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Biofouling
The Journal of Bioadhesion and Biofilm Research
ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20
Non-toxic plant metabolites regulate
Staphylococcus viability and biofilm formation:
a natural therapeutic strategy useful in the
treatment and prevention of skin infections
A. Morán, S. Gutiérrez, H. Martínez-Blanco, M.A. Ferrero, A. Monteagudo-
Mera & L.B. Rodríguez-Aparicio
To cite this article: A. Morán, S. Gutiérrez, H. Martínez-Blanco, M.A. Ferrero, A.
Monteagudo-Mera & L.B. Rodríguez-Aparicio (2014) Non-toxic plant metabolites regulate
Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful
in the treatment and prevention of skin infections, Biofouling, 30:10, 1175-1182, DOI:
10.1080/08927014.2014.976207
To link to this article: http://dx.doi.org/10.1080/08927014.2014.976207
Published online: 14 Nov 2014. Submit your article to this journal
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Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural
therapeutic strategy useful in the treatment and prevention of skin infections
A. Morán
a
, S. Gutiérrez
a
, H. Martínez-Blanco
a,b
, M.A. Ferrero
a,b
, A. Monteagudo-Mera
a
and L.B. Rodríguez-Aparicio
a,b
*
a
Departamento de Biología Molecular, Facultad de Veterinaria, Universidad de León, León, Spain;
b
Instituto de Biología Molecular,
Genómica y Proteómica (INBIOMIC). Universidad de León, León, Spain
(Received 12 June 2014; accepted 7 October 2014)
In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating
the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the
strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤50 μgml
−1
.
Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range
(SIC = 50–1,000 μgml
−1
), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract
were the most biologically compatible molecules (SIC ≥1000 μgml
−1
). Genistein, protocatechuic acid, cranberry extract,
p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis
in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations
of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory
effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.
Keywords: biofilm; non-toxic plant metabolites; Staphylococcus; antimicrobial; skin infection
Introduction
Many bacterial infections that cause morbidity and
mortality both in hospitals and in the community can be
attributed to species of the genus Staphylococcus. Among
them, S. aureus, a coagulase-positive microorganism, is
well known due to its capacity to cause chronic infections
that persist in medical implants or host tissues (Chambers
& DeLeo 2009; DeLeo & Chambers 2009). In addition,
S. epidermidis, a coagulase-negative strain long consid-
ered an innocuous commensal microorganism, is also
involved in nosocomial infections (Cerca et al. 2005;
Rogers et al. 2009; Christensen & Brüggemann 2013). In
both cases, the capacity to produce biofilms has been con-
sidered a crucial virulence factor that facilitates bacterial
adhesion and resistance to the host’s defences and to anti-
biotics (Kiedrowski & Horswill 2011; Gomes et al. 2013).
However, S. epidermidis is also considered a mutualistic
skin microorganism that maintains a benign relationship
with its host and helps to prevent colonisation by other
sometimes potentially more harmful organisms (Otto
2009). Moreover, S. epidermidis plays an important
role in balancing the epithelial microbiota (Otto 2009;
Christensen & Brüggemann 2013). A well-balanced skin
microbiota is fundamental for protection against
pathogens and for the prevention of epithelial infections.
However, an imbalance in the microbiota causes skin bar-
rier dysfunctions and is associated with the development
of various epithelial diseases such as acne vulgaris,
rosacea, seborrhoeic dermatitis or atopic dermatitis (Grice
& Segre 2011; Murillo & Raoult 2013). For example,
colonisation of the skin by S. aureus is very common in
atopic dermatitis (Wolf & Wolf 2012; Murillo & Raoult
2013); furthermore, the density of S. aureus has been
shown to correlate with cutaneous inflammation and
severity of eczema (Williams et al. 1990), and epithelial
dysbiosis can even lead to a skin commensal such as
S. epidermidis causing infection and disease, especially
when the flora invades other sites (Grice & Segre 2011).
Until now, the most common approach to treating
skin infection has been the administration of topical or
systemic antibiotics (Huang et al. 2009; Friedman &
Goldman 2011), but these can modify the defensive
functions of the epithelial microbiota and can result in
the development and spread of antimicrobial resistance.
It is crucial to develop non-aggressive therapeutic strate-
gies that contribute to maintaining and restoring the
healthy epithelial microbiota which protect against patho-
gens and prevent skin infections. In this respect, it has
been demonstrated that S. epidermidis inhibits S. aureus
biofilm formation (Iwase et al. 2010) and that plant
extracts can show anti-biofilm activity and eradicate the
biofilm formed by P. acnes (Coenye et al. 2012). Based
on these findings, a study was conducted to determine
whether non-toxic antimicrobial plant metabolites can
regulate the growth and biofilm formation by S. aureus
and S. epidermidis. The results may useful in the
*Corresponding author. Email: leandro.rodriguez@unileon.es
© 2014 Taylor & Francis
Biofouling, 2014
Vol. 30, No. 10, 1175–1182, http://dx.doi.org/10.1080/08927014.2014.976207
Downloaded by [University of Reading] at 10:33 28 October 2015
development of natural therapeutic strategies to maintain
skin microbiota balance, but also in the prevention and
treatment of epithelial infections.
Materials and methods
Strains and culture conditions
The bacteria used in this study were the commensal,
non-invasive Staphylococcus epidermidis CECT 231
(ATCC1798) and the enteropathogenic S. aureus CECT
59 (ATCC 9144), both obtained from the Spanish
Collection of Type Culture (CECT).
Both strains were grown in Trypticasein Soy Agar
Broth (TSA) and Trypticasein Soy Broth containing 0.6%
(w/v) yeast extract (TSB-YE) and cultured at 37°C for
12 h. The strains were then maintained in a 50–50 mixture
of TSB and glycerol at –80°C until use. For antimicrobial
and biofilm studies, cells from TSB-glycerol were
previously grown in slant-TSA and incubated overnight at
37°C in tubes containing 3 ml of TSB-YE.
Natural plant compounds
In this study, 11 non-toxic plant metabolites from five
families were selected for their antibacterial and/or anti-
biofilm activities (Table 1), from five families: (1) terpe-
noids (thymol, carvacrol and eugenol); (2) flavonoids
(catechin and genistein); (3) phenols/polyphenols (gallic
acid, protocatechuic acid, hydroquinone and cranberry
extract as Urell
R
); (4) one mono-hydroxybenzoic acid
(p-hydroxybenzoic acid); and (5) resveratrol, a stilbenoid.
All plant metabolites were commercially obtained from
Sigma Chemical Co. (St Louis, MO, USA) and Urell
R
from Pharmatoka (Rueil-Malmaison, Paris, France). For
antimicrobial and anti-biofilm studies, 50 μl in 70% etha-
nol of each plant metabolite (except for cranberry extract,
which was dissolved in water) were used and compared
with the respective controls containing ethanol (or water)
but not the plant compound being tested.
Determination of minimum inhibitory concentration
(MIC) and subinhibitory concentration (SIC)
MIC, defined as the lowest plant metabolite concentra-
tion that inhibits bacterial growth by > 90%, and SIC,
defined as the highest plant metabolite concentration
below the MIC (NCCLS 2002), were determined by
incubating the microorganisms in the presence or
absence of each plant compound. Briefly, 50 μlofthe
overnight bacterial culture (adjusted with sterile TSB-YE
to 1.0 at OD
540
nm
,
equivalent to 5 × 10
6
CFU), were
used to inoculate 5 ml of TSB-YE unsupplemented or
supplemented with the corresponding plant metabolite.
After incubation for 16 h at 37°C, growth was measured
by absorbance at OD
540
nm using a spectrophotometer
(Beckman DU640). The data from at least five indepen-
dent replicates were evaluated and modal results were
calculated.
Biofilm determination
To quantify biofilm formation, the method described by
Kubota et al. (2008) and Monteagudo-Mera et al.
(2012) was employed, using polystyrene microtiter
plates (DeltaLab, Barcelona, Spain). Briefly, the over-
night bacterial culture, diluted to 10
5
CFU ml
−1
in
TSB-YE was dispensed into each well of 96-well
microtiter plates (200 μl each) in the presence or
absence of the lowest SIC concentration (see Table 2)
of each plant metabolite tested. After incubation for
24 h at 37°C, planktonic cells were eliminated by
washing and the biofilm generated was evaluated, after
staining with crystal violet and elution with 95% etha-
nol, by absorption at OD
570
nm using a microtiter spec-
trophotometer (Anthos 2020, Biochrom, Cambridge,
UK). When necessary, planktonic and sessile cells
were directly evaluated by absorption at OD
540
nm.
The data from at least three independent replicates were
evaluated and modal results were calculated.
Statistical analysis
The results are presented as means ± SEM. Significant
differences between means were calculated with the
Student’st-test. Pvalues of 0.05 or less were considered
statistically significant.
Results and discussion
The growing antibiotic resistance of pathogenic bacterial
species poses a serious problem for public health. Many
commonly used antibiotics are no longer effective and
modify the natural defensive function of microbiota (Levy
1998; Wright 2010). In epithelial infections such as atopic
dermatitis, the long-term use of systemic or topical antibi-
otics is not recommended and should be reserved only for
cases in which explicit signs of infection are present
(Friedman & Goldman 2011). There is an urgent need to
study and develop new antibacterial therapeutics that
control bacterial growth without generating resistance or
altering the defensive capacity of the microbiota. The tra-
ditional use of plant metabolites to treat infectious
diseases may represent a good alternative. Many plant-
derived products have been tested for potential antimicro-
bial activity (Cowan 1999), but there is relatively little
information pertaining to the control of staphylococcal
infections. Therefore, the effects of different non-toxic
antimicrobial plant compounds (Table 1) on the growth
and biofilm formation by S. aureus and S. epidermidis
were studied.
1176 A. Morán et al.
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Antimicrobial activity of non-toxic plant-derived
compounds
Based on previous data (Parkar et al. 2008) reporting
that plant polyphenols can be present in a range of 50 to
1,000 μgml
−1
without showing colon toxicity, the anti-
Staphylococcus activity of the selected plant metabolites
was tested (see Materials and methods) using concentra-
tions of 2–2,000 μgml
−1
. Table 2shows the respective
Table 1. Non-toxic plant metabolites used in this work.
Compound Biological source Biological action Reference
Thymol (terpenoid) Origanum (spice) and
thyme
(Thymus sp.) extracts
Antibacterial activity against Streptococcus
mutans. Antibiofilm activity and inactivating
mature biofilms of Listeria monocytogenes
Botelho et al. (2007)
Upadhyay et al. (2013)
Carvacrol (thymol isomere)
(terpenoid)
Origanum (spice) and
thyme
(Thymus sp.) extracts
Antibacterial activity against B. cereus and S.
mutans.
Ultee et al. (1999)
Antibiofilm activity and inactivating mature
biofilms of L. monocytogenes
Botelho et al. (2007)
Upadhyay et al. (2013)
Eugenol (terpenoid) Cinnamon and clove
(Syzygium aromaticum and
Eugenia caryophillis)
extracts
Antimicrobial action against Helicobacter
pylori. B. cereus,S. aureus,S. epidermidis,
Streptococcus pneumoniae,S. pyogenes,
Salmonella typhimurium DT104 and E. coli
O157:H7
Ali et al. (2005)
Antibiofilm activity and inactivating mature
biofilms of L. monocytogenes
Kamatou et al. (2012)
Upadhyay et al. (2013)
(+)-Catechin hydrate
(flavonoid)
Tea ( Camellia sinensis)
and Vitis rotundifolia
extracts
Antibacterial activity against Vibrio cholerae
O1, S. mutans and Shigella
Toda et al. (1991)
Antibiofilm activity against Eikenella
corrodens
Shimamura et al.
(2007)
Matsunaga et al. (2010)
Genistein (flavonoid) Soybean (Glycine max)
extracts
Antibacterial activity against S. aureus,B.
anthracis and Vibrio fluvialis
Hong et al. (2006)
Gallic acid (phenolic acid) Vitis rotundifolia extracts Antimicrobial activity against P. aeruginosa
ATCC 9027. Antibiofilm activity against
E. coli,P. aeruginosa,L. monocytogenes and
S. aureus.
Rauha et al. (2000)
Borges et al. (2012)
Protocatechuic acid
(phenolic acid)
Scrophularia frutescens
extracts
Bacteriostatic effect against Micrococcus
luteus YMBL and P. aeruginosa ATCC 9027
Fernández et al. (1996)
Antimicrobial activity against L.
monocytogenes
Rauha et al. (2000)
Hydroquinone (phenol ) Bilberry (Vaccinium
myrtillus)
Slightly inhibited the growth of
Mycobacterium leprae
Drea (1944)
Alters the agglutination of S. mutans induced
by sucrose
Himejima et al. (2004)
Cranberry (polyphenols) American cranberry
(Vaccinium macrocarpon)
marketed as Urell
R
Inhibition of E. coli adherence to mucosal
surfaces.
Schmidt and Sobota
(1987)
Inhibited both the growth and biofilm
production by Staphylococcus spp.
LaPlante et al. (2012)
p-Hydroxybenzoic acid
(monohydroxibenzoic
acid)
Scrophularia frutescens
and Scrophularia
sambucifolia extracts
Antimicrobial effect against of Ganoderma
boninense
Fernández et al. (1996)
Chong et al. (2009)
Resveratrol (stilbenoid) Vitis vinifera extract Antimicrobial effect against B. cereus ATCC
11778, S. aureus ATCC 25923 and
Enterococcus faecalis ATCC 29212.
Antibiofilm action against P. acnes, and
E. coli O157:H7
Paulo et al. (2010)
Coenye et al. (2012)
Lee et al. (2013)
Biofouling 1177
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values obtained for the SIC and MIC. The results reflect a
variable degree of antibacterial activity against S. aureus
and S. epidermidis. Comparing the SIC and MIC, all the
plant compounds tested showed a dose-dependent anti-
microbial effect and could be classified into three groups,
as follows. (1) Highly active, consisting of the terpenoids
tested: thymol, carvacrol (a thymol isomer) and eugenol,
at an SIC ≤50 μgml
−1
. It is possible that the high sensi-
tivity shown by this group of molecules is a consequence
of the disruption of the bacterial membrane caused by
the presence of terpenoids, which inhibit ATP synthesis
(Ultee et al. 1999; Halcón & Milkus 2004; Gyawali &
Ibrahim 2012). (2) Moderately active, consisting of geni-
stein (from soybean), hydroquinone ( from bilberry) and
resveratrol (from black grapes), which also showed an
antimicrobial effect (Table 2) but with a wide concentra-
tion range (SIC = 50–1,000 μgml
−1
). (3) Weakly active,
consisting of catechin, gallic acid, protocatechuic acid,
p-hydroxybenzoic acid (from Scrophularia) and
cranberry extract present in berries and grapes, which
were the most biologically compatible products
(SIC ≥1,000 μgml
−1
).
Despite the observed concentration variability, the
analysed compounds showed similar antimicrobial
activity against the two Staphylococcus strains studied.
Nevertheless, S. epidermidis was more sensitive to the
presence of carvacrol (10-fold) and resveratrol (5-fold).
The compounds showing the strongest antimicrobial
action against both bacteria were eugenol (MIC =
100 μgml
−1
) and thymol (MIC = 200 μgml
−1
). These
results indicate that these natural generally recognised as
safe (GRAS) terpenoids, traditionally used as dietary
constituents (Gyawali & Ibrahim 2012; Venkitanarayanan
et al. 2013), possess strong antimicrobial properties and
can be used at low concentrations as a natural alternative
or complementary strategy for preventing S. aureus and
S. epidermidis proliferation. Moreover, this anti-
Staphylococcus activity opens the possibility of using
eugenol and thymol in the prevention and treatment of
skin infections and other opportunistic nosocomial
pathologies caused by S. aureus or S. epidermidis
without concerns about toxicity.
Effect of non-toxic plant-derived compounds on biofilm
formation
It is generally understood that staphylococci cause
chronic infections because they have the ability to adhere
to many types of surfaces, both abiotic and biotic, and
develop sessile communities (biofilms) that generate
resistance to host defences and antibiotics (Kiedrowski
& Horswill 2011). Plant polyphenols can inhibit the
adhesion of periodontopathogenic or gut pathogenic bac-
teria while enhancing the proliferation and adhesion of
probiotic microorganisms (Parkar et al. 2008; Matsunaga
et al. 2010; Rafsanjany et al. 2013). Furthermore, it has
been reported that cranberry (Vaccinium macrocarpon)
polyphenols selectively inhibit the adhesion of intestinal
and urinary pathogens (Puupponen-Pimiä et al. 2005;
Guay 2009). Consequently, cranberry is used pharmaco-
logically to avoid and prevent urinary tract infection
(Urell
R
and Inurec
R
are trademarks). Therefore, the
potential anti-Staphylococcus biofilm activity of the non-
toxic plant metabolites selected in this work was tested.
It was confirmed that under the assay conditions
used in the present work (see Materials and methods),
S. aureus and S. epidermidis were able to bind to poly-
styrene microtiter plates with an efficiency of 2.47% and
2.53%, respectively. Moreover, it was observed that the
addition of plant metabolites at the lowest SIC concen-
trations (Table 2) modified the biofilm forming ability of
both the Staphylococcus species tested. As shown in
Figure 1, the presence of genistein (500 μgml
−1
), proto-
catechuic acid (1,500 μgml
−1
), cranberry extract
(1,700 μgml
−1
), p-hydroxybenzoic acid (1,000 μgml
−1
)
and resveratrol (100 μgml
−1
) inhibited S. aureus biofilm
formation by between 20% and 45%, and the addition of
carvacrol (5 μgml
−1
), eugenol (50 μgml
−1
), or gallic
acid (1,800 μgml
−1
) enhanced S. aureus biofilm forma-
tion by between 5% and 40%. However, thymol
(50 μgml
−1
), catechin (2,000 μgml
−1
) and hydroquinone
(200 μgml
−1
) did not show a significant effect on bio-
film formation. In the case of S. epidermidis, the results
showed that all the non-toxic plant metabolites assayed
caused an increase in biofilm formation (35% to
> 1,300%). Although in all cases, the biofilm effect was
concentration dependent (data not shown), when the
results obtained between the two bacteria studied were
compared, it was observed that the presence of
Table 2. Effects of the non-toxic plant metabolites on the
viability of S. aureus and S. epidermidis.
S. aureus S. epidermidis
MIC
1
SIC
2
MIC
1
SIC
2
Thymol 200 50 200 50
Carvacrol >2,000 50 >2,000 5
Eugenol 100 50 100 50
Catechin >2,000 2,000 >2,000 2,000
Genistein >2,000 500 >2,000 500
Gallic acid >2,000 1,800 >2,000 >2,000
Protocatechuic acid >2,000 1,500 >2,000 >2,000
Hydroquinone >2,000 200 >2,000 200
Cranberry extract >2,000 1,700 >2,000 1,700
p-Hydroxybenzoic acid >2,000 1,000 >2,000 1,500
Resveratrol >2,000 500 >2,000 100
1
Minimun inhibitory concentration (MIC) = the lowest plant metabolite
concentration (µg ml
−1
) that inhibited bacterial growth by > 90%
compared with the control incubated in absence of the metabolites.
2
Subinhibitory concentration (SIC) = the highest metabolite concentra-
tion (µg ml
−1
) below the MIC that did not inhibit bacterial growth.
1178 A. Morán et al.
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protocatechuic acid, p-hydroxybenzoic acid, resveratrol
and especially genistein and cranberry extract stimulated
S. epidermidis biofilm formation (by up to > 1,300%)
while inhibiting S. aureus biofilm formation by up to
45% (Figure 1). The apparently opposite effect observed
with the same metabolite on biofilm formation by differ-
ent species can be a consequence of the multiple mole-
cules and factors (adhesion factors, quorum sensing,
capsular polymers, etc) that are involved in biofilm for-
mation and that indeed can differ between bacterial spe-
cies (Karatan & Watnick 2009; Otto 2013). Thus, the
biofilm effect caused by the plant metabolites tested in
this work can be considered to be molecule- and bacte-
rial species-specific. In addition, it is possible that the
effect caused by these metabolites could be modified by
using combinations of them. Binary studies carried out
by using a combination of the plant metabolites that
showed opposite biofilm effects (Figure 1), revealed that
the presence of genistein or p-hydroxybenzoic acid in
combination with cranberry extract or with resveratrol
caused an additional stimulation of S. epidermidis bio-
film formation (Table 3). Also, a partial additive effect
was observed when protocatechuic acid was combined
with cranberry extract or with resveratrol but no stimula-
tion of biofilm formation was detected when the
combinations tested were genistein with protocatechuic
acid or p-hydroxybenzoic acid, protocatechuic with
Thymol
Carvacrol
Eugenol
Catechin
Genistein
Gallic acid
Protocatechuic acid
Hydroquinone
Cranberry extract
p-Hydroxybenzoic acid
Resveratrol
Biofilm (%)
0
100
200
300
400
500
600
700
1200
1300
1400
S. aureus
S. epidermidis
Control
** **
** *
**
Figure 1. Effect of non-toxic plant compounds on S. aureus
(□) and S. epidermidis (■) biofilm formation. The con-
centrations (μgml−1) used were: thymol, 50; carvacrol,
5; eugenol, 50; catechin, 2,000; genistein, 500; gallic
acid, 188; protocatechuic acid 1,500; hydroquinone, 200;
cranberry extract 1,700; p-hydroxybenzoic acid, 1,000
and resveratrol, 100. The values are expressed as per-
centage bacterial biofilm formed in the presence of the
non-toxic plant compounds, compared with the control
(without plant compounds), expressed as means ± SEM
of three assays in duplicate.
* = no significant differences (p < 0.05) with respect to the
control.
** = no significant differences (p < 0.05) between the two
bacteria.
Table 3. Effect of binary combinations of non-toxic plant compounds on biofilm formation
1
by S. aureus and S. epidermidis.
Combination (A + B)
% Activity/S. epidermidis biofilm formation % Activity/S. aureus biofilm formation
A
(% ± SEM)
B
(% ± SEM) A + B (%)
Interpretation of
activation
effect (AEF)
2
A (% ± SEM) B (% ± SEM) A + B (%)
Interpretation of
inhibition effect
(AEF)
2
Genistein + protocatechuic acid 1,314 ± 35 386 ± 29 928 ± 10 No synergy 54 ± 14 71 ± 5 179 ± 9 No synergy
Genistein + cranberry extract 1,314 ± 35 663 ± 27 2,231 ± 13 Additive 54 ± 14 79 ± 16 52 ± 5* No synergy
Genistein + p-hydroxybenzoic acid 1,314 ± 35 171 ± 25 556 ± 5 No synergy 54 ± 14 70 ± 20 177 ± 5 No synergy
Genistein + resveratrol 1,314 ± 35 152 ± 24 3,627 ± 26 Additive 54 ± 14 66 ± 6 182 ± 31 No synergy
Protocatechuic acid + cranberry extract 386 ± 29 663 ± 27 956 ± 29 Partial synergy 71 ± 5 79 ± 16 59 ± 4 Partial synergy
Protocatechuic acid + p-hydroxybenzoic acid 386 ± 29 171 ± 25 395 ± 2* No synergy 71 ± 5 70 ± 20 166 ± 4 No synergy
Protocatechuic acid + resveratrol 386 ± 29 152 ± 24 414 ± 2 Partial synergy 71 ± 5 66 ± 6 199 ± 12 No synergy
Cranberry extract + p-hydroxybenzoic acid 663 ± 27 171 ± 25 1,164 ± 10 Additive 79 ± 16 70 ± 20 57 ± 12 Partial synergy
Cranberry extract + resveratrol 663 ± 27 152 ± 24 366 ± 2 No synergy 79 ± 16 66 ± 6 84 ± 2* No synergy
p-hydroxybenzoic + resveratrol 171 ± 25 152 ± 24 450 ± 1 Additive 79 ± 20 66 ± 6 85 ± 2* No synergy
1
The results are presented as percentage of activity biofilm genesis by the presence of non-toxic plant compounds, compared with the control (without plant metabolites).
2
AEF = additive effect factor (activation or inhibition) was defined as the ratio of the combined effects (A + B) and a summatory of the individual effects (A and B) and the interpretation of the results
was carried out according to Sato et al. (2004).
*
Indicates no significant differences (p< 0.05) among A and A + B.
Biofouling 1179
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p-hydroxybenzoic acid and cranberry extract with resvera-
trol (Table 3). With respect to S. aureus, test compound
anti-biofilm activity (Figure 1) was only enhanced when
cranberry extract was combined with p-hydroxybenzoic
acid or protocatechuic acid (Table 3). Surprisingly, the
biofilm inhibitory effect in the presence of genistein
(Figure 1) disappeared in the presence of resveratrol, pro-
tocatechuic acid or p-hydroxybenzoic acid (Table 3).
Moreover, the anti-biofilm effect caused by protocateuchic
acid (Figure 1) disappeared in the presence of resveratrol,
p-hydroxybenzoic acid or resveratrol (Table 3). In these
cases, an antagonistic anti-biofilm effect may be the cause.
Nevertheless, these results confirm that the activity of the
molecules involved in bacterial biofilm metabolism can be
modified by the presence of plant metabolites and confirm
that this effect is molecule- and species-specific.
Moreover, these data support the hypothesis of Lee
et al. (2013) that plants have developed diverse mecha-
nisms of action to regulate biofilm formation and to
control bacterial proliferation.
The ability of some non-toxic plant molecules to
inhibit or enhance biofilm synthesis by the pathogen
S. aureus or the commensal S. epidermidis, respectively
(see Figure 1), creates the possibility of developing
non-aggressive and non-toxic therapeutic strategies to
modulate bacterial proliferation. As described in the
Introduction, a well-balanced microbiota is essential to
protect against pathogens and to prevent infections. In
this sense, S. epidermidis, which is recognised as a
mutualistic skin microorganism, plays an important role
in balancing the epithelial microbiota (Otto 2009;
Christensen & Brüggemann 2013). On the other hand,
skin colonisation by S. aureus is very common in atopic
dermatitis (Wolf & Wolf 2012; Murillo & Raoult 2013),
and it is correlated with cutaneous inflammation and
severity of eczema (Williams et al. 1990). In general, an
imbalance of the skin microbiota is associated with the
development of various epithelial diseases such as acne
vulgaris, rosacea, seborrhoeic dermatitis or atopic derma-
titis (Grice & Segre 2011; Murillo & Raoult 2013). The
results presented in this work reveal that, as a prebiotic
function, the presence of specific non-toxic plant metabo-
lites can regulate epithelial Staphylococcus proliferation,
and can be useful also in the maintenance of a balanced
skin microbiota and in the prevention and treatment
of specific epithelial dysbiosis related with S. aureus
and S. epidermidis. Moreover, since the presence of
S. epidermidis inhibits nasal colonisation and biofilm for-
mation by S. aureus (Iwase et al. 2010), the addition of
these non-toxic plant metabolites may enhance the effec-
tiveness of this bacterial interference mechanism.
Similar to the potential application of plant extracts
to eradicate the biofilm generated by P. acnes (Coenye
et al. 2012), it is possible to promote the proliferation of
S. epidermidis and to inhibit the growth of S. aureus by
the presence of genistein, protocatechuic acid, cranberry
extract, p-hydroxybenzoic acid or resveratrol (individu-
ally or in different combinations; Figure 1and Table 3),
to maintain or to restore the balance of skin microbiota
and to prevent skin bacterial infections.
In summary, the present study confirms the anti-
staphylococcal activity and biofilm regulation effects
shown by GRAS, non-toxic plant metabolites, which
have been traditionally used as dietary constituents due
to their health-promoting properties (eg antioxidant, anti-
inflammatory, analgesic, anticarcinogenic, antimutagenic,
antigenotoxic, antiulcerogenic, and antidiarrheic) (Cowan
1999; Gyawali & Ibrahim 2012; Kamatou et al. 2012).
Moreover, their potential use is proposed for both the
maintenance of a balanced skin microbiota and the treat-
ment or prevention of nosocomial and epithelial infec-
tions such as atopic dermatitis. Nevertheless, more
investigation is currently in progress to confirm the
in vivo efficacy of these plant molecules.
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