RESEARCH ARTICLE

Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana

Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibio¢lm
producing and antiquorum sensing in Streptococcus pyogenes
Surasak Limsuwan1 & Supayang Piyawan Voravuthikunchai2
1
Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkla, Thailand; and 2Natural Products Research Center and
Department of Microbiology, Faculty of Science, Prince of Songkla University, Songkla, Thailand

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Correspondence: Supayang Piyawan Abstract
Voravuthikunchai, Natural Products Research
Center and Department of Microbiology,
Biofilm formation has been demonstrated as a potentially important mechanism
Faculty of Science, Prince of Songkla contributing to antibiotic treatment failure on Streptococcus pyogenes. It could play
University, Hat Yai, Songkla 90112, Thailand. a significant role in recurrent and chronic infections. Boesenbergia pandurata
Tel.: 166 7444 6661; fax: 166 7444 6661; (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton)
e-mail: [email protected] Hassk. have been previously reported from our laboratory as effective agents
against S. pyogenes. Therefore, in the present study, we observed the effect of these
Received 22 December 2007; revised 29 May plants on biofilm formation. The bacterial biofilms were quantified by safranin
2008; accepted 2 June 2008.
staining and absorbance at 492 nm. The results clearly demonstrated that all
First published online 9 July 2008.
subinhibitory concentrations [1/32–1/2 minimal inhibitory concentration (MIC)]
DOI:10.1111/j.1574-695X.2008.00445.x
of E. americana (7.81–125 mg mL 1) and R. tomentosa (0.24–7.81 mg mL 1) extracts
significantly prevented biofilm formation while 1/2MIC (7.81 mg mL 1) of
Editor: Ewa Sadowy B. pandurata extract produced this effect. The issue of antiquorum sensing of this
pathogenic bacterium has been further explored. A correlation between antiquor-
Keywords um-sensing and antibiofilm-producing activities was demonstrated. Strong in-
antibiofilm; antiquorum sensing; Streptococcus hibition on quorum sensing was displayed with the extract of R. tomentosa.
pyogenes ; Boesenbergia pandurata ; Eleutherine americana extract showed partial inhibition, while B. pandurata did
Eleutherine americana ; Rhodomyrtus not show this activity. By contrast, an assay of microbial adhesion to hydrocarbon
tomentosa .
revealed no changes in the cell-surface hydrophobicity of the treated organisms.
Active organisms with the ability to inhibit quorum sensing and biofilm formation
are worth studying as they may provide complimentary medicine for biofilm-
associated infections.

implanted devices remain a major medical problem (Hoyle

Introduction & Costerton, 1991; Costerton et al., 1999; Pajkos et al., 2004;
Many pathogenic bacteria with the ability to form biofilms Vickery et al., 2004).
are responsible for acute and chronic infections. Examples Streptococcus pyogenes is one of the most important
of biofilm-associated diseases are dental caries, gingivitis, human pathogens associated with extensive human morbid-
periodontitis, endocarditis and prostatitis (Hall-Stoodley ity worldwide. It causes primary infections of skin, throat
et al., 2004). In addition, implanted medical devices includ- and mucosal surfaces. Even though the infections are
ing intravenous catheters, artificial joints and cardiac pace- normally self-limited, antibiotic treatment is usually em-
makers can become rapidly coated with human extracellular ployed to relief discomfort, minimize transmission and
matrix and plasma proteins which are prime targets for reduce complications (Bisno et al., 1997). The newly emer-
bacterial biofilm formation (Costerton et al., 1999; Donlan ging S. pyogenes virulence trait potentially renders this
& Costerton, 2002; Parsek & Singh, 2003). Because of the important pathogen more resistant to antibiotic therapy
sharply decreased susceptibility of biofilm-forming bacteria and to innate as well as adaptive immune responses. Biofilm
to host defenses and antibiotic treatments, biofilms on formation is known to allow bacteria to become more

FEMS Immunol Med Microbiol 53 (2008) 429–436

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
430 S. Limsuwan & S.P. Voravuthikunchai

resistant to antibiotic treatment, and significantly impairs materials were dried at 60 1C overnight. They were crushed
antimicrobial therapy even in those cases caused by strains and soaked with extractive solvents for 7 days. The solvent
that are not resistant to the relevant antibiotics (Macris was then distilled under reduced pressure in a rotary
et al., 1998; Kuhn et al., 2001). Many forms of streptococcal evaporator until it became completely dry. The extracts were
infections, especially recurrent and chronic ones, are asso- dissolved in dimethyl sulfoxide (DMSO, Merck, Germany)
ciated with the formation of bacterial biofilm (Lembke before use.
et al., 2006).
Penicillin is the antibiotic of choice for S. pyogenes
infections based on its narrow spectrum of effect, efficacy, Bacterial strains
good safety profile and low cost (Bisno et al., 1997, 2002). Eleven clinical isolates of S. pyogenes (NPRC 101–111) were
However, penicillin treatment failure of S. pyogenes infec-

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isolated from patients with tonsillitis. A throat swab of each
tions has been demonstrated to be associated with biofilm patient was individually plated onto Columbia blood agar
formation (Conley et al., 2003). In patients with known or base (Oxoid) containing 5% sheep red blood cells.
suspected allergy to penicillin, erythromycin and other b-Haemolytic streptococcal-like colonies were subjected to
macrolides are considered as alternative treatment (Hooton, appropriate biochemical testing as described previously
1991; Stein et al., 1991; Adam & Scholz, 1996). Recently, (Forbes et al., 2002). All isolates were negative for catalase
macrolide resistance in S. pyogenes has been reported in using 3% H2O2, susceptible to bacitracin disc (0.04 units per
several countries (Eisner et al., 2006; Hsueh et al., 2006; disk), positive for pyrrolidonyl aminopeptidase reagent and
Littauer et al., 2006). Because of the increasing resistance of produced no growth on 6.5% NaCl agar and bile esculin
S. pyogenes to antibiotics, much effort is being exerted to hydrolysis agar. The isolates were stored in brain heart
identify novel compounds with antibacterial activity and to infusion (BHI) broth (Difco Laboratories, Detroit, MI)
analyse their mechanisms of action. Specifically, there is a containing 5% glycerol at 70 1C until use. Chromobacter-
critical need to identify therapeutic strategies which are ium violaceum DMST 21761 was purchased from the
directed towards the inhibition of biofilm formation and National Institute of Health (NIH), Department of Medical
effective treatment of biofilms once they have formed. Sciences, Ministry of Public Health, Thailand. All strains
Quorum sensing molecules have been shown to be essential were routinely grown in BHI broth (Difco, France) or
for biofilm formation (Chen et al., 2004; Hornby & Nick- trypticase soy agar (TSA, Difco, France) plates.
erson, 2004). Quorum sensing is a strategy of cell–cell
communication favouring the biofilm community by reg-
ulating unnecessary overpopulation and nutrient competi- Determination of minimal inhibitory
tion with important implications for the infectious process concentration (MIC)
(Davey & O’Toole, 2000; Douglas, 2003). A modified broth microdilution method outlined by the
Many plants have been reported to demonstrate anti- Clinical and Laboratory Standards Institute (CLSI, 2006)
quorum-sensing activity (Rasmussen et al., 2005; Adonizio was performed. The bacterial suspensions (105 CFU mL 1)
et al., 2006; Choo et al., 2006). Our preliminary screening on were added to BHI broth supplemented with the plant
a wide range of Thai plant species demonstrated that extracts serially diluted twofold to give final concentrations
Boesenbergia pandurata (Roxb.) Schltr., Eleutherine ameri- ranging from 0.5 to 1000 mg mL 1 and incubated at 37 1C for
cana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. had 20 h. The MIC was recorded as the lowest concentration that
good antibacterial activities on S. pyogenes (Voravuthi- produced complete suppression of visible growth.
kunchai et al., 2007). The aim of the present study was
to investigate the effect of these effective plants on bio-
Biofilm assays
film development and on quorum-sensing activity. Aspects
on their effects on cell-surface hydrophobicity (CSH) of All S. pyogenes isolates were screened for biofilm production
S. pyogenes were also examined. according to the method of Lembke et al. (2006). Glass test
tubes containing BHI broth were supplemented with bacter-
ial suspensions (105 CFU mL 1), and the remaining plank-
Materials and methods tonic bacteria were removed by aspiration of the liquid after
desired incubation time points. The test tubes with biofilms
Medicinal plants
were washed four times with 0.85% normal saline solution
Three medicinal plants were used in this study. Classified (NSS) and stained in a 0.1% safranin solution for 30 min.
reference voucher specimens were deposited at the Herbarium The stained biofilms were washed four times with NSS and
of Faculty of Pharmaceutical Sciences, Prince of Songkla allowed to dry. The stained biofilms were removed from the
University, Hat Yai, Songkhla, Thailand. All of the plant tube surface by adding ethanol with vigorous vortexing, and

c 2008 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 53 (2008) 429–436
Published by Blackwell Publishing Ltd. All rights reserved
Antibiofilm agents for Streptococcus pyogenes 431

quantified in a spectrophotometer (Shimadzu UV-1601 Statistical analysis

Spectrophotometer, Japan) at 492 nm. Tubes containing
Statistical analysis was performed using SPSS. Values were
BHI were used as controls.
expressed as mean  SD. A Duncan-ANOVA test was used to
The effect of the plant extracts on biofilm formation of
compare the parameters between the groups and a Dunnett-
S. pyogenes was investigated by adding the extracts at
ANOVA test to compare between the tests and control.
subinhibitory concentrations into glass test tubes containing
BHI broth. Subsequently, the tubes were supplemented with
the bacterial suspension. After incubation, biofilm staining Results
was performed as described above. The tubes containing the
media and the extracts were tested as control. In parallel Screening for biofilm formation
experiments, unstained biofilm and planktonic bacteria

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Eleven isolates of S. pyogenes were preliminarily screened for
were mixed by vigorous vortexing, and bacterial growth biofilm production (Fig. 1). Most strains produced high
was quantified in a spectrophotometer at 660 nm. levels of biofilm during 24–48 h. Streptococcus pyogenes
For visualisation, biofilms were allowed to grow on glass NPRC 109, NPRC 110 and NPRC 111 were efficient
pieces in glass test tubes supplemented with the extracts. biofilm-producers (Duncan test, P o 0.05). Streptococcus
Following incubation, the glass pieces were washed four pyogenes NPRC 109 was the most efficient strain in terms
times with NSS and stained with 0.1% safranin solution. of biofilm production at 24 and 48 h, whereas S. pyogenes
Stained glass pieces were observed at a magnification of 40 NPRC 110 and 111 showed this efficacy at 24 h. However, we
using an Olympus CX-31 light microscope. decided to select 48 h for further study because the organ-
isms have recovered from any inhibitory effect. Differences
Antiquorum-sensing activity in biofilm levels indicated that there was a spectrum of
ability of different S. pyogenes isolates to form biofilms.
The paper disc agar diffusion method was used to detect
antiquorum-sensing activity of the extracts (Adonizio et al.,
2006). The extracts were dissolved in DMSO, 10 mL Biofilm assay with subinhibitory concentrations
(250 mg mL 1) of the crude extracts was applied to sterile of plant extracts
filter paper discs (Whatman no. 1; 6 mm in diameter) so The MICs of the plant extracts on S. pyogenes are shown in
that each disc was saturated with 2.5 mg of the extract. Table 1. Boesenbergia pandurata and R. tomentosa demon-
Dry discs (dried at 37 1C overnight) were applied to strated antibacterial activity with MIC values (7.81 mg mL 1)
the surface of TSA seeded with 3–5 h tripticase soy broth while E. americana inhibited the growth at an MIC value of
(TSB) culture of C. violaceum. The plates were incubated 250 mg mL 1. Plant extracts at subinhibitory concentrations
overnight at 37 1C and examined for violacein production. (1/2, 1/4, 1/8, 1/16 and 1/32MIC) were used for biofilm assay.
Quorum-sensing inhibition was detected by a colourless, Their effects on biofilm production are shown in Fig. 2.
opaque halo around the disc. DMSO was used as control. Eleutherine americana and R. tomentosa extracts at all

0.25
CSH testing
0.20 g
The effect on CSH of S. pyogenes was measured by microbial
adhesion to hydrocarbon (MATH) (Rozenberg et al., 1980).
A492 nm

0.15 f
Briefly, the bacterial cells grown in BHI broth with sub-
0.10 e
inhibitory concentrations of the plant extracts were washed d
c cd
twice and suspended in NSS so that their OD600 nm was 0.3. 0.05 ab ab ab b
a
The cell suspension (3 mL) was placed in tubes and 0.25 mL
0.00
of toluene was added. The tubes were agitated uniformly in a
PR 01

PR 2

PR 3

PR 4

PR 5

PR 6

PR 07

PR 08

PR 9

PR 0

1
10

10

10

10

10

10

11

11
1

vortex mixer for 2 min and allowed to equilibrate at room

C

C
PR

temperature for 10 min. After the toluene phase was sepa-

N

rated from the aqueous phase, the OD of the aqueous phase 24 h 48 h 72 h 96 h

was determined spectrophotometrically. Controls consisted
Fig. 1. Biofilm quantification of different strains of Streptococcus pyo-
of cells incubated with 1% DMSO. The hydrophobicity
genes (NPRC 101–111) to glass surfaces by standard safranin staining of
index (HPBI) was calculated as: OD initial OD final/OD potential biofilms and measuring absorbance at 492 nm. The mean
initial 100%. Streptococcus pyogenes with a HPBI 4 70% values of duplicate independent experiments and deviations are shown.
was arbitrarily classified as hydrophobic (Martin et al., 1989; Duncan test demonstrates significant difference in biofilm production
Nostro et al., 2004). (P o 0.05).

FEMS Immunol Med Microbiol 53 (2008) 429–436

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
432 S. Limsuwan & S.P. Voravuthikunchai

Table 1. MIC of crude plant extracts on Streptococcus pyogenes NPRC 109

Specimen no. Botanical species Plant part tested % Extract yield MIC (mg mL 1)
NPRC0007 Boesenbergia pandurata (Roxb.) Schltr. (Zingiberaceae) Rhizome 1.58 (chloroform) 7.81
NPRC0044 Eleutherine americana Merr. (Iridaceae) Bulb 4.80 (ethanol) 250.00
NPRC0057 Rhodomyrtus tomentosa (Aiton) Hassk. (Myrtaceae) Leaf 7.40 (ethanol) 7.81

0.18 contrast, the control (1% DMSO) revealed a strong and

0.16 dense adherence after 48 h of incubation (Fig. 4g).
0.14
0.12
A492 nm

0.10 Antiquorum sensing

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0.08
0.06 Chromobacterium violaceum synthesizes the violet pigment
0.04
0.02 violaceine as a result of quorum sensing (Lichstein & Van
0.00 De Sand, 1946). Loss of purple pigment violaceine in
1/2MIC 1/4MIC 1/8MIC 1/16MIC 1/32MIC 1%DMSO
C. violaceum is an indicative of quorum-sensing inhibition
by the plant extracts (Fig. 5). The yellowish zone of inhibi-
Fig. 2. Effects of subinhibitory concentrations (1/2–1/32 MIC) of the tion observed was opaque rather than transparent, indicat-
plant extracts on the biofilm production by S. pyogenes NPRC 109 at
ing that the halo around the disc was caused by inhibition of
48 h, quantified by safranin staining and subsequently by measuring
quorum sensing, not inhibition of cell growth. The anti-
absorbance at 492 nm. The mean values of duplicate independent
experiments and SDs are shown. Dunnett test demonstrates significant quorum-sensing activity was dose-dependent (data not
difference between the tests and the control (P o 0.05). shown). Strong quorum-sensing inhibition was observed in
the extract of R. tomentosa (Fig. 5c). Eleutherine americana
showed partial inhibition with an incomplete zone (Fig. 5b).
1.20
By contrast, B. pandurata did not demonstrate this activity
1.00 ∗ (Fig. 5a).
0.80 ∗
A660 nm

0.60
CSH
0.40

0.20 The effect of subinhibitory concentrations of the plant

0.00 extracts on CSH of S. pyogenes was investigated (Fig. 6).
1/2MIC 1/4MIC 1/8MIC 1/16MIC 1/32MIC 1%DMSO
The growth of S. pyogenes NPRC 109 cells in the presence of
subinhibitory concentrations of all plant extracts resulted in
high CSH (HPBIs were 95.8–98.8%). However, no signifi-
Fig. 3. Subinhibitory (1/2–1/32 MIC) effects of the plant extracts on the
cant differences in HPBIs were observed between the tests
growth of S. pyogenes NPRC 109. Bacterial growth was quantified by
measuring absorbance at 660 nm at 48 h. The mean values of duplicate
and control (HPBI was 97.3%).
independent experiments and SDs are shown. Dunnett test demon-
strates significant difference between the tests and the control
(P o 0.05). Discussion
Very few S. pyogenes are nonbiofilm formers, up to 90% (289
concentrations significantly inhibited biofilm formation of strains) (Baldassarri et al., 2006) and 100% (99 strains)
S. pyogenes (Dunnett-ANOVA test, P o 0.05). In contrast, B. (Conley et al., 2003) were able to form biofilm. Biofilm
pandurata extract demonstrated this effect only at 1/2 MIC. production by this organism is a recent discovery and has
At almost all subinhibitory concentrations, growth of S. not been well documented. Lembke et al. (2006) reported
pyogenes was at the same level as that of the control (Fig. 3). that serotype M2 strain was connected by thread-like
This result suggested that inhibition of biofilm formation structures of an as-yet-unknown chemical composition.
did not result from inhibition of cell growth. There was no other report on extracellular polymeric sub-
stance or exopolysaccharide production.
Many factors influence biofilm production, including the
Evaluation and detection of biofilm formation
ability to adhere to the substratum (Donlan, 2002), quorum
by microscopy
sensing (Li et al., 2001) growth rate (Sissons et al., 1995;
Streptococcus pyogenes NPRC 109 did not form dense Rozen et al., 2001) and anaerobiosis (Baldassarri et al., 2006).
biofilm layers when treated with subinhibitory concentra- Antibiofilm activities have been demonstrated in a number of
tions (1/2 and 1/4 MIC) of the extracts (Fig. 4a–f). In medicinal plants (Limsong et al., 2004; Duarte et al., 2006;

c 2008 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 53 (2008) 429–436
Published by Blackwell Publishing Ltd. All rights reserved
Antibiofilm agents for Streptococcus pyogenes 433

(a) (b) (c)

(d) (e) (f)

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(g)

Fig. 4. Inspection of biofilm development of S. pyogenes NPRC 109 on glass surfaces by light microscopy at a magnification of  40. Bacterial biofilms
grown in (a–c) 1/2 MIC and (d–f) 1/4 MIC of the plant extracts at 48 h. (a,d) Boesenbergia pandurata, (b,e) Eleutherine americana, (c,f) Rhodomyrtus
tomentosa and (g) 1% DMSO.

(a) (b)

(c) (d)

Fig. 5. Inhibition of violacein production by

(a) Boesenbergia pandurata, (b) Eleutherine
americana and (c) Rhodomyrtus tomentosa
extracts using Chromobacterium violaceum
DMST 21761 biomonitor strain and agar disc
diffusion method. The inhibition was detected
by a colourless, opaque halo around the discs.
(d) DMSO was used as control.

FEMS Immunol Med Microbiol 53 (2008) 429–436

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
434 S. Limsuwan & S.P. Voravuthikunchai

100 have demonstrated that many plant extracts can affect CSH
80 of Gram-negative bacteria including Escherichia coli (Turi
et al., 1997; Voravuthikunchai & Limsuwan, 2006), Acineto-
HPBI (%)

60
bacter baumannii (Turi et al., 1997), Helicobacter pylori
40
(Annuk et al., 1999) and Salmonella typhimurium (Das &
20 Devaraj, 2006), and Gram-positive bacteria such as Strepto-
0 coccus mutans (Nostro et al., 2004; Prabu et al., 2006; Rahim
1/2MIC 1/4MIC 1/8MIC 1/16MIC 1/32MIC 1%DMSO & Khan, 2006) Streptococcus sanguinis, Streptococcus mitis
Boesenbergia pandurata Eleutherine americana Rhodomyrtus tomentosa and Actinomyces sp. (Razak et al., 2006). Changes in
bacterial hydrophobicity resulted in a significant decrease
Fig. 6. The effect of subinhibitory concentrations (1/2–1/32 MIC) of the
in adhesion ability (Fonseca et al., 2004; Das & Devaraj,

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plant extracts on CSH of S. pyogenes NPRC 109 by microbial adhesion to
hydrocarbon (MATH) method. The hydrophobicity index (HPBI) was 2006; Razak et al., 2006) and may be associated with biofilm
calculated as: OD initial OD final/OD initial  100%. Streptococcus formation. However, our results showed no effect from all
pyogenes with a hydrophobic index 4 70% was arbitrarily classified as plant extracts on the CHS of S. pyogenes, irrespective of the
hydrophobic. significant decrease in biofilm production.

Cartagena et al., 2007; Kuzma et al., 2007). Berberine

sulphate, an alkaloid extracted from the roots
Conclusions
and bark of various plants, had been reported to interfere Our study described the inhibition of biofilm formation of
with the adherence of S. pyogenes by releasing the adhesin S. pyogenes by B. pandurata, E. americana and R. tomentosa.
lipoteichoic acid from the streptococcal cell surface and A correlation between antiquorum-sensing and antibiofilm-
directly preventing or dissolving lipoteichoic acid–fibronectin producing activities was demonstrated. Active principles
complexes (Sun et al., 1988). In the present study, the plant from these effective plant species are worth studying in
extracts possibly interfered at any step of S. pyogenes biofilm order to solve problems with biofilm-associated infections.
formation, but obviously did not inhibit growth at all
subinhibitory concentrations tested. Observation of the
architecture of S. pyogenes biofilms by light microscopy Acknowledgements
further confirmed that the bacteria did not form dense
This work was supported by the Thailand Research Fund:
biofilm layers when treated with subinhibitory concentra-
Royal Golden Jubilee, PhD grant (PHD/0029/2548), fiscal
tions of the extracts.
year 2005–2010 and Basic Research Grant (BRG 4880021),
Quorum sensing consists of the release and the reception
fiscal year 2005–2008.
of signalling molecules produced by bacteria within a given
population. This exchange of signals subsequently leads to
the induction of more signalling molecules and finally to the
acquisition of defined cellular characteristics. The signalling
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