 biofilm formation
by threefold. Therefore, IAN was an eight times more
effective biofilm inhibitor than indole for E. coli O157:H7 at
37C. Also, the addition of IAN (100 mg ml-1) was still more
potent for E. coli O157:H7 biofilm reduction than that
(0.125 0.05 OD570/620) of the higher concentration of
indole (200 mg ml-1). Furthermore, IAN decreased E. coli
O157:H7 biofilm in a dose-dependent manner from 0 to
150 mg ml-1 (Fig. 1B).
To investigate if IAN and I3CA are biofilm inhibitors for
pseudomonads, too, IAN and I3CA were tested with P.
aeruginosa PAO1. IAN and I3CA caused a significant
decrease in the biofilm formation for P. aeruginosa (2.3-fold
for IAN and 1.9-fold for I3CA, Fig. 2), whereas indole and
7-hydroxyindole stimulated P. aeruginosa biofilm formation
(Lee et al., 2009). Therefore, IAN and I3CA were biofilm
inhibitors for both E. coli O157:H7 and P. aeruginosa.
To confirm the lack of metabolism of IAN in E. coli
O157:H7 and P. aeruginosa, the extracellular IAN concentration was measured in the supernatant of cell culture
using HPLC. The IAN concentration (110 mg ml-1 for E. coli
O157:H7 and 70 mg ml-1 for P. aeruginosa) was stably
maintained over 24 h without any degradation or evaporation in the presence of E. coli O157:H7 and P. aeruginosa
(Fig. 3A). This result suggests that IAN could not be
metabolized and transported into E. coli O157:H7 and P.
aeruginosa. It is notable that neither pathogenic bacteria
could metabolize IAN, whereas P. aeruginosa rapidly
degraded indole and 7-hydroxyindole (Lee et al., 2009).
Hence, IAN from plants was a relatively stable and potent
biofilm inhibitor for both E. coli O157:H7 and P. aeruginosa.

Differential gene expression of E. coli O157:H7

cells with IAN
To investigate the global genetic basis of IAN regulation
of E. coli O157:H7 biofilm reduction, DNA microarrays
were first used to determine differential gene expression

A
140

E. coli O157:H7

120
100
80

P. aeruginosa PAO1

60
40
20
0

10
15
Culture time (h)

20

25

B E. coli O157:H7

C P. aeruginosa PAO1
Cell growth (OD600)

0.8

IAN (mg ml-1)

1.0

Cell growth (OD600)

Biofilm formation (OD570/620)

64 J.-H. Lee, M. H. Cho and J. Lee

6
5
4
3
2
1
0

None

IAN

4
8
Culture time (h)

12

5
4
3

None

IAN

1
0
0

4
8
Culture time (h)

12

Fig. 3. Stability of 3-indolylacetonitrile (IAN) in the presence of

E. coli O157:H7 and P. aeruginosa PAO1 (A), and cell growth of
E. coli O157:H7 (B) and P. aeruginosa PAO1 (C) in the presence
of IAN. The initial turbidity of cells was 0.05 at 600 nm, and the
cells were cultured at 37C and 250 r.p.m. in LB medium. Each
experiment was performed using two independent cultures, and
one representative data set is shown.

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

3-Indolylacetonitrile inhibits E. coli O157:H7 biofilm 65

Table 1. Partial list of the genes that were repressed and induced
from E. coli O157:H7 cells upon addition of 100 mg ml-1
3-indolylacetonitrile (IAN).

Gene name

Fold
change

Curli genes
csgB
csgA
z2200

-3.6
-2.6
-2.2

Curlin minor subunit

Cryptic curlin major subunit
Type-1 fimbrial protein, A
chain precursor

Glycerol-related genes
glpD

-2.0

Glycerol-3-phosphate
dehydrogenase
Glycerol facilitator protein
Sn-glycerol-3-phosphate
transporter
Glycerol kinase

glpF
glpT

-2.3
-2.3

glpK

-3.2

Prophage genes
z2978
z3345
Indole-related genes
tnaC

3.7
3.0

2.3

wrbA

2.7

Others
flaB

2.4

z2254

2.4

entE

2.1

marR

2.0

ynfK

-2.0

afuA

-2.0

fruB

-2.0

yhcI

-2.0

focA
ilvL
napD

-2.0
-2.1
-2.1

uhpT
narK
yfiD

-2.2
-3.1
-3.5

z3921
z2148

2.9
2.2

Description

Putative replication protein

for prophage
Antitermination protein Q for
prophage
Tryptophanase leader
peptide
TrpR binding protein WrbA
Fructose-bisphosphate
aldolase
H-repeat-containing Rhs
element
Enterobactin synthase
subunit E
Repressor of multiple
antibiotic resistance
Predicted dethiobiotin
synthetase
Periplasmic ferric
iron-binding protein
Bifunctional fructose-specific
PTS IIA/HPr protein
N-acetylmannosamine
kinase
Formate transporter
ilvG operon leader peptide
Assembly protein for
periplasmic nitrate
reductase
Sugar phosphate antiporter
Nitrite extrusion protein
Autonomous glycyl radical
cofactor GrcA
Hypothetical protein
Hypothetical protein

The cells were grown until the absorbance of 1.0 in LB medium at

37C at 250 r.p.m. with and without IAN. The full data are available
using GEO accession number GSE19842.

for E. coli O157:H7 cells with and without IAN

(100 mg ml-1). It was found that 25 genes were significantly regulated (more than twofold); 8 genes were
induced and 17 were repressed by IAN (Table 1). Most
noticeably, two curli genes (csgA and csgB), a fimbria
gene (z2200) and glycerol-related genes (glpD, glpF,

glpK and glpT) were most repressed (2.0- to 3.6-fold),

while two prophage genes (z2978 and z3345) were
most induced (3.0- to 3.7-fold) (Table 1). Additionally,
tnaC, which encoded the leader peptide of tna operon,
and TrpR binding protein WrbA were also induced. The
impact of IAN was probably a chronic process rather
than an acute process because only 25 genes were differentially (above twofold) changed, which was a small
number compared with the biofilm reduction (Fig. 1). The
full data are available using GEO accession number
GSE19842.
IAN reduces the curli formation in E. coli O157:H7
Curli formation is important for the biofilm formation of E.
coli O157:H7 (Ryu and Beuchat, 2005; Uhlich et al.,
2006). Since the microarray data showed that two curli
genes (csgA and csgB) were most repressed by IAN
(Table 1), curli production was measured using a Congo
red plate and SEM. The Congo red plate was used to
observe curli production of colonies that were grown
under static conditions, whereas SEM were used to
observe curli production from the planktonic cells that
were used for the DNA microarrays and biofilm cells
grown in a nylon filter.
Clearly, IAN decreased the curli production on the
Congo red plate (Fig. 4A) and in the SEM analysis for
planktonic cells (Fig. 4B) and biofilm cells (Fig. 4C).
Notably, curli formation in the absence of IAN was more
distinctive in biofilm cells than planktonic cells. The result
can be explained by the previous results that curli production is regulated via the level of c-di-GMP (Weber et al.,
2006) and that a high level of c-di-GMP results in higher
biofilm formation in E. coli (Mndez-Ortiz et al., 2006).
Since Congo red binds both curli and cellulose (Zogaj
et al., 2003), a cellulose-specific assay using calcofluor
was also used to investigate the cellulose production
upon the addition of IAN. Unlike the curli production
with colonies, the cellulose production with planktonic
cells slightly increased upon the addition of IAN
(1.6 0.1-fold). Therefore, these results indicated that
the reduction of Congo red binding was caused by a
decrease in the curli production (Fig. 4A).
Under SEM, polymerized curli appeared as 4- to
7-nm-wide fibres of varying lengths, and a large amount
of curli appeared as a tangled and amorphous matrix
surrounding bacterial cells (Chapman et al., 2002). IAN
clearly decreased the curli production from planktonic
cells (Fig. 4B). The results were in good agreement
with the microarray data (Table 1). Therefore, IAN inhibited curli production, which further decreased E. coli
O157:H7 biofilm formation. Additionally, IAN also
reduced the production of polymeric matrix in P. aeruginosa (Fig. 4D).

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

66 J.-H. Lee, M. H. Cho and J. Lee

Quantitative real-time reverse transcription polymerase
chain reaction (qRT-PCR) was used to investigate the
gene expression for csgA (encoding curlin major subunit)
and tnaA (encoding indole-synthesizing tryptophanase)
with and without IAN. The qRT-PCR result was comparable to the DNA microarray data in expression of csgA
with IAN (-4.6 0.7-fold in qRT-PCR versus -2.6-fold in
microarrays at a cell absorbance of 1.0). Additionally, the
qRT-PCR results showed 3.4 2.1-fold induction of tnaA
with IAN at a cell absorbance of 2.0, which corroborated
the increased indole production with IAN.
Differential gene expression of P. aeruginosa cells
with IAN

Fig. 4. Effect of 3-indolylacetonitrile (IAN) on polymeric matrix

production in E. coli O157:H7 and P. aeruginosa PAO1. Curli
production in E. coli O157:H7 observed by (A) a Congo red plate
after 24 h, (B) SEM for planktonic cells and (C) SEM for biofilm
cells grown in a nylon filter. Polymeric matrix production in P.
aeruginosa PAO1 was also observed by SEM for biofilm cells
grown in a nylon filter (D). For the SEM analysis, planktonic cells
were grown in LB medium at a cell absorbance of 1.0 and were
directly fixed through the addition of glutaraldehyde and
formaldehyde and filtered with a 0.45 mm Nylon filter. Biofilm cells
were cultured on nylon filter in 96-well plate at 37C for 24 h.

IAN increases indole production in E. coli O157:H7

Indole decreases the biofilm formation of E. coli
O157:H7 (Bansal et al., 2007; Lee et al., 2007b). The
whole-transcriptome analysis here showed that IAN
induced tnaC (Table 1), which positively regulates the
indole production (Gong and Yanofsky, 2002; Lee et al.,
2007a). Hence, the extracellular indole concentrations
were measured in the presence of IAA, IAN and I3CA.
Confirming the microarray data, IAN produced twofold
more extracellular indole than the control, whereas IAA
did not significantly affect indole production (Fig. 5A).
These results were distinctive because isatin (double
oxidized indole) decreased indole production and
increased biofilm formation (Lee et al., 2007b). Hence,
the results partially explained the biofilm reduction of E.
coli O157:H7 for IAN.

To investigate the global genetic basis of IAN regulation of

P. aeruginosa biofilm reduction, DNA microarrays were
used to determine differential gene expression for P.
aeruginosa cells with and without IAN (100 mg ml-1). It
was found that 50 genes were significantly regulated
(more than 1.7-fold); 29 genes were induced and 21 were
repressed by IAN (Table 2). Most noticeably, two
virulence-related genes (pqsE and pvcC), a fimbrial gene
(z2200) and three motility-related genes (type IV twitching
motility protein pilI, flagellar biosynthesis protein flhF and
flagellar motor protein motD) were most repressed, while
11 small molecule transport genes were most induced
(Table 2). The full data are available using GEO accession
number GSE21508.
Flagellar-mediated motility and type IV pili protein are
conditionally necessary for P. aeruginosa biofilm development (OToole and Kolter, 1998; Klausen et al., 2003).
Hence, IAN probably reduces P. aeruginosa biofilm development partially by repressing these motility-related
genes (pilI, flhF and motD) (Table 2). Also, the PQS
system plays a role in P. aeruginosa biofilm formation
under diverse conditions (Diggle et al., 2003; Yang et al.,
2009). Recently, another trasncriptomic analysis revealed
that PqsE is required for swarming motility, biofilm development and virulence, and PqsE is a key regulator in
facilitating the environmental adaptation of P. aeruginosa
to plant and animal hosts (Rampioni et al., 2010). Since
plant auxin IAN most repressed pqsE (Table 2), our data
support the role of PqsE in controlling the adaptive behaviour of P. aeruginosa to plant.
qRT-PCR was also used to investigate the gene
expression for pqsE, pvcC and pilI with and without IAN
treatment in P. aeruginosa. The qRT-PCR result was
comparable to the DNA microarray data in expression of
pqsE (-2.9 0.5-fold in qRT-PCR versus -2.1-fold in
microarrays), pvcC (-1.5 0.2-fold in qRT-PCR versus
-1.8-fold in microarrays), and pilI (-3.6 0.6-fold
in qRT-PCR versus -1.8-fold in microarrays) with IAN
treatment.

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

3-Indolylacetonitrile inhibits E. coli O157:H7 biofilm 67

50
40
30
20
10
0

None

IAA

I3CA

IAN

C. P. aeruginosa
PQS production
(fold change)

1.2
1.0
0.8
0.6
0.4
0.2
0.0

None

IAA 7HI I3CA IAN

Pyocyanine production
(fold change)

B. P. aeruginosa
1.2
1.0
0.8
0.6
0.4
0.2
0.0

None IAA

7HI I3CA IAN

D. P. aeruginosa

Pyoverdine production
(fold change)

Indole production
(mg ml-1)

A. E. coli O157:H7

1.2
1.0
0.8
0.6
0.4

Fig. 5. Effect of indole and indole derivatives

on (A) the extracellular indole concentration in
E. coli O157:H7 and (B) pyocyanin
production, (C) PQS production (a picture of
thin layer chromatography is also shown) and
(D) pyoverdine production in P. aeruginosa
PAO1. Indole (I), 7-hydroxyindole (7HI),
3-indoleacetic acid (IAA),
indole-3-carboxyaldehyde (I3CA) and
3-indolylacetonitrile (IAN) at 100 mg ml-1 were
dissolved in DMSO. DMSO and indole were
used as the negative and positive controls
respectively. The extracellular indole
concentration was measured at E. coli
O157:H7 cell absorbance of 2.0 in LB
medium. Production of pyocyanin and PQS
was measured in LB medium and production
of pyoverdine was measured in minimal
succinate medium from planktonic P.
aeruginosa PAO1 cells after 24 h. At least two
independent experiments were conducted,
and the error bars indicate one standard
deviation.

0.2
0.0

None I

IAA 7HI I3CA IAN

IAN reduces virulence factor production in P. aeruginosa

Since the microarray data showed that IAN repressed two
virulence-related genes (pqsE and pvcC) (Table 2), we
assayed the production of four virulence factors, pyocyanin, PQS, siderophore pyoverdine and rhamnolipid, in P.
aeruginosa for 24 h. Most distinctively, indole decreased
the pyocyanin production by 29-fold, which was similar
to the previous results (Lee et al., 2009), and IAN
decreased the pyocyanin production by sevenfold,
whereas the same concentration of plant auxin IAA (as a
structural control) did not affect the pyocyanin production
(Fig. 5B). Similarly, indole decreased the PQS production
significantly, which was similar to the previous results (Lee
et al., 2009), and IAN decreased the pyocyanin production by threefold (Fig. 5C). Hence, IAN exhibited a smaller
impact than indole on the production of pyocyanine and
PQS in P. aeruginosa. However, IAN decreased more
pyoverdine production than that of indole (Fig. 5D). Additionally, both indole and IAN did not significantly affect
rhamnolipid production after 24 h growth (data not
shown).
Discussion
In this study, we demonstrated that IAN inhibited the
biofilm formation of both E. coli O157:H7 and P. aeruginosa PAO1 and reduced the production of virulence
factors from P. aeruginosa without affecting their growth.
We have sought the genetic mechanisms of IAN in both E.
coli O157:H7 and P. aeruginosa using the transcriptomic
assays and the phenotypic assays.

Cruciferous (or Brassica) vegetables, including

broccoli, cauliflower, brussels and cabbage, are rich
sources of glucosinolates including indole derivatives,
and a high intake of cruciferous vegetables lowered risk
of the lung and colorectal cancer in some epidemiological studies (Kutcek et al., 1960; Higdon et al., 2007).
Previous studies have shown that I3C and MI (Fig. 1)
from cruciferous vegetables have antimicrobial,
antiviral and anticancer activities (Higdon et al., 2007;
Fan et al., 2009). Cruciferous vegetables also produce
ascorbigen and IAN (Kutcek et al., 1960). Like the plant
auxin IAA, IAN is synthesized from tryptophan in cruciferous vegetables (Kutcek et al., 1960) and is a naturally occurring plant growth hormone (auxin) (Jones
et al., 1952). IAN can be converted into another plant
auxin IAA by nitrilases in plants (Kobayashi et al., 1993).
Although the function of IAN as an auxin is obvious in
plant cells (Kutcek et al., 1960), a few studies of IAN
have examined in microbial ecology. Only a few plant
pathogenic bacteria, such as Agrobacterium tumefaciens, Rhizobium sp. (Kobayashi et al., 1995) and
Pseudomonas syringae, contain nitrilase to use IAN as a
nitrogen source (Howden et al., 2009). However, P.
aeruginosa and E. coli do not have nitrilase (Howden
et al., 2009), so they do not degrade IAN (Fig. 3A). Additionally, unlike other glucosinolates, IAN shows no antimicrobial activity for P. aeruginosa (Aires et al., 2009).
Interestingly, plant auxin IAA did not show any activity on
E. coli O157:H7 and P. aeruginosa, whereas another
plant auxin IAN showed significant effects on both
strains (Figs 1, 2, 4 and 5).

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68 J.-H. Lee, M. H. Cho and J. Lee

Table 2. Partial list of the genes that were induced and repressed from P. aeruginosa PAO1 cells upon addition of 100 mg ml-1 3-indolylacetonitrile
(IAN).
Gene name

Fold change

Description

Virulence genes
PA1000
PA2256

-2.1
-1.8

Quinolone signal response protein PqsE

Pyoverdine biosynthesis protein PvcC

Motility-related genes
PA0410
PA1453
PA1461

-1.8
-1.8
-1.7

Type IV twitching motility protein PilI

Flagellar biosynthesis protein FlhF
Flagellar motor protein MotD

Other repressed genes

PA0525
PA1060
PA1063
PA1178
PA1632
PA1797
PA1832
PA1877
PA1882
PA2345
PA2655
PA2778
PA3075
PA3595
PA3715
PA4377

-1.7
-1.8
-1.8
-1.7
-1.7
-1.8
-1.7
-1.8
-1.8
-1.9
-1.7
-1.7
-1.8
-1.7
-1.8
-1.9

Probable dinitrification protein NorD

Hypothetical protein
Hypothetical protein
PhoP/Q and low Mg2+ inducible outer membrane protein H1 precursor
K+-transporting ATPase KdpF
Hypothetical protein
Probable protease
Probable secretion protein
Probable transporter
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Major facilitator superfamily (MFS) transporter
Hypothetical protein
Hypothetical protein

Induced small molecule transport genes

PA0119
PA0138
PA0196
PA0203
PA0204
PA0206
PA2135
PA2914
PA3376
PA3407
PA3448

1.7
1.8
2.1
1.7
2.1
1.7
2.0
1.7
1.7
1.9
2.3

Dicarboxylate transporter
Permease of ABC transporter
Pyridine nucleotide transhydrogenase PntB
Binding protein component of ABC transporter
Permease of ABC transporter
ATP-binding component of ABC transporter
Probable transporter
Probable transporter
ATP-binding component of ABC transporter
Haem acquisition protein HasAp
Permease of ABC transporter

Other induced genes

PA0193
PA0238
PA0239
PA0422
PA0522
PA0653
PA1235
PA1360
PA1489
PA1929
PA2903
PA3413
PA3492
PA3501
PA4886
PA5144
PA5417
PA5420

1.7
2.1
1.8
1.7
1.7
1.7
1.8
1.7
2.0
1.9
1.8
1.9
1.8
2.4
1.9
2.1
1.7
2.7

Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein
Probable transcriptional regulator
Hypothetical protein
Hypothetical protein
Hypothetical protein
Precorrin-3 methylase CobJ
Hypothetical protein
Hypothetical protein
Hypothetical protein
Probable two-component sensor
Hypothetical protein
Sarcosine oxidase delta subunit, SoxD
Formyltetrahydrofolate deformylase PurU2

The cells were grown until the absorbance of 2.0 in LB medium at 37C at 250 r.p.m. with and without IAN. The full data are available using GEO
accession number GSE21508.

Current study demonstrated for the first time that IAN

acted as a biofilm inhibitor for E. coli O157:H7, as well as
an antivirulence compound for P. aeruginosa without
affecting their growth. Importantly, while indole and

7-hydroxyindole were degraded by P. aeruginosa (Lee

et al., 2009), IAN was stable in the presence of either E.
coli O157:H7 or P. aeruginosa (Fig. 3), probably because
IAN was less prevalent in the microbial community, so that

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

3-Indolylacetonitrile inhibits E. coli O157:H7 biofilm 69

two pathogenic bacteria did not possess a defence
system to degrade IAN. Furthermore, since IAN is not
toxic to bacteria, it has a smaller selection pressure for
drug resistance than antibiotics that are toxic to bacteria.
Since humans contain a large amount of indole in the
intestine, the addition of indole (1 mM corresponding to
117 mg ml-1) into human epithelial (HCT-8) cells was not
toxic (Bansal et al., 2010). While toxicity data of IAN in
human cells is not yet available, IAN (up to 200 mg kg-1
dose) was not teratogenic to the rat foetus, but IAN in a
high dose (300 mg kg-1) induced sedation and ataxia in
rats (Nishie and Daxenbichler, 1980).
By studying the whole transcriptomic profiling, it was
shown that IAN repressed curli genes and induced indole
and prophage genes in E. coli O157:H7 (Table 1). Curli
are fibres that are produced by E. coli and have the same
biochemical and biophysical properties of human amyloid
that are associated with Alzheimers and prion disease
(Chapman et al., 2002). Additionally, curli formation is
important for the biofilm formation of pathogenic E. coli
O157:H7 (Ryu and Beuchat, 2005; Uhlich et al., 2006) as
well as non-pathogenic E. coli (Prigent-Combaret et al.,
2000; Reisner et al., 2006). Using a Congo red assay and
SEM, this study confirmed that IAN inhibited the biofilm
formation of E. coli O157:H7 by reducing curli production
(Fig. 4A, B and C). This result suggested that the inhibition of curli formation could be a way to eradicate the
biofilm formation of E. coli O157:H7.
The genetic mechanism of the biofilm formation of E.
coli O157:H7 is a complex process that is now being
unveiled. In addition to curli formation mentioned above,
intercellular signal molecules, such as autoinducer-2
(Yoon and Sofos, 2008) and indole (Bansal et al., 2007;
Lee et al., 2007b), are also involved in the biofilm formation of E. coli O157:H7. Since IAN increased indole production and decreased the biofilm formation of E. coli
O157:H7, IAN partially interfered with the indole signalling. Therefore, it was confirmed that the elevated indole
production was responsible for the reduction of biofilm
formation, and IAN triggered the indole production.
Recently, a report showed that prophage genes played
a role in E. coli biofilm formation (Wang et al., 2009). For
example, the deletion of 35 prophage genes, such as
CP4-57 and DLP12, increased biofilm formation up to
17-fold in E. coli. The current study showed that IAN most
induced two putative prophage genes, z2978 and z3345
(Table 1). Although it is highly speculative, these prophage genes may also be involved in controlling the biofilm
formation of E. coli O157:H7, which must be further
studied.
Previously, we reported that indole affects biofilm formation of non-pathogenic E. coli and pathogenic E. coli
O157:H7 via different mechanisms; for example, indole
decreased E. coli K-12 biofilm formation mediated by

SdiA (Lee et al., 2007a; Lee et al., 2008), and indole

decreased E. coli O157:H7 biofilm by reducing cell motility
and attachment (Bansal et al., 2007). Hence, a role of
SdiA in IAN-mediated reduction of biofilm formation of E.
coli was explored by using E. coli K-12 BW25113 wildtype and its sdiA knockout mutant. As a result, IAN
decreased the biofilm formation of both wild-type and sdiA
knockout mutant in a same dose-dependent manner (data
not shown). Therefore, it appears IAN does not work
through SdiA in E. coli K-12.
Plants are the richest sources of bioactive molecules
including antimicrobial compounds. Several non-toxic,
plant-derived biofilm inhibitors exist for pathogenic bacteria, such as furanones that originate from the red macroalga Delisea pulchra by blocking the quorum sensing of
bacteria (Ren et al., 2001; Hentzer et al., 2003), garlic
extract as a quorum-sensing inhibitor (Rasmussen et al.,
2005; Shuford et al., 2005), both ursolic acid (Ren et al.,
2005) and corosolic acid (Garo et al., 2007) that are produced by the ebony tree Diospyros dendo, and asiatic
acid that is produced by the tropical medicinal plant Centella asiatica (Garo et al., 2007). Unlike other plant biofilm
inhibitors, IAN inhibited E. coli biofilm formation through
curli formation and indole production. Therefore, diverse
plants may have developed different means of coping with
environmental bacteria. Since various indole derivatives
from plants and numerous synthetic indole derivatives are
commercially available, work is currently in progress to
identify stronger and safer biofilm inhibitors.

Experimental procedures
Bacterial strains, materials and growth
rate measurements
Serotype enterohemorrhagic E. coli O157:H7 [ATCC43895,
EDL933 strain (Strockbine et al., 1986)] and P. aeruginosa
PAO1 [the sequenced Holloway strain (Stover et al., 2000)]
were used. LuriaBertani medium (Sambrook et al., 1989)
was used as the medium for the growth of E. coli and P.
aeruginosa. Indole, IAA, MI, I3PA, I3C, I3CA, IAN, crystal
violet, Congo red, Coomasie brilliant blue, b-mercapto
ethanol, sodium phosphate and trifluoroacetic acid (TFA)
were purchased from Sigma-Aldrich Co. (Missouri, USA). 7HI
was purchased from Fisher Scientific Co. (Pittsburg, USA).
The other chemicals (acetonitrile, amyl alcohol, formaldehyde, glutaraldehyde, ethyl alcohol, DMSO, hydrochloric
acid, OsO4 and p-dimethylamino-benzaldehyde) were purchased from Duksan Pure Chemical (Ansan, Korea). The
strains were initially streaked from -80C glycerol stock on a
LB plate and a fresh single colony was inoculated in LB
(25 ml) in 250 ml flasks and cultured at 37C and 250 r.p.m.
Human body temperature (37C) was chosen here because
this study focused on investigating the effects of indole
derivatives on human pathogenic bacteria, although the
impact of indole in E. coli was less significant at 37C than at
low temperatures (Lee et al., 2008). Overnight cultures were

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

70 J.-H. Lee, M. H. Cho and J. Lee

re-inoculated at 1:100 dilution in the medium. For the cell
growth measurements, the optical density was measured at
600 nm using a spectrophotometer (UV-160, Shimadzu,
Japan). Each experiment was performed with at least two
independent cultures.

Crystal-violet biofilm assay

A static biofilm formation assay was performed in 96-well
polystyrene plates (SPL life sciences, Korea) as previously
reported (Pratt and Kolter, 1998). Briefly, cells were inoculated with an initial turbidity of 0.05 at 600 nm and cultured for
24 h without shaking at 37C. Cell growth and total biofilm
were measured using crystal violet staining. Each data point
was averaged from at least 12 replicate wells (six wells from
each of at least two independent cultures).

Virulence factor assays of P. aeruginosa

Except for the pyoverdine assay, overnight cultures were
diluted 1:100 and contacted with indole and indole derivatives
or diluent DMSO (negative control). The pyocyanin assay of
P. aeruginosa was adapted (Essar et al., 1990); after growth
for 24 h, supernatants were extracted with chloroform and
analysed spectrophotometrically. The PQS assay was
adapted (Attila et al., 2008); after growth for 24 h, supernatants were extracted with acidified ethyl acetate and analysed
by thin layer chromatography. The pyoverdine assay was
adapted (Anyanful et al., 2005); after growth in minimal succinate medium, cells were diluted to a turbidity of 0.05 at
600 nm in fresh minimal succinate medium and were grown
for 24 h. The pyoverdine concentration was measured spectrophotometrically at 405 nm (Stintzi et al., 1998). The rhamnolipid assay was adapted (Wilhelm et al., 2007); after
growth for 24 h, supernatants were assayed for rhamnolipids
using the orcinol colorimetric assay. At least two independent
experiments were conducted.

Assay of indole and IAN

To measure the concentration of extracellular indole, E. coli
O157:H7 was grown with and without IAN in LB medium at
37C and 250 r.p.m. to an absorbance of 2.0 at 600 nm. The
absorbance of 2.0 represented the time point when E. coli
secreted the stationary signal indole at a concentration of
approximately 25 mg ml-1. The extracellular indole concentration was measured with the protocol using the Kovacs
reagent (Kawamura-Sato et al., 1999) and was corroborated
with HPLC. To measure the concentration of IAN, bacteria
were grown with and without IAN (110 mg ml-1 for E. coli and
70 mg ml-1 for P. aeruginosa) at 37C and 250 r.p.m. The
concentrations of indole or IAN were measured with reversephase HPLC using a 100 4.6 mm Chromolith Performance
RP-18e column (Merck KGaA, Darmstadt, Germany) (Lee
et al., 2007b) and elution with H2O-0.1% TFA and acetonitrile
as the mobile phases at a flow rate of 0.5 ml min-1 (50:50).
Under these conditions, the retention times and the absorbance maxima were 5.1 min/271 nm for indole and
4.3 min/298 nm for IAN. Each experiment was performed
with two independent cultures.

Total RNA isolation

For the microarray experiments and qRT-PCR experiments,
E. coli O157:H7 and P. aeruginosa PAO1 were inoculated in
25 ml of LB medium in 250 ml shake flasks with overnight
cultures (1:100 dilution). Either IAN (100 mg ml-1) that was
dissolved in 25 ml DMSO was added or 25 ml DMSO was
added as a control. Cells were cultured in LB at 37C with
shaking at 250 r.p.m. until an absorbance of 1.0 for E. coli
O157:H7 and 2.0 for P. aeruginosa PAO1 at 600 nm was
reached. Due to the low biofilm formation by IAN, planktonic
cells were utilized to investigate the effect of IAN on wholeglobal transcriptome. The cells were immediately chilled with
dry ice and 95% ethanol (to prevent RNA degradation) for
30 s before centrifugation at 13 000 g for 2 min. The cell
pellets were immediately frozen with dry ice and stored at
-80C. Total RNA was isolated using a Qiagen RNeasy mini
Kit (Valencia, CA, USA). RNA quality was assessed using an
Agilent 2100 bioanalyser and a RNA 6000 Nano Chip (Agilent
Technologies, Amstelveen, the Netherlands), and the quantity was determined using ND-1000 Spectrophotometer
(NanoDrop Technologies, DE, USA).

DNA microarray analysis

The E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N
900551, Santa Clara, USA) and the P. aeruginosa Genechip
Genome Array (Affymetrix, P/N 900339) were used in order
to study the differential gene expression profile of the cells
after the addition of IAN. From each sample, 10 mg of total
RNA was converted to cDNA using random primers. The
purified cDNA was fragmented using 0.6 U/mg of DNase I and
end-labelled by terminal transferase reaction incorporating a
biotinylated dideoxynucleotide. Hybridization was performed
for 16 h at 45C for the E. coli Genechip and at 50C for P.
aeruginosa Genechip and 60 r.p.m. as described in the Gene
Chip Expression Analysis Technical Manual (Affymetrix).
After the hybridization, the chips were stained and washed in
a Genechip Fluidics Station 450 (Affymetrix) and scanned by
using a Genechip Array scanner 3000 7G (Affymetrix). The
probe array images were inspected for any image artifacts.
Background values, noise values and scaling factors of both
arrays were examined and were comparable. The intensities
of the polyadenosine RNA controls of Bacillus subtilis (lys,
phe, thr and dap) at different concentrations were used in
order to monitor the labelling and scanning process. A gene
was considered differentially expressed when the P-value for
comparing two chips was lower than 0.05 (to assure that the
change in gene expression was statistically significant and
that any false positives arise less than 5%) and when the
expression ratio was higher (twofold for the E. coli Genechip
and 1.7-fold for P. aeruginosa Genechip). Gene functions
were obtained from the AffymetrixNetAffx Analysis Center
(https://www.affymetrix.com/analysis/netaffx/index.affx).

Quantitative real-time reverse transcription polymerase

chain reaction
To corroborate the DNA microarray data and curli assay and
to confirm the induction of indole production with IAN, qRT-

2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 6273

3-Indolylacetonitrile inhibits E. coli O157:H7 biofilm 71

PCR was used to investigate the transcription level of csgA
(encoding curlin major subunit) and tnaA (encoding
indole-synthesizing tryptophanase), in E. coli O157:H7 pqsE
(quinolone signal response protein), pvcC (pyoverdine biosynthesis protein) and pilI (twitching motility protein) in P.
aeruginosa with and without IAN treatment. Two primers for
csgA (forward primer 5-AGATGTTGGTCAGGGCTCAG-3
and reverse primer 5-CGTTGTTACCAAAGCCAACC-3),
two primers for tnaA (forward primer 5-TACACCATTCCG
ACTCACCA-3 and reverse primer 5-CCGTATCGAAGGCTT
CTTTG-3), two primers for pqsE (forward primer 5-GAC
ATGGAGGCTTACCTGGA-3 and reverse primer 5-CTC
AGTTCGTCGAGGGATTC-3), two primers for pvcC (forward
primer 5-GACCAGGGTATCGTCGTCAG-3 and reverse
primer 5-AGCGGATAGTCGAAGGGACT-3), and two primers for pilI (forward primer 5-ATCATGGACCTCTGCG
GTTT-3 and reverse primer 5-GAAGACGCCATGAATG
AAGG-3) were used. The expression level of the housekeeping gene rrsG (16S rRNA, forward primer 5-TATTGCA
CAATGGGCGCAAG-3 and reverse primer 5-ACTTAACA
AACCGCCTGCGT-3) for E. coli O157:H7 and proC
(pyrroline-5-carboxylate reductase, forward primer 5-CAG
GCCGGGCAGTTGCTGTC-3 and reverse primer 5-GGTC
AGGCGCGAGGCTGTCT-3) for P. aeruginosa were used to
normalize the expression data of the genes of interest. The
qRT-PCR method was adapted from a previous study (Lee
et al., 2008). qRT-PCR was performed using a SYBR Green
master mix (Applied Biosystems, Foster City, USA) and a ABI
7500 Real-Time PCR System (Applied Biosystems) with two
independent cultures.

and the nylon filter were incubated together to form biofilm

cells at 37C for 24 h without shaking. After fixation with
glutaraldehyde and formaldehyde, either planktonic cells or
biofilm cells grown on a nylon filter were washed three times
with a 0.2 M sodium phosphate buffer (pH 7.2) for 20 min
each time at 4C and stored in the same buffer at 4C overnight. Then, the samples were washed twice with a 0.2 M
sodium phosphate buffer for 20 min before they were postfixed for 90 min with an Osmium solution [containing 1.5 ml
of a sodium phosphate buffer (0.2 M), 3 ml of 2% OsO4 and
3 ml deionized water]. The cells were washed again four
times with 1.5 ml of a sodium phosphate buffer (0.2 M) for
20 min. In the next step, cells were dehydrated through successive 20 min incubations in 50%, 70%, 80%, 90% and
95% ethanol, and then two successive 20 min incubations in
100% ethanol. After the dehydration process, the cells were
incubated twice in isoamyl acetate for 20 min. These filter
pieces, which contained the cells, were dried using a criticalpoint dryer (HCP-2, Hitachi, Japan). Then, the dried filters
were affixed to SEM stubs, and coated with white gold for
200 s using an Ion-sputter (E-1030, Hitachi, Japan). The
specimens were examined using SEM S-4100 (Hitachi,
Japan) at a voltage of 15 kV and magnifications ranging
from 2000 to 25 000.

Acknowledgements
This research was supported by the Yeungnam University
research grant (to J. Lee). J-H. Lee was supported by the
Brain Korea 21 Project from the Ministry of Education and
Human Resources, Korea.

Curli assay with a Congo red plate and SEM

To measure the curli production, LB agar medium containing
20 mg ml-1 Congo red (Sigma), 10 mg ml-1 Coomassie brilliant
blue (Sigma), 15 g l-1 agar was used as previously described
to visualize E. coli curli expression after 24 h incubation at
37C (Reisner et al., 2006). IAN (100 mg ml-1) was added to
the Congo red plate. Since Congo red binds both curli and
cellulose (Zogaj et al., 2003), a quantitative cellulose assay
using cellulose-specific binding calcofluor (Ma and Wood,
2009) was used to determine if curli or cellulose was identified by the Congo red assay. Cells (2 ml) at a turbidity of 4
were centrifuged and resuspended in 1 ml of 1% tryptone
with 16 mg ml-1 calcofluor and incubated for 2 h at 250 r.p.m.
Bacterial bound calcofluor was removed by centrifugation for
5 min at 17 000 g, and the amount of unbound calcofluor to
cellulose was determined by measuring the absorbance of
the supernatant at 350 nm. In order to corroborate the plate
assay, SEM was used with a modified protocol (Hossain
et al., 1996). Briefly, for planktonic cells, when cell growth
reached at optical density of 1.0, the cells were directly fixed
through the addition of glutaraldehyde (2.5% in the final concentration) and formaldehyde (2% in the final concentration)
and incubated at 4C overnight. Then, the cells were collected by filtering with a 0.45 mm Nylon filter (Nalgene, New
York, USA) under vacuum. The filter, which contained the
cells, was cut into 0.5 0.5 mm squares and placed in clean
ampoules. For biofilm cells, a nylon filter was cut into
0.5 0.5 mm square and placed in 96-well plates with
300 ml of cells with an initial turbidity of 0.05 at 600 nm. Cells

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