ORIGINAL RESEARCH

published: 21 December 2016

doi: 10.3389/fmicb.2016.02052

Cinnamaldehyde Inhibits
Staphylococcus aureus Virulence
Factors and Protects against
Infection in a Galleria mellonella
Model
Thiago A. F. Ferro 1 , Jéssica M. M. Araújo 1 , Bruna L. dos Santos Pinto 1 ,
Jéssica S. dos Santos 1 , Eliene B. Souza 1 , Bruna L. R. da Silva 1 ,
Valderlane L. P. Colares 1 , Tânia M. G. Novais 1 , Clovis M. B. Filho 2 , Carsten Struve 3 ,
João B. Calixto 4 , Valério Monteiro-Neto 1,5 , Luís C. N. da Silva 1 and
Elizabeth S. Fernandes 1*
1
Programa de Pós-graduação, Universidade CEUMA, São Luís, Brazil, 2 Universidade Federal de Pernambuco,
Edited by: Pernambuco, Brazil, 3 Statens Serum Institut, Copenhagen, Denmark, 4 Centro de Inovação e Estudos Pré-clínicos,
Octavio Luiz Franco, Florianópolis, Brazil, 5 Universidade Federal do Maranhão, São Luís, Brazil
Universidade Católica de Brasília,
Brazil
Bacterial resistance to the available marketed drugs has prompted the search of
Reviewed by:
Atte Von Wright,
novel therapies; especially in regards of anti-virulence strategies that aim to make
University of Eastern Finland, Finland bacteria less pathogenic and/or decrease their probability to become resistant to
Andre Moraes Nicola,
therapy. Cinnamaldehyde is widely known for its antibacterial properties through
University of Brasilia, Brazil
mechanisms that include the interaction of this compound with bacterial cell walls.
*Correspondence:
Elizabeth S. Fernandes However, only a handful of studies have addressed its effects on bacterial virulence,
elizabeth.soares@ceuma.br especially when tested at sub-inhibitory concentrations. Herein, we show for the
first time that cinnamaldehyde is bactericidal against Staphylococcus aureus and
Specialty section:
This article was submitted to Enterococcus faecalis multidrug resistant strains and does not promote bacterial
Antimicrobials, Resistance tolerance. Cinnamaldehyde actions were stronger on S. aureus as it was able to
and Chemotherapy,
a section of the journal
inhibit its hemolytic activity on human erythrocytes and reduce its adherence to latex.
Frontiers in Microbiology Furthermore, cinnamaldehyde enhanced the serum-dependent lysis of S. aureus. In
Received: 12 October 2016 vivo testing of cinnamaldehyde in Galleria mellonella larvae infected with S. aureus,
Accepted: 07 December 2016
showed this compound improves larvae survival whilst diminishing bacterial load in their
Published: 21 December 2016
hemolymph. We suggest that cinnamaldehyde may represent an alternative therapy to
Citation:
Ferro TAF, Araújo JMM, control S. aureus-induced bacterial infections as it presents the ability to reduce bacterial
dos Santos Pinto BL, virulence/survival without promoting an adaptive phenotype.
dos Santos JS, Souza EB,
da Silva BLR, Colares VLP, Keywords: essential oil, cinnamaldehyde, infection, bacterial virulence, S. aureus
Novais TMG, Filho CMB, Struve C,
Calixto JB, Monteiro-Neto V,
da Silva LCN and Fernandes ES
(2016) Cinnamaldehyde Inhibits
INTRODUCTION
Staphylococcus aureus Virulence
Factors and Protects against Infection
Bacterial pathogens have evolved several mechanisms to acquire resistance to drug and hereby
in a Galleria mellonella Model. survive antibiotic treatment in eukaryotic hosts, including mutations, plasmid acquisition,
Front. Microbiol. 7:2052. amongst others (Blair et al., 2015; Lin et al., 2015). In fact, multidrug resistant strains have been
doi: 10.3389/fmicb.2016.02052 observed with increasing frequency and their spreading has been recognized as one of the most

Frontiers in Microbiology | www.frontiersin.org 1 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

alarming issues for the global health system, resulting in high 2015) and Enterococcus faecalis (Chang et al., 2001). Although,
levels of morbidity and mortality (Wilson et al., 2016). Infections the ability of this compound to interact with the cell walls
caused by staphylococcal and enterococcal are reported as a of these bacteria is well-studied, little is known of its
major problem in hospitalized patients especially those using effects on bacterial virulence, especially when tested at sub-
indwelling medical devices such as urinary catheters, feeding inhibitory concentrations. Anti-virulence strategies have
tubes, and peripherally inserted central catheters (Padmavathy gained attention in the recent years as a novel therapeutic
et al., 2015; Tong et al., 2015). In order to cause infection, paradigm (Rasko and Sperandio, 2010; Kong et al., 2016).
these pathogens produce a range of virulence factors which in These approaches aim to inhibit the synthesis of bacterial
turn, promote host tissue damage and contribute to bacterial virulence factors that are essential for bacterial survival
evasion from the host’s immune response and their subsequent within the host; thus, making the bacteria less pathogenic
survival in the bloodstream (Bhatty et al., 2015; Thammavongsa and/or decreasing the probability of resistance development
et al., 2015; Theilacker et al., 2015). This scenario coupled with rather than targeting bacterial viability (Heras et al.,
a diminished antibiotic pipeline has lead to serious social and 2015).
economic complications and it has prompted the search of Here, we investigated the antimicrobial and anti-virulence
novel compounds and therapies to combat bacterial infections properties of cinnamaldehyde against S. aureus and E. faecalis,
(Barriere, 2015). including multidrug resistant strains. Additionally, we evaluated
The antimicrobial properties of plant-derived products have the ability of cinnamaldehyde to protect against S. aureus-
been tested against several pathogens. Cinnamaldehyde is the induced infection in G. mellonella larvae, an alternative model of
predominant active compound found in the cinnamon oil bacterial infection.
from the stem bark of Cinnamomum cassia. It is well-known
for its wide spectrum antimicrobial activity at concentrations
higher than 500 mg/ml (Chen et al., 2015; Shen et al., MATERIALS AND METHODS
2015; Utchariyakiat et al., 2016). The antimicrobial actions
of cinnamaldehyde are related to inhibition of cell division Bacterial Strains
through FtsZ (filamentation temperature sensitive protein Z; All tested bacteria were kindly provided by the bacterial collection
Domadia et al., 2007), reduction of energy generation and sector of the Universidade CEUMA and included: six strains of
glucose uptake or expenditure (Gill and Holley, 2004) and effects S. aureus (standard strains ATCC 25923 and ATCC 6538; clinical
on bacterial cell membrane permeability and integrity (Gill isolates SA01, SA02, SA03, SA04); four strains of E. faecalis
and Holley, 2004; Shen et al., 2015). Recently, cinnamaldehyde (standard strain ATCC 19443; clinical isolates EF01, EF02,
was shown to protect against the systemic inflammatory EF03). Susceptibility to antimicrobials was determined in an
response syndrome (SIRS) induced by the Gram-negative automated VITEK 2 system (BioMérieux Clinical Diagnostics,
R

bacteria cell wall component lipopolysaccharide (LPS) in mice USA) and data interpretation was performed as recommended
(Mendes et al., 2016). A similar effect was observed when by the Clinical Laboratory Standards Institute [CLSI] (2015). The
cinnamaldehyde was administered to Galleria mellonella infected multiple antibiotic resistance (MAR) index was calculated using
with Listeria monocytogenes (Upadhyay and Venkitanarayanan, the formula MAR = x/y, where “x” was the number of antibiotics
2016). to which the isolate demonstrated resistance; and “y” was the total
Previous reports described the antibacterial effects of number of antibiotics tested. The antibiotic susceptibility profile
cinnamaldehyde against Staphylococcus aureus (Shen et al., of each strain is shown at Table 1.

TABLE 1 | Antibiotic susceptibility profiles of Enterococcus faecalis and Staphylococcus aureus strains.

Strain Antibiotic MAR

PEN VAN OXA GEN CLI CIP SUT

E. faecalis ATCC 19443 S S – S – S – 0

E. faecalis 1 S S – S – S – 0
E. faecalis 2 R S – S – S – 0.25
E. faecalis 3 R S – R – R – 0.75
S. aureus ATCC 25923 S S S S S S S 0
S. aureus ATCC 6538 S S S S S S S 0
S. aureus 1 S S S S S S S 0
S. aureus 2 R S R S R S R 0.50
S. aureus 3 R S R S R S R 0.50
S. aureus 4 R S R S R S S 0.43

Experiments were performed three times in duplicate.

PEN, penicillin; VAN, vancomycin; OXA, oxacillin; GEN, gentamicin; CLI, clindamycin, CIP, ciprofloxacin; SUT, sulfamethoxazole/trimethoprim. Multiple antibiotic resistance
(MAR) index.

Frontiers in Microbiology | www.frontiersin.org 2 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

Antimicrobial Assays the manufacturer’s instructions. Cell viability is expressed as

The antimicrobial activity of trans-cinnamaldehyde (Sigma- absorbance in nm.
Aldrich ; 99% purity) was determined by the microdilution
R

method (Clinical Laboratory Standards Institute [CLSI], 2015). Studies with Human Samples
Briefly, each strain was grown on Müeller-Hinton Agar Blood samples were collected from three healthy volunteers with
(MHA) plates at 37◦ C for 24 h, and suspended in saline no recent history of taking either antibiotic or anti-inflammatory
solution (∼1.5 × 108 CFU/ml). For the determination of drugs, and/or infectious or inflammatory diseases in the last
minimum inhibitory concentrations (MICs), 10 µl of bacterial 3 weeks prior to sample collection; after a written informed
suspension (approximately 1.5 × 108 CFU/ml) were incubated consent was obtained. The study was reviewed and approved
in Müeller-Hinton (MH) broth containing cinnamaldehyde at by the Human Research Ethics Committee of the Universidade
different concentrations (62.5–2,000 µg/ml). Serial dilutions of CEUMA (CEP-UNICEUMA) and was performed in accordance
ciprofloxacin (0.06–256 µg/ml) were used as positive controls, with the Declaration of Helsinki 1975, as revised in 2008.
while sterile dimethyl sulfoxide (DMSO; 2% in phosphate-
buffered saline; PBS) was used as negative control. Samples were Hemolysis Assay
then, incubated for 24 h at 37◦ C. The MIC was defined as the Samples (2.5 ml of blood) were collected in heparinised tubes and
lowest concentration at which no bacterial growth was observed. the erythrocytes were immediately isolated by centrifugation at
For determining the minimum bactericidal concentrations 1,500 rpm for 10 min. After removal of plasma, the erythrocytes
(MBCs), just after the MIC experiments, the cultures were seeded were washed three times with PBS (pH 7.4) and then suspended
on MHA and incubated for 24 h at 37◦ C. The MBC corresponded in BHI broth. In parallel, bacterial suspensions were obtained as
to the lowest concentration of the compound to which no viable described for MIC determination (∼1.5 × 108 CFU/ml). Aliquots
bacteria was observed. of 10 µl of each bacterial suspension were added into 200 µl
of BHI broth supplemented with human erythrocytes (2%) and
incubated with sub-inhibitory concentrations of cinnamaldehyde
Analysis of Bacterial Tolerance to Drug (MIC/2 and MIC/4) or vehicle (2% DMSO in PBS). After
In order to investigate whether cinnamaldehyde is able to 24 h of incubation at 37◦ C, the tubes were centrifuged and
induce bacterial tolerance to drug, we performed serial passage the supernatant (100 µl/per sample/well) was transferred to a
experiments, using the standard strains of S. aureus (ATCC 96-well plate. Absorbance was read at 550 nm and taken as
25923) and E. faecalis (ATCC 19443). For this, bacterial an indicative of hemolytic activity. Results are expressed as
suspensions (1 ml, ∼1.5 × 108 CFU/ml) were added to six-well percentage (%) in relation to the hemolytic activity of each
tissue culture plates containing MH broth and sub-inhibitory bacterial strain incubated with vehicle (2% DMSO in PBS;
concentrations (MIC/2) of cinnamaldehyde or ciprofloxacin vehicle-controls).
(positive control). After 24 h at 37◦ C, the culture growing at one
dilution below the MIC was used to inoculate the subsequent Analysis of Bacterial Survival following Incubation
passage, and this process was repeated for a total of 10 passages. with Human Serum
The compound concentration range of each new passage was This assay was performed according to the method previously
based on the MIC calculated for the previous passage. Vehicle- described by Ismail et al. (1988), modified. Blood samples
treated bacteria (2% DMSO in PBS) were used as negative (2.5 ml) were collected in tubes containing no anticoagulant.
controls. Serum was separated by centrifugation at 1,500 rpm for 10 min.
Aliquots (10 µl/well) of the bacterial suspensions (∼1.5 × 108
Anti-biofilm Activity CFU/ml) were mixed with 140 µl/well of BHI broth containing
Biofilm formation was quantified according to the method sub-inhibitory concentrations of cinnamaldehyde (MIC/2 and
previously described by Stepanović et al. (2004). For this, 10 µl MIC/4) or vehicle (2% DMSO in PBS) and 60 µl/well of serum.
of bacterial suspension (prepared as described above) were After incubation for 24 h at 37◦ C, the absorbance was read at
added per well in to a 96-well cell culture plate containing 600 nm and taken as bacterial growth index. Results are expressed
sub-inhibitory concentrations of cinnamaldehyde (MIC/2 and as percentage (%) in relation to the bacterial growth registered for
MIC/4) and 200 µl of Luria-Bertani (LB) broth. Vehicle (2% each bacterial strain incubated with vehicle (2% DMSO in PBS;
DMSO in PBS)-treated bacteria and broth without bacteria were vehicle-controls).
used as positive and negative controls, respectively. Samples
were incubated at 37◦ C and after 24 h, and then, the wells Bacterial Adherence to Latex
were washed three times with PBS. Biofilm was stained with 5% As previously described (Chandra et al., 2008), siliconized latex
crystal violet for 10 min at room temperature, and immediately catheter segments (4 mm) were placed into tubes containing
solubilised with methanol (200 µl, 100%). The absorbance was 4.5 ml of LB medium with and without sub-inhibitory
read at 570 nm. Relative biofilm mass results are expressed as concentrations of cinnamaldehyde or vehicle (2% DMSO in PBS).
percentage (%) in relation to control (vehicle-treated wells). In Then, 225 µl of bacterial suspension (∼1.5 × 108 CFU/ml) were
a different set of experiments, the effects of cinnamaldehyde added to each tube. Tubes were incubated at 37◦ C for 3 h and
on bacterial viability were assessed and calculated by addition then, the latex segments were washed three times with PBS and
of PrestoBlue reagent (1:10; Life Technologies), according to
R
plated on LB Agar. Following incubation for 24 h, at 37◦ C, plates

Frontiers in Microbiology | www.frontiersin.org 3 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

were analyzed for CFU counting. The results are expressed as resistant to different antibiotics: S. aureus strains 2 (SA02)
CFU/ml. and 3 (SA03) were resistant to penicillin-oxacillin-clindamycin-
sulfamethoxazole/trimethoprim (MAR index: 0.57) and S. aureus
S. aureus-Induced Infection in strain 4 (SA04) was resistant to penicillin-oxacillin-clindamycin
G. mellonella (MAR index: 0.43).
The in vivo antimicrobial actions of cinnamaldehyde were Cinnamaldehyde was active against all strains of E. faecalis and
evaluated in an in vivo model of infection induced by S. aureus, including those with a multidrug resistance phenotype
S. aureus in G. mellonella larvae. Briefly, G. mellonella larvae (Table 2). MIC values were of 0.25 mg/ml for all tested strains,
(∼200 mg) were randomly distributed in two experimental except for the S. aureus standard strain ATCC 25923 (MIC value
groups (n = 10/group), and were then infected by injection of 0.5 mg/ml). MBC values were of 1.0 mg/ml to all strains, 2–
of 10 µl of bacterial suspension (S. aureus ATCC 25923; 4-fold higher than each respective MIC, indicating a bactericidal
1.0 × 105 CFU/ml in PBS) in to the last left proleg. The larvae action for cinnamaldehyde (Table 2). It is important to highlight
were incubated at 37◦ C. After 2 h, the larvae received either that at the used concentration, the vehicle (2% DMSO in PBS) did
cinnamaldehyde at different doses (2.5–5.0 µg /100 mg of larvae) not affect bacterial growth.
or vehicle (PBS, 5 µl/100 mg), and were incubated at 37◦ C. Additionally, when incubated in vitro with cinnamaldehyde,
Mortality rate was observed over 4 days post-infection. neither S. aureus (ATCC 25923) nor E. faecalis (ATCC 19443)
In order to assess the bacterial load in the hemolymph, developed adaptive phenotypes even after 10 sequential passages.
in a separate set of experiments, the larvae were infected In contrast, both strains became tolerant to the clinically used
with S. aureus as described above and then received either antibiotic ciprofloxacin as MIC values increased from 0.0625
cinnamaldehyde (5.0 µg/100 g of larvae; n = 5/day) or vehicle to 0.5 µg/ml for S. aureus, and from 0.125 to 0.5 µg/ml for
(PBS; n = 5/day). Larvae were incubated at 37◦ C for up to 4 days. E. faecalis.
Five larvae of each group were culled per day and analyzed for
bacterial load. Briefly, at each time point, the larvae were cut Cinnamaldehyde Sub-inhibitory
through in a cephalocaudal direction with a scalpel blade and Concentrations Do Not Affect Biofilm
squeezed to remove the hemolymph. Serial dilutions (10x) of the Formation by E. faecalis
hemolymph of each larvae were made in PBS and 4 µl of each We attempted to analyze the effects of sub-inhibitory
dilution were incubated in MHA and cultured for 24 h at 37◦ C. concentrations of cinnamaldehyde (MIC/4 or MIC/2) on
After this period, the plates were analyzed for CFU counting. The the ability of E. faecalis and S. aureus to form biofilm. As depicted
results are expressed as CFU/ml. on Figures 1A and 2A, cinnamaldehyde did not diminish biofilm
formation by these bacteria at any of the tested concentrations.
Statistical Analysis However, cinnamaldehyde treatment increased biofilm mass
Statistical analyses were performed using the software GraphPad for some strains of S. aureus (S. aureus ATCC 6538, SA01 and
Prism version 5.01 . Data from were analyzed by two-way analysis SA03). In order to assess whether cinnamaldehyde-induced
of variance (ANOVA) and Tukey test. A p-value of <0.05 was increase in biofilm formation is due to accumulation of dead
considered as statistically significant. Differences in G. mellonella cells, we evaluated the viability of the S. aureus (ATCC 6538) cells
larvae survival were determined using the Kaplan–Meier method composing the biofilm. We found that cinnamaldehyde (MIC/2)
to calculate survival fractions and log-rank test was used to decreases S. aureus viability (32.1 ± 3.9%; Figure 1A, inset box),
compare survival curves. indicating reduction in the number of viable cells.

RESULTS TABLE 2 | Antimicrobial activity of cinnamaldehyde against

Staphylococcus aureus and Enterococcus faecalis.
Cinnamaldehyde Inhibits the Growth of Strain MIC1 MBC2
S. aureus and E. faecalis without
E. faecalis ATCC 19443 0.25 1
Inducing an Adaptive Phenotype
E. faecalis 1 0.25 1
We initially analyzed the antimicrobial effects of cinnamaldehyde
E. faecalis 2 0.25 1
against clinical isolates and ATCC standard strains of S. aureus
E. faecalis 3 0.25 1
and E. faecalis. The strains showed different susceptibility profiles
S. aureus ATCC 25923 0.5 1
to clinically available antibiotics (Table 1). Amongst the three
S. aureus ATCC 6538 0.25 1
tested E. faecalis clinical isolates, two were resistant to at least
S. aureus 1 0.25 1
one antibiotic: E. faecalis strain 2 (EF02) was resistant to
S. aureus 2 0.25 1
penicillin (MAR index: 0.25) and E. faecalis strain 3 (EF03)
S. aureus 3 0.25 1
was resistant to penicillin, gentamicin and ciprofloxacin (MAR
S. aureus 4 0.25 1
index: 0.75). Of the four tested S. aureus strains, three were
Experiments were performed three times in duplicate.
1 Minimum Inhibitory Concentration (MIC) and 2 Minimum Bactericidal
1
www.graphpad.com
Concentration (MBC) are expressed in mg/ml.

Frontiers in Microbiology | www.frontiersin.org 4 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

FIGURE 1 | Effect of cinnamaldehyde (MIC/2 and MIC/4) on virulence factors of Staphylococcus aureus strains. (A) Biofilm mass production;
(B) Hemolytic activity; (C) Serum resistance; (D) Adhesion to latex (catheter). Inset box indicates bacterial viability. ∗ p < 0.05, compared with vehicle-treated
controls. Experiments were performed three times in duplicate. Each bar represents mean + SD.

Cinnamaldehyde Sub-inhibitory E. faecalis caused hemolysis, only the strain EF02 was inhibited
Concentrations Inhibit the Hemolytic by cinnamaldehyde at the MIC/4 (56.5%; Figure 2B).
Activity of S. aureus but not E. faecalis Cinnamaldehyde Sub-inhibitory
Three clinical isolates of S. aureus (SA02, SA03, SA04) and the
standard S. aureus strain ATCC25923 were hemolytic. Hemolysis
Concentrations Decrease S. aureus
was reduced by cinnamaldehyde when tested at MIC/2 (p < 0.05) Survival in the Presence of Human
(Figure 1B). Percentage of inhibitions were of 99.9, 81.4, 90.3, Serum
and 55.7%, for SA02, SA03, SA04, and ATCC25923; respectively. We next determined whether cinnamaldehyde is able to enhance
The strains ATCC25923 and SA03 were also significantly the lysis of S. aureus and E. faecalis in the presence of human
inhibited (p < 0.05) by cinnamaldehyde at MIC/4 (54.4 and serum. Our results show that when treated with cinnamaldehyde
76.7%, respectively). On the other hand, although all strains of at MIC/2 and MIC/4, S. aureus strains were less able to survive

Frontiers in Microbiology | www.frontiersin.org 5 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

FIGURE 2 | Effect of cinnamaldehyde (MIC/2 and MIC/4) on virulence factors of Enterococcus faecalis strains. (A) Biofilm mass production; (B) Hemolytic
activity; (C) Serum resistance; (D) Adhesion to latex (catheter). ∗ p < 0.05, compared with vehicle-treated controls. Experiments were performed three times in
duplicate. Each bar represents mean + SD.

when incubated with freshly isolated human serum (p < 0.05), and E. faecalis strains were able to adhere to latex. The
except SA01 at MIC/4. Inhibitions ranged from 23.5% (SA01) sub-inhibitory concentrations of cinnamaldehyde were able to
to 66.9% (SA03) for cinnamaldehyde at MIC/2, and from reduce the adherence to latex by all tested S. aureus strains
22.9% (SA01) to 53.5% (SA03) for cinnamaldehyde at MIC/4 (Figure 1D). When tested at MIC/2, cinnamaldehyde maximum
(Figure 1C). Amongst the E. faecalis strains, only EF02 showed inhibitory effects were observed for S. aureus ATCC 25923
a slight reduction on its serum resistance when incubated with (94.2%), and the clinical isolates SA03 (93.1%) and SA01 (91.3%).
cinnamaldehyde (9.1 and 9.4% at MIC/4 and MIC/2, respectively) Also importantly, the same concentration of cinnamaldehyde
(Figure 2C). diminished latex adhesion by S. aureus ATCC 6538 (67.4%),
SA04 (59.6%), and SA02 (48.7%). The adhesion of S. aureus
ATCC 25923 was also the most reduced by cinnamaldehyde
Cinnamaldehyde Sub-inhibitory at MIC/4 (93.0%), followed by SA01 (79.6%), SA03 (69.0%),
Concentrations Decrease the Ability of SA02 (58.6%), SA04 (46.7%), and SA01 (44. 7%). On the
S. aureus to Adhere to Latex other hand, this compound only affected the adherence to
We also evaluated whether the sub-inhibitory concentrations latex of E. faecalis ATCC 19443 with reductions of 79.7 and
of cinnamaldehyde were able to affect bacterial adhesion to 69.8% by cinnamaldehyde at MIC/2 and MIC/4, respectively
latex, using a catheter model. As expected, all tested S. aureus (Figure 2D).

Frontiers in Microbiology | www.frontiersin.org 6 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

Cinnamaldehyde Increases G. mellonella

Larvae Survival and Reduces Bacterial
Load in the Hemolymph
As cinnamaldehyde exhibited stronger actions on S. aureus, we
performed an in vivo infection assay using G. mellonella larvae.
Cinnamaldehyde or PBS (vehicle) did not induce any toxicity
on larvae. Cinnamaldehyde (2.5–5.0 µg/100 mg of larvae) effects
were compared to those of vehicle-treated larvae infected with
S. aureus ATCC 25923. At 2 days post-infection, 80% of the
vehicle-treated larvae had died, with no survivals on the third
day.
In contrast, cinnamaldehyde enhanced G. mellonella larvae
survival, as >50% remained alive on day 4 (p < 0.05; Figure 3A).
This effect was more pronounced for the highest dose of the
compound (5.0 µg/100 mg of larvae). By analyzing the bacterial
load, it was observed that cinnamaldehyde significantly reduces
the number of S. aureus in G. mellonella hemolymph samples
in comparison with vehicle-treated larvae, as indicated by CFU
counting (4-log reduction in bacterial survival). This effect
was noted from 48 h post-infection and remained significant
throughout the rest of the assay (Figure 3B).

DISCUSSION
Cinnamaldehyde Inhibits the Growth of
S. aureus and E. faecalis without
Inducing an Adaptive Phenotype
Cinnamaldehyde presented with antimicrobial actions on clinical
isolates of S. aureus and E. faecalis, in addition to ATCC standard
strains. This compound was effective on all strains of E. faecalis
and S. aureus, including those with a multidrug resistance
phenotype. The antimicrobial properties of cinnamaldehyde
have been demonstrated against a range of Gram-positive and
Gram-negative pathogens including S. aureus and E. faecalis
(Cox and Markham, 2007; Shen et al., 2015; Upadhyay and
Venkitanarayanan, 2016). Cinnamaldehyde actions against these
FIGURE 3 | Effect of cinnamaldehyde on survival (A) and hemolymph
pathogens are related to changes in their cell membrane polarity bacterial load (B) of Galleria mellonella larvae infected with S. aureus.
and permeability (Hammer and Heel, 2012). Importantly, we G. mellonella received either cinnamaldehyde (CNM; 2.5–5.0 µg/100 mg of
show for the first time that although becoming tolerant to larvae) or vehicle (PBS, 5 µl/100 mg) and were evaluated for 4-days
ciprofloxacin, neither S. aureus (ATCC 25923) or E. faecalis post-infection. n = 10/group for survival experiments; n = 5/group/day for
bacterial load quantification. Data for bacterial load is represented as
(ATCC 19443) develop an adaptive phenotype when incubated mean + SD. ∗ p < 0.05, compared with vehicle-treated larvae. Experiments
with cinnamaldehyde in vitro. were performed twice in duplicate.

Cinnamaldehyde Sub-inhibitory
Concentrations Do Not Affect Biofilm These results oppose to those of previously published reports
Formation by E. faecalis in that this compound was suggested to reduce biofilm
Biofilm formation is an important virulence factor involved formation by these bacteria. Recently, Budri et al. (2015) showed
in staphylococcal and enterococcal infections (Claessen et al., that cinnamaldehyde strongly diminishes biofilm formation
2014). In a recent report, cinnamaldehyde was shown to by S. aureus ATCC 35983 on both polystyrene and stainless
inhibit the expression of sarA (a positive regulator of biofilm steel surfaces, when tested at 0.199 mg/ml. This effect was
formation) in S. aureus at sub-inhibitory concentrations (Jia also observed when cinnamaldehyde was associated with either
et al., 2011). Surprisingly, cinnamaldehyde not only had no biodegradable polymers or nanoparticles (Zodrow et al., 2012;
effects on the ability of E. faecalis to form biofilm, but Duncan et al., 2015). Similarly, a Cinnamomum zeylanicum
enhanced biofilm formation by some strains of S. aureus. essential oil, rich in cinnamaldehyde, was shown to reduce

Frontiers in Microbiology | www.frontiersin.org 7 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

biofilm formation by E. faecalis (Abbaszadegan et al., 2016). of these pathogens in the bloodstream is due to their ability
It is possible that the discrepancies found between our to express different virulence factors that target components
results and the above discussed are due to differences on of the host’s immune system (Foster et al., 2014; Hall et al.,
the strains tested which may present different virulence 2015; Richards et al., 2015). We show that cinnamaldehyde
patterns. enhances S. aureus but not E. faecalis killing when incubated with
Additionally, different antimicrobial agents may be able freshly isolated human serum. To the best of our knowledge,
to induce biofilm formation at sub-inhibitory concentrations this study presents the first evidence on that cinnamaldehyde
(Kaplan et al., 2012; Schilcher et al., 2016). This effect is impairs S. aureus resistance to human serum. This may represent
suggested to be strain-specific and related to the induction of an additional mechanism by which cinnamaldehyde confers
stress pathways in S. aureus that in turn, lead to the expression protection to infection in vivo.
of biofilm-associated genes (Schilcher et al., 2016). Herein, as
in other reports (Kaplan et al., 2012; Schilcher et al., 2016), Cinnamaldehyde Sub-inhibitory
biofilm formation was evaluated by the crystal violet assay. This Concentrations Decrease the Ability of
method is widely used for this purpose, however, crystal violet S. aureus to Adhere to Latex
stains both viable and dead cells, in addition to the extracellular Catheterization is a potential risk factor for bacterial colonization
matrix (Xu et al., 2016). In order to determine whether biofilm and infection (Padmavathy et al., 2015; Tong et al., 2015).
formation by S. aureus was associated with cell survival, we S. aureus and E. faecalis are both capable of adhering to abiotic
evaluated the viability of biofilm-forming S. aureus (ATCC surfaces (such as catheter) due to the expression of surface
6538) cells incubated with cinnamaldehyde at MIC/2. We found proteins (Foster et al., 2014), such as the S. aureus protein
that at this concentration, cinnamaldehyde reduces S. aureus A (SpA) and the enterococcal surface protein (Esp) (Elhadidy
metabolic activity, indicating loss of viable cells. These results and Elsayyad, 2013; Zapotoczna et al., 2016). Cinnamaldehyde
allow us to suggest that the increased biofilm mass observed in strongly inhibited S. aureus adherence to latex, an effect that was
cinnamaldehyde-treated S. aureus is due to the accumulation of observed when this compound was tested at MIC/2 and MIC/4
dead cells rather than increase in S. aureus virulence. They also on all strains. When tested on E. faecalis strains, cinnamaldehyde
support the applicability of cinnamaldehyde in the treatment of only diminished latex adherence by the standard strain ATCC
S. aureus-induced infections. 19443.
The use of essential oils or other plant-derived material to
Cinnamaldehyde Sub-inhibitory prevent bacterial adhesion to catheters and other medical devices
Concentrations Inhibit the Hemolytic has been pointed as an interesting approach for the medical field
Activity of S. aureus but not E. faecalis (Silva et al., 2016). These strategies involve the modification of
Hemolytic toxins are secreted virulence factors expressed by the surface by the incorporation of the anti-adhesive compounds,
some strains of S. aureus and E. faecalis which increase resulting in functionalized surfaces with improved resistance to
pathogenicity (Van Tyne et al., 2013; Tabor et al., 2016). Our microbial colonization (Grumezescu, 2013; Trentin et al., 2015).
data show that cinnamaldehyde diminishes S. aureus-induced Our results show that cinnamaldehyde may be useful for the
hemolysis, but is only able to inhibit this parameter in one of the development of surface-modified materials in order to prevent
tested E. faecalis strains. The effects of cinnamaldehyde on cell S. aureus adhesion.
survival have been widely studied in different cells lines, including
immune cells (Roth-Walter et al., 2014), neurones (Pyo et al., Cinnamaldehyde Increases the
2013), erythrocytes (Theurer et al., 2013), amongst others. Of G. mellonella Larvae Survival and
importance, this compound was shown to cause hemolysis per se Reduce Bacterial Load in the
when incubated for 48 h with human erythrocytes (Theurer et al.,
2013). On the other hand, we show that the hemolysis caused by
Hemolymph
S. aureus is markedly inhibited by sub-inhibitory concentrations Overall, cinnamaldehyde in vitro antimicrobial actions
of cinnamaldehyde. It is possible that in this experimental were more pronounced on S. aureus. In vivo-testing of
setting, cinnamaldehyde targets bacteria rather than erythrocytes. cinnamaldehyde in G. mellonella larvae infected with S. aureus
Similarly, Amalaradjou et al. (2014) reported a protective effect showed this compound augments larvae survival whilst reducing
for cinnamaldehyde in Cronobacter sakazakii-induced intestinal bacterial load in their hemolymph. In vivo protection of infection
epithelial cell death. by cinnamaldehyde has been previously reported. Recently,
cinnamaldehyde was shown to protect G. mellonella larvae
Cinnamaldehyde Sub-inhibitory against L. monocytogenes-induced infection (Upadhyay and
Venkitanarayanan, 2016). This action was attributed to its ability
Concentrations Decrease S. aureus to up-regulate the expression of antimicrobial peptide genes in
Survival in the Presence of Human G. mellonella (Upadhyay and Venkitanarayanan, 2016). The
Serum immunomodulatory properties of cinnamaldehyde were also
Staphylococcus aureus and E. faecalis are common etiological evaluated in a mouse model of LPS-induced SIRS. The authors
agents of bacteraemia which often lead to septic shock and showed that cinnamaldehyde protection is related to the ability
endocarditis (Dahl et al., 2016; Yahav et al., 2016). The survival of this compound in modulating the immune response through

Frontiers in Microbiology | www.frontiersin.org 8 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

transient receptor potential ankyrin 1 (TRPA1)-dependent and infections as it presents the ability to diminish bacterial
independent mechanisms (Mendes et al., 2016). virulence/survival in addition to improve the host’s immune
These results are rather promising as cinnamaldehyde is response to infection.
effective against bacteria and also improves the immune response
to infection by these pathogens. Here, cinnamaldehyde protected
against S. aureus infection in G. mellonella, in doses equivalent AUTHOR CONTRIBUTIONS
to 25–50 mg/kg. On the other hand, further studies are necessary
in order to establish cinnamaldehyde safety and effectiveness in TF, JA, BdSP, JdS, ES, BdS, VC, TN, CF, CS, JC, VM-N, LdS, and
humans when given by oral route. Indeed, reports suggest that EF contributed to conception, design, data acquisition, analysis,
cinnamaldehyde presents both genotoxic and irritative effects, and interpretation, drafted and critically revised the manuscript.
although these are noted when this compound is administered at All authors gave final approval and agree to be accountable for all
much higher concentrations/doses than the ones investigated in aspects of the work.
our study, such as >500 mg/kg (systemically) or >3% (topically
applied to the skin) (for review see, Bickers et al., 2005).
Data obtained from animal studies suggest cinnamaldehyde is ACKNOWLEDGMENTS
safe by oral route when administered as either a single dose
(2,220 mg/kg) or repeatedly for even over 2 years (up to This work was supported by the Coordenação de
550 mg/kg/day). Importantly, cinnamaldehyde excretion rate at Aperfeiçoamento de Pessoal de Nivel Superior (CAPES; Brazil;
24 h after administration varies between 70 and 98% in rodents, 3325/2013), Conselho Nacional de Desenvolvimento Científico
depending on the route of administration; and reaches 100% e Tecnológico (CNPq; Brazil; 474999/2012-2), Fundação de
within 8 h when given orally to healthy human volunteers (for Amparo à Pesquisa e Desenvolvimento Científico do Maranhão
review see, Bickers et al., 2005; Cocchiara et al., 2005). Thus, (FAPEMA; Brazil; 00740/13 and 03619/13) and Fundação de
implementation of dose schemes may also consider the excretion Amparo à Ciência e Tecnologia de Pernambuco (FACEPE).
rate of cinnamaldehyde. JA, BdSP, JdS, ES, and BdS are M.Sc. students receiving grants
Overall, we suggest that cinnamaldehyde may represent from FAPEMA. CF is a Ph.D. student receiving grant from
an alternative therapy to control S. aureus-induced bacterial FACEPE.

REFERENCES Chen, W., Golden, D. A., Critzer, F. J., and Davidson, P. M. (2015). Antimicrobial
activity of cinnamaldehyde, carvacrol, and lauric arginate against Salmonella
Abbaszadegan, A., Dadolahi, S., Gholami, A., Moein, M. R., Hamedani, S., Tennessee in a glycerol-sucrose model and peanut paste at different fat
Ghasemi, Y., et al. (2016). Antimicrobial and cytotoxic activity of Cinnamomum concentrations. J. Food Prot. 78, 1488–1495. doi: 10.4315/0362-028X.JFP-
zeylanicum, Calcium Hydroxide, and triple antibiotic paste as root canal 14-599
dressing materials. Contemp. Dent. Pract. 17, 105–113. doi: 10.5005/jp-journ Claessen, D., Rozen, D. E., Kuipers, O. P., Søgaard-Andersen, L., and van Wezel,
als-10024-1811 G. P. (2014). Bacterial solutions to multicellularity: a tale of biofilms, filaments
Amalaradjou, M. A., Kim, K. S., and Venkitanarayanan, K. (2014). Sub-inhibitory and fruiting bodies. Nat. Rev. Microbiol. 12, 115–124. doi: 10.1038/nrmicro3178
concentrations of trans-cinnamaldehyde attenuate virulence in Cronobacter Clinical Laboratory Standards Institute [CLSI] (2015). Performance Standards for
sakazakii in vitro. Int. J. Mol. Sci. 15, 8639–8655. doi: 10.3390/ijms15058639 Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement.
Barriere, S. L. (2015). Clinical, economic and societal impact of antibiotic Wayne PA: Clinical and Laboratory Standards Institute, 1–240.
resistance. Expert Opin. Pharmacother. 16, 151–153. doi: 10.1517/14656566. Cocchiara, J., Letizia, C. S., Lalko, J., Lapczynski, A., and Api, A. M. (2005).
2015.983077 Fragrance material review on cinnamaldehyde. Food Chem. Toxicol. 43, 867–
Bhatty, M., Cruz, M. R., Frank, K. L., Laverde Gomez, J. A., Andrade, F., Garsin, 923. doi: 10.1016/j.fct.2004.09.014
D. A., et al. (2015). Enterococcus faecalis pCF10-encoded surface proteins PrgA, Cox, S. D., and Markham, J. L. (2007). Susceptibility and intrinsic tolerance
PrgB (aggregation substance) and PrgC contribute to plasmid transfer, biofilm of Pseudomonas aeruginosa to selected plant volatile compounds. J. Appl.
formation and virulence. Mol. Microbiol. 95, 660–677. doi: 10.1111/mmi.12893 Microbiol. 103, 930–936. doi: 10.1111/j.1365-2672.2007.03353.x
Bickers, D., Calow, P., Greim, H., Hanifin, J. M., Rogers, A. E., Saurat, J. H., Dahl, A., Lauridsen, T. K., Arpi, M., Sørensen, L. L., Østergaard, C., Sogaard, P.,
et al. (2005). A toxicologic and dermatologic assessment of cinnamyl alcohol, et al. (2016). Risk factors of endocarditis in patients with Enterococcus faecalis
cinnamaldehyde and cinnamic acid when used as fragrance ingredients. Food bacteremia: external validation of the NOVA score. Clin. Infect. Dis. 63, 771–
Chem. Toxicol. 43, 799–836. doi: 10.1016/j.fct.2004.09.013 775. doi: 10.1093/cid/ciw383
Blair, J. M., Webber, M. A., Baylay, A. J., Ogbolu, D. O., and Piddock, L. J. (2015). Domadia, P., Swarup, S., Bhunia, A., Sivaraman, J., and Dasgupta, D. (2007).
Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13, 42–51. Inhibition of bacterial cell division protein FtsZ by cinnamaldehyde. Biochem.
doi: 10.1038/nrmicro3380 Pharmacol. 74, 831–840. doi: 10.1016/j.bcp.2007.06.029
Budri, P. E., Silva, N. C. C., Bonsaglia, E. C. R., Fernandes, A., Araújo, J. P., Duncan, B., Li, X., Landis, R. F., Kim, S. T., Gupta, A., Wang, L. S., et al. (2015).
Doyama, J. T., et al. (2015). Effect of essential oils of Syzygium aromaticum and Nanoparticle-stabilized capsules for the treatment of bacterial biofilms. ACS
Cinnamomum zeylanicum and their major components on biofilm production Nano. 9, 7775–7782. doi: 10.1021/acsnano.5b01696
in Staphylococcus aureus strains isolated from milk of cows with mastitis. Dairy Elhadidy, M., and Elsayyad, A. J. (2013). Uncommitted role of enterococcal surface
Sci. 98, 5899–5904. doi: 10.3168/jds.2015-9442 protein, Esp, and origin of isolates on biofilm production by Enterococcus
Chandra, J., Mukherjee, P. K., and Ghannoum, M. A. (2008). In vitro growth and faecalis isolated from bovine mastitis. Microbiol. Immunol. Infect. 46, 80–84.
analysis of Candida biofilms. Nat. Protoc. 3, 1909–1924. doi: 10.1038/nprot. doi: 10.1016/j.jmii.2012.02.002
2008.192 Foster, T. J., Geoghegan, J. A., Ganesh, V. K., and Höök, M. (2014). Adhesion,
Chang, S. T., Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of invasion and evasion: the many functions of the surface proteins of
leaf essential oils and their constituents from Cinnamomum osmophloeum. Staphylococcus aureus. Nat. Rev. Microbiol. 12, 49–62. doi: 10.1038/nrmicr
J. Ethnopharmacol. 77, 123–127. doi: 10.1016/S0378-8741(01)00273-2 o3161

Frontiers in Microbiology | www.frontiersin.org 9 December 2016 | Volume 7 | Article 2052

Ferro et al. Cinnamaldehyde-Induced Protection against Staphylococcus aureus

Gill, A. O., and Holley, R. A. (2004). Mechanisms of bactericidal action Stepanović, S., Cirković, I., Ranin, L., and Svabić-Vlahović, M. (2004).
of cinnamaldehyde against Listeria monocytogenes and of eugenol against Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic
L. monocytogenes and Lactobacillus sakei. Appl. Environ. Microbiol. 70, 5750– surface. Lett. Appl. Microbiol. 38, 428–432. doi: 10.1111/j.1472-765X.2004.
5755. doi: 10.1128/AEM.70.10.5750-5755.2004 01513.x
Grumezescu, A. M. (2013). Essential oils and nanotechnology for Tabor, D. E., Yu, L., Mok, H., Tkaczyk, C., Sellman, B. R., Wu, Y., et al.
combating microbial biofilms. Curr. Org. Chem. 17, 90–96. doi: (2016). Staphylococcus aureus Alpha-toxin is conserved among diverse hospital
10.2174/1385272811317020003 respiratory isolates collected from a global surveillance study and is neutralized
Hall, J. W., Yang, J., Guo, H., and Ji, Y. (2015). The AirSR two-component by monoclonal antibody MEDI4893. Antimicrob. Agents Chemother. 60, 5312–
system contributes to Staphylococcus aureus survival in human blood and 5321. doi: 10.1128/AAC.00357-16
transcriptionally regulates sspABC operon. Front. Microbiol. 6:682. doi: 10. Thammavongsa, V., Kim, H. K., Missiakas, D., and Schneewind, O. (2015).
3389/fmicb.2015.00682 Staphylococcal manipulation of host immune responses. Nat. Rev. Microbiol.
Hammer, K. A., and Heel, K. A. (2012). Use of multiparameter flow cytometry to 13, 529–543. doi: 10.1038/nrmicro3521
determine the effects of monoterpenoids and phenylpropanoids on membrane Theilacker, C., Diederich, A. K., Otto, A., Sava, I. G., Wobser, D., Bao, Y., et al.
polarity and permeability in staphylococci and enterococci. Int. J. Antimicrob. (2015). Enterococcus faecalis glycolipids modulate lipoprotein-content of the
Agents 40, 239–245. doi: 10.1016/j.ijantimicag.2012.05.015 bacterial cell membrane and host immune response. PLoS ONE 10:e0132949.
Heras, B., Scanlon, M. J., and Martin, J. L. (2015). Targeting virulence not viability doi: 10.1371/journal.pone.0132949
in the search for future antibacterials. Br. J. Clin. Pharmacol. 79, 208–215. Theurer, M., Shaik, N., and Lang, F. (2013). Stimulation of suicidal erythrocyte
doi: 10.1111/bcp.12356 death by trans-cinnamaldehyde. Phytomed. 20, 1119–1123. doi: 10.1016/j.phym
Ismail, G., Razak, N., Mohamed, R., Embi, N., and Omar, O. (1988). Resistance ed.2013.05.006
of Pseudomonas pseudomallei to normal human serum bactericidal action. Tong, S. Y., Davis, J. S., Eichenberger, E., Holland, T. L., and Fowler, V. G. (2015).
Microbiol. Immunol. 32, 645–652. doi: 10.1111/j.1348-0421.1988.tb01426.x Staphylococcus aureus infections: epidemiology, pathophysiology, clinical
Jia, P., Xue, Y. J., Duan, X. J., and Shao, S. H. (2011). Effect of cinnamaldehyde on manifestations, and management. Clin. Microbiol. Rev. 28, 603–661. doi: 10.
biofilm formation and sarA expression by methicillin-resistant Staphylococcus 1128/CMR.00134-14
aureus. Lett. Appl. Microbiol. 53, 409–416. doi: 10.1111/j.1472-765X.2011. Trentin, D. S., Silva, D. B., Frasson, A. P., Rzhepishevska, O., da Silva, M. V.,
03122.x Pulcini Ede, L., et al. (2015). Natural green coating inhibits adhesion of clinically
Kaplan, J. B., Izano, E. A., Gopal, P., Karwacki, M. T., Kim, S., Bose, J. L., et al. important bacteria. Sci. Rep. 2015, 8287. doi: 10.1038/srep08287
(2012). Low levels of β-lactam antibiotics induce extracellular DNA release and Upadhyay, A., and Venkitanarayanan, K. (2016). In vivo efficacy of trans-
biofilm formation in Staphylococcus aureus. MBio. 3:e198–e112. doi: 10.1128/ cinnamaldehyde, carvacrol, and thymol in attenuating Listeria monocytogenes
mBio.00198-12 infection in a Galleria mellonella model. J. Nat. Med. 70, 667–672. doi: 10.1007/
Kong, C., Neoh, H. M., and Nathan, S. (2016). Targeting Staphylococcus aureus s11418-016-0990-4
toxins: a potential form of anti-virulence therapy. Toxins 8, E72. doi: 10.3390/ Utchariyakiat, I., Surassmo, S., Jaturanpinyo, M., Khuntayaporn, P., and
toxins8030072 Chomnawang, M. T. (2016). Efficacy of cinnamon bark oil and cinnamaldehyde
Lin, J., Nishino, K., Roberts, M. C., Tolmasky, M., Aminov, R. I., and Zhang, L. on anti-multidrug resistant Pseudomonas aeruginosa and the synergistic effects
(2015). Mechanisms of antibiotic resistance. Front. Microbiol. 6:34. doi: 10. in combination with other antimicrobial agents. BMC Complement. Altern.
3389/fmicb.2015.00034 Med. 16:158. doi: 10.1186/s12906-016-1134-9
Mendes, S. J., Sousa, F. I., Pereira, D. M., Ferro, T. A., Pereira, I. C., Silva, B. L., Van Tyne, D., Martin, M. J., and Gilmore, M. S. (2013). Structure, function, and
et al. (2016). Cinnamaldehyde modulates LPS-induced systemic inflammatory biology of the Enterococcus faecalis cytolysin. Toxins 5, 895–911. doi: 10.3390/
response syndrome through TRPA1-dependent and independent mechanisms. toxins5050895
Int. Immunopharmacol. 34, 60–70. doi: 10.1016/j.intimp.2016.02.012 Wilson, B. A., Garud, N. R., Feder, A. F., Assaf, Z. J., and Pennings, P. S. (2016).
Padmavathy, K., Praveen, S., Madhavan, R., Krithika, N., and Kiruthiga, A. J. The population genetics of drug resistance evolution in natural populations of
(2015). Clinico-microbiological investigation of catheter associated urinary viral, bacterial and eukaryotic pathogens. Mol. Ecol. 25, 42–66. doi: 10.1111/me
tract infection by Enterococcus faecalis: vanA genotype. Clin. Diagn. Res. 9, c.13474
DD05–DD6. doi: 10.7860/JCDR/2015/13856.6378 Xu, Z., Liang, Y., Lin, S., Chen, D., Li, B., Li, L., et al. (2016). Crystal Violet and
Pyo, J. H., Jeong, Y. K., Yeo, S., Lee, J. H., Jeong, M. Y., Kim, S. H., et al. (2013). XTT Assays on Staphylococcus aureus biofilm quantification. Curr. Microbiol.
Neuroprotective effect of trans-cinnamaldehyde on the 6-hydroxydopamine- 73, 474–482. doi: 10.1007/s00284-016-1081-1
induced dopaminergic injury. Biol. Pharm. Bull. 36, 1928–1935. doi: 10.1248/ Yahav, D., Yassin, S., Shaked, H., Goldberg, E., Bishara, J., Paul, M., et al. (2016).
bpb.b13-00537 Risk factors for long-term mortality of Staphylococcus aureus bacteremia.
Rasko, D. A., and Sperandio, V. (2010). Anti-virulence strategies to combat Eur. J. Clin. Microbiol. Infect. Dis. 35, 785–790. doi: 10.1007/s10096-016-
bacteria-mediated disease. Nat. Rev. Drug Dis. 9, 117–128. doi: 10.1038/nrd3013 2598-8
Richards, R. L., Haigh, R. D., Pascoe, B., Sheppard, S. K., Price, F., Jenkins, D., et al. Zapotoczna, M., O’Neill, E., and O’Gara, J. P. (2016). Untangling the diverse
(2015). Persistent Staphylococcus aureus isolates from two independent cases and redundant mechanisms of Staphylococcus aureus biofilm formation. PLoS
of bacteremia display increased bacterial fitness and novel immune evasion Pathog. 12:e1005671. doi: 10.1371/journal.ppat.1005671
phenotypes. Infect. Immun. 83, 3311–3324. doi: 10.1128/IAI.00255-15 Zodrow, K. R., Schiffman, J. D., and Elimelech, M. (2012). Biodegradable polymer
Roth-Walter, F., Moskovskich, A., Gomez-Casado, C., Diaz-Perales, A., Oida, K., (PLGA) coatings featuring cinnamaldehyde and carvacrol mitigate biofilm
Singer, J., et al. (2014). Immune suppressive effect of cinnamaldehyde due formation. Langmuir 28, 13993–13999. doi: 10.1021/la303286v
to inhibition of proliferation and induction of apoptosis in immune cells:
implications in cancer. PLoS ONE 9:e108402. doi: 10.1371/journal.pone Conflict of Interest Statement: The authors declare that the research was
.0108402 conducted in the absence of any commercial or financial relationships that could
Schilcher, K., Andreoni, F., Haunreiter, V. D., Seidl, K., Hasse, B., and Zinkernagel, be construed as a potential conflict of interest.
A. S. (2016). Modulation of Staphylococcus aureus biofilm matrix by
subinhibitory concentrations of clindamycin. Antimicrob. Agents Chemother. Copyright © 2016 Ferro, Araújo, dos Santos Pinto, dos Santos, Souza, da Silva,
60, 5957–5967. doi: 10.1128/AAC.00463-16 Colares, Novais, Filho, Struve, Calixto, Monteiro-Neto, da Silva and Fernandes.
Shen, S., Zhang, T., Yuan, Y., Lin, S., Xu, J., and Ye, H. (2015). Effects of This is an open-access article distributed under the terms of the Creative Commons
cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane. Food Attribution License (CC BY). The use, distribution or reproduction in other forums
Control. 47, 196–202. doi: 10.1016/j.foodcont.2014.07.003 is permitted, provided the original author(s) or licensor are credited and that the
Silva, L. N., Zimmer, K. R., Macedo, A. J., and Trentin, D. S. (2016). Plant natural original publication in this journal is cited, in accordance with accepted academic
products targeting bacterial virulence factors. Chem. Rev. 116, 9162–9236. doi: practice. No use, distribution or reproduction is permitted which does not comply
10.1021/acs.chemrev.6b00184 with these terms.

Frontiers in Microbiology | www.frontiersin.org 10 December 2016 | Volume 7 | Article 2052