Food Control 75 (2017) 255e261

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Carvacrol as potential quorum sensing inhibitor of Pseudomonas

aeruginosa and biofilm production on stainless steel surfaces
Melvin R. Tapia-Rodriguez a, Adrian Hernandez-Mendoza a,
Gustavo A. Gonzalez-Aguilar a, Miguel Angel Martinez-Tellez a, Claudia Miranda Martins b,
J. Fernando Ayala-Zavala a, *
a
Centro de Investigacion en Alimentacion y Desarrollo, Hermosillo, Sonora, Mexico
b
, Pici Campus, Block 909, 60455-760,
Laboratory of Environmental Microbiology, Department of Biology, Sciences Center, Federal University of Ceara
Fortaleza, CE, Brazil

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

Article history: Pseudomonas aeruginosa biofilm confers resistance to antibiotics and biocides; therefore, it represents a
Received 20 October 2016 problem to clinical and industrial settings. This bacterial organization is controlled by quorum sensing
Received in revised form (QS), which depends of autoinducer molecules, e.g. acyl-homoserine-lactones that regulate production
6 December 2016
on virulence factors as pyocyanin. As a solution to this problem, carvacrol, present in most of the anti-
Accepted 8 December 2016
Available online 9 December 2016
bacterial essential oils could be a potential agent to inhibit QS for its ability to interact with cell mem-
brane and protein receptors involved in biofilm formation. Therefore, this work evaluated the effect of
carvacrol on pyocyanin production and biofilm formation of P. aeruginosa. Minimal inhibitory concen-
Keywords:
Terpene
tration (MIC) of carvacrol against planktonic P. aeruginosa was 7.9 mM; in addition, carvacrol was tested
Antibacterial against Chromobacterium violaceum as model for anti-QS agents, showing a MIC of 0.7 mM. Lower
Intercellular communication concentrations of carvacrol to observed MICs were applied to observe changes in QS activity and biofilm
Pyocyanin production to avoid effect of cell death on mentioned parameters. Carvacrol inhibited P. aeruginosa
Biosensor strain biofilms (1.5e3 Log CFU/cm2) at 0.9e7.9 mM, compared to non-treated bacteria on stainless steel surface.
Pyocyanin production by P. aeruginosa was reduced up to 60% at 3.9 mM of carvacrol. Higher doses of
carvacrol affected P. aeruginosa viability. Similar results were obtained for violacein production that is
related to QS of C. violaceum, where carvacrol reduced up to 50% at 0.7 mM without affecting cell
viability. These results showed that the inhibition of QS could be related with reduction of bacterial
virulence and biofilm formation on stainless steel surfaces exposed to carvacrol.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction homoserine lactones as autoinducer molecules; which in turn play

Biofilm association of Pseudomonas aeruginosa causes resistance
to antibiotic and biocide treatments, resulting in human infections.
Biofilm forms a protective barrier of polymeric substances that
protects bacterial cell against environmental stress, including an-
tibiotics (Chuanchuen, Karkhoff-Schweizer, & Schweizer, 2003;
Smith & Hunter, 2008). Formation of biofilms are regulated by a cell
density dependent communication system named quorum sensing
(QS) (Husain, Ahmad, Asif, & Tahseen, 2013). It has been reported
that P. aeruginosa uses specific receptor proteins that recognize acyl
an important role in the expression of virulence factors during
biofilm formation (Burt, Vlielander, Haagsman, & Veldhuizen,
2005; Soni et al., 2013), like the production of pyocyanin, a blue-
green toxin pigment that is a signaling factor in QS and biofilm
development (El-Shouny, Al-Baidani, & Hamza, 2011; Mavrodi
et al., 2001). For this reason, this signal pathway could be a novel
and potential target to inhibit biofilm formation and virulence
factors of P. aeruginosa.
Terpenes from several plant extracts, have been effective to
inhibit QS in Chromobacterium violaceum, a model bacteria used to

* Corresponding author. Laboratorio de Tecnologias Emergentes, Coordinacion de Tecnologia de Alimentos de Origen Vegetal, Centro de Investigacion en Alimentacion y
Desarrollo, Hermosillo, Sonora, 83000, Mexico.
E-mail address: jayala@ciad.mx (J.F. Ayala-Zavala).

http://dx.doi.org/10.1016/j.foodcont.2016.12.014
0956-7135/© 2016 Elsevier Ltd. All rights reserved.
256 M.R. Tapia-Rodriguez et al. / Food Control 75 (2017) 255e261
study Gram negative communication systems, affecting acyl
homoserine lactones and violacein production (Teplitski,
Mathesius, & Rumbaugh, 2010; Vattem, Mihalik, Crixell, &
McLean, 2007). Terpenes compounds like cinnamaldehyde,
eugenol and citronellol reduced P. aeruginosa, P. fluorescens and
P. putida biofilms (Niu & Gilbert, 2004). Furthermore, carvacrol is
the oregano essential oil major terpene, which has been tested to
inhibit production of Staphylococcus spp. biofilms, reporting that
this effect could be attributed to hydrophobic interactions with
membrane proteins, leading to an alteration of bacterial cell sur-
faces and affecting the initial adhesion involved during biofilm
development (Nostro et al., 2007). In this context, another research
reported that carvacrol decrease biofilm formation of S. Thypimu-
rium and S. aureus, but does not affect P. aeruginosa preformed
biofilms (Burt, Ojo-Fakunle, Woertman, & Veldhuizen, 2014).
However, this work did not prove the impact of this compound
during biofilms formation, where QS could be interrupted.
Published evidence reported that terpenes similar to carvarol
interrupted QS in Gram negative bacteria; e.g. cinnamaldehyde was
proposed as inhibitor of QS in a Vibrio spp. model, interrupting LuxR
protein receptors binding to acyl homoserine lactones, reducing
virulence factors and production of polymeric substances involved
during biofilm formation (Galloway, Hodgkinson, Bowden, Welch,
& Spring 2010). On the other side, it has been reported that
carvacrol reduced QS in C. violaceum, without affecting cell viability
(Ojo-Fakunle, Woertman, Veldhuizen, & Burt, 2013). With this in
mind, the aim of this study was to evaluate the role of carvacrol as
interrupter of pyocyanin production as indicator of QS during
P. aeruginosa biofilm development.
2. Materials and methods
2.1. Bacterial strains and growth conditions
Pseudomonas aeruginosa (ATCC 10154) and Chromobacterium
violaceum (ATCC 12472) were grown aerobically in Cetrimide
(Fluka®) and Luria Bertani (Difco™) broth, incubated at 37 and
25
C, respectively for 24 h to obtain 1  108 CFU/mL. This pro-
cedure was performed before every assay.
2.2. Minimal inhibitory (MIC) and bactericidal concentrations
(MBC) of carvacrol against P. aeruginosa and C. violaceum
These concentrations were determined to establish doses that
only affect QS and biofilm development, without affecting cell
viability, evading the effect of cell dead on the mentioned param-
eters. MIC values were obtained using a micro well dilution assay
technique (Burt et al., 2005). This assay was made by triplicate for
each bacteria, 5 mL of bacteria suspension (1  108 CFU/mL) were
transferred to a 96 well microplate (COSTAR) containing 295 mL of
cetrimide or LB broth with carvacrol (Sigma Aldrich, Toluca-
Me
xico) at different concentrations (0e15 mM). Microplates were
incubated at 25 and 37
C for 24 h for C. violaceum and P. aeruginosa,
respectively (Burt et al., 2005). MIC values were determined as the
lowest dose at which the well showed no turbidity by bacterial
growth. For MBC determination, cultures of those wells where the
MIC was selected and the next three higher concentrations of
carvacrol were plated in Pseudomonas and LB agar (Difco™), incu-
bated at 25 and 37
C for 24 h to observe total inhibition of bacteria
growth and expressed in mM of carvacrol.
2.3. Biofilm formation of P. aeruginosa exposed to carvacrol
The effect of carvacrol on P. aeruginosa biofilm formation on
stainless steel was determined using the protocol described by Hui
and Dykes (2012) with modifications. Stainless steel type 304 was
cut into coupons (1  1  0.1 mm), that were soaked in acetone for
30 min, rinsed in distilled water and sterilized by autoclaving
(15 min, 121
C). Coupons were placed within cell suspension
(1  108 CFU/mL) with different concentrations of carvacrol
(0e15 mM, doses determined considering the MIC values) incu-
bated at 37
C for 48 h. Surface attached biofilms were stained with
300 mL of 0.1% crystal violet solution (Husain et al., 2015). After
45 min, crystal violet solution was discarded and coupons were
immersed within 300 mL of 20% acetic acid to solubilize the stained
biomass. The biofilm was then quantified by measuring optical
density at 595 nm in a microplate reader Fluostar Omega (BMG
Labtech, Chicago, IL, USA). In addition, a similar experimental
design was performed to count biofilm embedded bacteria; cou-
pons were introduced in sterile saline solution tubes (5 mL), then
an ultrasonic bath (5 min) was used to remove bacteria from the
biofilm. These bacteria were quantified by plating in Pseudomonas
agar and incubated at 37
C for 24 h, results were expressed as
bacterial density (Log CFU/cm2) (Kerekes et al., 2013). This experi-
ment was carried out three times, and there were three replicates
per assay.
2.4. Effect of carvacrol on production of P. aeruginosa and
C. violaceum QS related pigments
Pyocyanin produced by P. aeruginosa exposed to different con-
centrations of carvacrol (doses determined considering MIC values)
was measured with a spectrophotometric method (Adonizio,
Downum, Bennett, & Mathee, 2006; Huerta, Mihalik, Crixell, &
Vattem, 2008). Six milliliters of P. aeruginosa planktonic cultures
treated with carvacrol as explained in the biofilm formation assay
were centrifuged (5000g for 5 min) with 2 mL of chloroform; the
organic layer was separated and solubilized in 2 mL of chlorhydric
acid (HCl) 0.2 M. Then, OD of each supernatant was measured at
520 nm in a spectrophotometer UVeVisible (JENWAY). Pyocyanin
production of control bacteria, without carvacrol, was set as 100%;
the blank used was bidistilled water þ HCl. Additionally, viability of
P. aeruginosa treated with different carvacrol concentrations was
registered using serial dilutions of treated bacteria counting in agar
plates expressed as Log CFU/mL. This experiment was carried out
three times, and there were three replicates per assay.
In addition, violacein production by C. violaceum exposed to
carvacrol (doses determined considering the MIC values) was car-
ried out as indicator of intercellular communication (Choo,
Rukayadi, & Hwang, 2006). One mL of culture from each treat-
ment was centrifuged (15,800g for 10 min) to precipitate violacein.
Then, the culture supernatant was discarded, and the pellet was
solubilized in 1 mL of dimethylsulfoxide, vortexed and centrifuged
(15,800g for 10 min) to remove cells. OD of each tube containing
supernatant was measured in a microplate reader Fluostar Omega
(BMG Labtech, Chicago, IL, USA) at 585 nm; results were expressed
as percentage of violacein production. Pigment production of non-
treated bacteria was set as 100%. Cell viability of C. violaceum was
registered and expressed as log CFU/mL, this experiment was per-
formed by triplicate.
Table 1
Minimum inhibitory (MIC) and bactericidal concentration (MBC) of carvacrol (mM)
against C. violaceum and P. aeruginosa.
C. violaceum (mM) P. aeruginosa (mM)
MIC 0.7 7.9
MBC 1.5 15
Values are mean of three replicates.
 M.R. Tapia-Rodriguez et al. / Food Control 75 (2017) 255e261 257

2.4.1. Statistical analysis 3. Results

A completely randomized experimental design was applied to
all experiments. The effects of carvacrol against P. aeruginosa and
C. violaceum, biofilm, pyocyanin and violacein productions were
evaluated. An analysis of variance (ANOVA) was performed to
determine statistical differences, and a mean comparison using
Tukey Kramer test was executed. The level of significance was set at
P  0.05, using the software Number Cruncher Statistical Systems
(NCSS) 2007.
3.1. Minimum inhibitory and bactericidal concentration
Table 1 shows antibacterial activity of carvacrol against
C. violaceum and P. aeruginosa. Tested doses were effective to inhibit
bacterial growth at MICs of 0.7 and 7.9 mM and showed MBCs of 1.5
and 15 mM, respectively for bacteria.

0.6

a
Biofilm formation(OD 595nm)

0.5

0.4 b

c
0.3 d

0.2 e

0.1

0.0
Control 0.9 1.9 3.9 7.9

Carvacrol (mM)
Fig. 1. Biofilm production by P. aeruginosa exposed to carvacrol at different concentrations. Different letters indicate statistical differences among bars (P  0.05).

c
8
P. aeruginosa cells attached

b b
Log CFU/cm2

6
a a a

0
Control 0.9 1.9 3.9 7.9

Carvacrol (mM)
Fig. 2. Effect of carvacrol on the number of P. aeruginosa biofilm embedded cells on stainless steel coupons. Different letters indicate statistical differences among bars (P  0.05).
258 M.R. Tapia-Rodriguez et al. / Food Control 75 (2017) 255e261

3.2. Biofilm formation of P. aeruginosa exposed to carvacrol
The inhibitory effect of carvacrol on P. aeruginosa biofilm is
shown in Fig. 1; these results showed a significant effect (P  0.05)
of all tested concentrations (0.9, 1.9, 3.9 and 7.9 mM) on biofilm
production, obtaining biomass reductions of 34, 47, 57 and 70%,
respectively to each tested concentration, thus showing that
7.9 mM was the most effective dose compared to un-treated bio-
film. Similarly, carvacrol decreased presence of cells in
P. aeruginosa biofilms (Fig. 2), obtaining significant reductions
(P  0.05) in all carvacrol concentrations (0.9, 1.9, 3.9 and 7.9 mM),
reporting inhibition values of 1.5e3 Log CFU/cm2, being 7.9 mM
the most effective concentration compared to un-treated biofilm
that showed 7.8 Log CFU/cm2. In summary, carvacrol caused a
diminution of biofilm production and a reduction of embedded
cells.
3.3. Effect of carvacrol on the production of P. aeruginosa and
C. violaceum QS related pigments
Fig. 3A shows the capacity of carvacrol to inhibit pyocyanin
production from P. aeruginosa; these data showed a significant
reduction with all different concentrations of tested carvacrol (0.9,
1.9, 3.9 and 7.9 mM). Pyocyanin was inhibited up to 60% at the
highest tested concentration; this reduction was statistically sig-
nificant (P  0.05) compared to all carvacrol treatments and un-
treated bacteria. In addition, cell viability was only affected
applying 7.9 mM (Fig. 3B) (P  0.05). On the other side, violacein
production by C. violaceum treated with carvacrol is depicted in
Fig. 4A. It was noticed, that higher the applied dose of carvacrol,
higher the inhibition of violacein production, showing a maximum
decrement of 40% at 0.7 mM (P  0.05). Finally, cell viability of
C. violaceum (Fig. 4B) showed no significant difference among

120
a A)
Pyocyanin production (%)

100

80

b
c
60 c
d
40

20

0
8
Control 0.9 1.9 3.9 7.9 B)
Carvacrol (mg/mL)
7
(Log CFU/mL)

*
Cell viability

3
Control 0.9 1.9 3.9 7.9
Carvacrol (mM)

Fig. 3. (A) Inhibition of pyocyanin production by carvacrol at different concentrations below MIC value against P. aeruginosa. Different letters indicate statistical differences among
bars (P  0.05). (B) Cell viability of P. aeruginosa after incubation in cetrimide broth with different concentrations of carvacrol.* significantly different when compared with other
treatments (P  0.05).
 M.R. Tapia-Rodriguez et al. / Food Control 75 (2017) 255e261
120
Violacein production (%)
100
a
80
60
40
20
0
10
8
(Log CFU/mL)
6
Cell viability
Control
Fig. 4. (A) Inhibition of violacein production of C. violaceum exposed to carvacrol at different concentrations. Different letters among bars indicated significant differences (P  0.05).
(B) Cell viability of C. violaceum after incubation in LB broth with carvacrol treatments. * Significantly different when compared with other treatments.
control and cultures treated with carvacrol (P  0.05). in this
context, carvacrol inhibited the production of both pigments that
are products of QS, related to acyl homoserine lactones presence,
indicating its interference with QS systems of these bacteria.
4. Discussion
Carvacrol MBCs values were twofold greater than MICs required
to inhibit bacteria growth; this effect could be attributed to its
reactivity and ability to degrade membrane proteins and cell
permeability in a concentration dependent way (Rodriguez-Garcia
et al., 2016). It has been reported that the antibacterial mechanism
of carvacrol against pathogenic bacteria is related to disturbance of
membrane embedded proteins, disruption of lipids, RNA synthesis,
ATPase activity and efflux pump (Simoes, Bennett, & Rosa, 2009). In
another research; antibacterial activity of carvacrol was widely
reported against pathogenic bacteria like L. monocytogenes, S.
typhimurium, E. coli O157:H7, B. cereus and S. aureus, showing MICs
at a range of 0e2 mM (Burt, 2004). As can be observed, carvacrol
MIC against P. aeruginosa was higher compared to other bacteria;
this could be attributed to P. aeruginosa resistance associated to its
mechanism of efflux pump and b-lactamase activity (Delgado,
Mayorgas, Rodríguez, Ibarra, & Roma
n, 2007). It has to be
259
A)
b
b
c
d
B)
0.09 0.19 0.39 0.79
Carvacrol (mM)
highlighted that the interest of this study was to evaluate carvacrol
effect on QS and biofilm development to propose more complete
and realistic mechanisms against cell communities that are the
nature way of bacterial organization instead individual planktonic
cells. It has to be mentioned that, lower concentrations to the MIC
values were applied to avoid the affection of bacteria viability.
P. aeruginosa biofilm formation on stainless steel exposed to
carvacrol was reduced up to 70% of biomass production, showing a
dose dependent response: higher the carvacrol dose, lower the
biofilm production. These data could be related to biofilm inhibi-
tion as the influence of carvacrol over QS related parameters. In
addition, these results proved that carvacrol is effective against
P. aeruginosa biofilm, contrasting with a previous study that re-
ported no effect up to 2 mM against preformed P. aeruginosa bio-
film, it has to be mentioned that they used four times less than the
dose used in our study (Burt et al., 2014). In addition, a significant
correlation between biomass production and embedded bacteria
within biofilm (r ¼ 0.7758; P ¼ 0.0007) was observed. These data
can be compared to recent studies showing the efficacy of carvacrol
to reduce Salmonella Saintpaul biofilms formed on stainless steel
surfaces, achieving reductions of 2 Log CFU/cm2 at concentrations
of 0.7 mM (Uchida et al., 2015). These results could be attributed to
carvacrol affecting QS of Gram negative bacteria, causing a delay or
260 M.R. Tapia-Rodriguez et al. / Food Control 75 (2017) 255e261
interruption of initial phase in biofilm formation, when their
adhesion is reversible, and synthesis of extracellular polymeric
substances are minimal, causing a reduction of bacterial attach-
ment and forming weaker biofilms (Vu, Chen, Crawford, & Ivanova,
2009).
In other works, the reduction in preformed biofilms of S. aureus
(46%) and S. epidermidis (58%) treated with carvacrol was reported
at 0.03 mM. However, in those studies only cell viability and
biomass production were evaluated, without contemplating its
effect on QS (Knowles, Roller, Murray, & Naidu, 2005; Nostro et al.,
2007). Therefore, carvacrol was effective to reduce biomass pro-
duction; however, deeper studies are needed to generate knowl-
edge about the different mechanisms of carvacrol to inhibit
biofilms, e.g. cell to cell communication related to acyl-
homoserine-lactones and protein involved in QS.
Carvacrol reduced both pyocyanin production and biofilm for-
mation, indicating the importance of their relation and a possible
disruption of QS. It is expected that pyocyanin inhibition will lead to
the attenuation of virulence factors to control biofilm production
that confers resistance to P. aeruginosa against antibacterial agents.
Our results showed that treated bacteria with carvacrol at 3.9 mM
caused the highest pyocyanin reduction (50%), without changes in
bacterial growth; remarking that a decrement of its synthesis can
be attributed to interfere in the pathway of cell communication that
involves receptor proteins and synthesis of pyocyanin precursor
called phenazine (Mavrodi et al., 2001). A previous study related
the decrease of extracellular polymeric substances with pyocyanin
reduction of P. aeruginosa exposed to clove oil (0.15 mg/mL) in a
dose dependant manner (Husain et al., 2013). In addition, it has
been showed that P. aeruginosa exposed to menthol (0.8 mg/mL), a
potential terpene of peppermint essential oil, inhibited 80% of
pyocyanin and reduced biofilm formation up to 69% (Husain et al.,
2015), giving higher inhibition results than in our study because of
the higher concentration used. Our results are in accordance with
recent reports and demonstrated that carvacrol can be applied to
decrease bacteria pathogenicity.
On the other hand, reduction of violacein production by
C. violaceum exposed to carvacrol was observed; it has previously
confirmed that this effect is attributed to inhibition of acyl homo-
serine lactone production owed to the interruption of QS. All tested
carvacrol concentrations showed a significant reduction in viola-
cein production even the lowest one (0.09 mM) which decreased
pigment production by more than 35%. Moreover, cell viability of
C. violaceum was not affected at all the tested concentrations.
Recently, it has been reported that oregano essential oil posses
antimicrobial activity against foodborne pathogens at 1.5 mM and
inhibition (90%) of QS in C. violaceum at 0.8 mM (Alvarez et al.,
2014). In addition, they reported that anti QS effect of oregano
essential oil could be attributed to its major compound carvacrol.
Choo et al. (2006) reported that any compound with the potential
of inhibit QS without decrease cell viability, could be acting
inhibiting molecules of the system e.g. synthase CviI, receptor CviR
or acyl-homoserine-lactones, which in turn could be possible
mechanisms of carvacrol to inhibit intercellular communication.
Our results showed that carvacrol has the potential to be a prom-
ising agent capable of inhibiting bacterial communication,
decreasing their pathogenesis and biofilm formation.
5. Conclusion
The present study demonstrated that carvacrol inhibited biofilm
formation of P. aeruginosa. Biofilm inhibition could be related to QS
interruption as evidenced by inhibition of pyocyanin and violacein
production in P. aeruginosa and C. violaceum, respectively. These
findings suggest that carvacrol could be exploited to develop new
and safe broad spectrum antibiofilm and anti-QS compounds.
Conflict of interest statement
Authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Acknowledgements
Authors thank the financial support of the Mexican council for
science and technology CONACYT (Project number CB-2013-01-
222691).
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