International Journal of Antimicrobial Agents 36 (2010) 129131

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International Journal of Antimicrobial Agents

journal homepage: http://www.elsevier.com/locate/ijantimicag

Antimicrobial activity of the green tea polyphenol ()-epigallocatechin-3-gallate

(EGCG) against clinical isolates of Stenotrophomonas maltophilia
Nicola C. Gordon a , David W. Wareham a,b,
a

Division of Infection, Barts & The London NHS Trust, London, UK

Centre for Immunology and Infection, Blizard Institute of Cell and Molecular Science, Barts & The London School of Medicine and Dentistry,
Queen Mary University of London, London, UK
b

a r t i c l e

i n f o

Article history:
Received 5 March 2010
Accepted 17 March 2010
Keywords:
Stenotrophomonas maltophilia
EGCG

a b s t r a c t
Stenotrophomonas maltophilia is increasingly recognised as an important nosocomial pathogen. Treatment options are limited due to intrinsic resistance to many antibiotics as well as concerns over toxicity of
the mainstay of treatment, co-trimoxazole. Epigallocatechin-3-gallate (EGCG), the major catechin found
in green tea, has been shown to have antimicrobial effects against a number of bacterial pathogens. We
evaluated the in vitro activity of this compound against 40 clinical isolates of S. maltophilia. MIC50/90 values
(minimal inhibitory concentrations for 50% and 90% of the organisms, respectively) were 256 mg/L when
determined by agar dilution and 512 mg/L by broth microdilution. MBC50/90 values (minimal bactericidal
concentrations for 50% and 90% of the organisms, respectively) were 512 mg/L. In timekill assays, the
bactericidal activity of EGCG was analysed by viable colony counts as well as a colorimetric assay for bacterial reduction of XTT. EGCG was slowly bactericidal at 4 MIC, with a 2.5 log reduction in viable bacteria
at 24 h. EGCG has promising in vitro antimicrobial activity against S. maltophilia. Although the mechanism
of action is not yet clear, further studies to evaluate its clinical potential and role in combination with
other antimicrobial agents are warranted.
2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction
Stenotrophomonas maltophilia is a non-fermentative Gramnegative bacillus that has gained increasing importance as an
important nosocomial pathogen in recent years. It is a recognised
cause of skin and soft-tissue, respiratory, bloodstream and prosthetic device infections, especially in immunocompromised and
critically ill patients [1]. The organism has also been implicated in a
number of outbreaks secondary to environmental sources [24].
Although S. maltophilia is considered to have relatively limited
pathogenic potential, when infection does occur treatment is complicated by intrinsic resistance to a wide range of antimicrobials
[5] and methodological difculties in performing susceptibility
testing [1]. At present, the mainstay of treatment is trimethoprim/sulfamethoxazole (co-trimoxazole), an antibiotic whose use
for other infections has declined due to problems with toxicity
and intolerance. Surveillance studies are few, but the emergence of
resistance to co-trimoxazole has been reported, in part mediated by
the acquisition of genes encoding sulphonamide resistance (sul1/2)
which reside on mobile genetic elements [6]. Alternative treatment

options are limited to ticarcillin/clavulanic acid, minocycline, the

newer uoroquinolones (moxioxacin) and possibly tigecycline
[7]. As with all multidrug-resistant (MDR) Gram-negative bacteria, there is an urgent need to identify new agents with promising
activity that can be exploited and developed further.
Epigallocatechin-3-gallate (EGCG) is the major polyphenol
found in the leaves of Camellia sinensis (tea), present in particularly high concentrations in green (unfermented) tea. It has
been reported to have a variety of health benets, including
anticancer and antimicrobial properties [8,9]. Signicant antimicrobial activity in vitro has been demonstrated against a variety
of Gram-positive, Gram-negative and fungal pathogens, including
meticillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii [10]. Consequently, it is a possible candidate for
development as a treatment for infections caused by other MDR
organisms. In this study, we evaluated the antimicrobial activity of
EGCG against a collection of S. maltophilia clinical isolates.

2. Methods
2.1. Characterisation of bacterial isolates

Corresponding author. Present address: Centre for Immunology and Infection,

Blizard Institute of Cell and Molecular Science, 4 Newark Street, Whitechapel,
London E1 2AT, UK. Tel.: +44 20 7882 2317; fax: +44 20 7882 2181.
E-mail address: d.w.wareham@qmul.ac.uk (D.W. Wareham).

Forty isolates of S. maltophilia were obtained from clinical specimens of patients treated at Barts and The London NHS Trust
(London, UK) over an 18-month period. The collection included

0924-8579/$ see front matter 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2010.03.025

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N.C. Gordon, D.W. Wareham / International Journal of Antimicrobial Agents 36 (2010) 129131

strains involved in respiratory, wound and bloodstream infections. Isolates were identied by biochemical proling using API
20E (bioMrieux, France) and the MicroScan WalkAway system
(Siemens Healthcare Diagnostics, Deereld, IL). Susceptibility testing to routine antimicrobials was performed by the British Society
for Antimicrobial Chemotherapy (BSAC) disk diffusion method
[11] and with the Negative Combo 42 panel on the MicroScan.
Stenotrophomonas maltophilia ATCC 10258 was used as a representative type strain.
2.2. In vitro susceptibility testing of EGCG
Stock solutions (10 000 mg/L) of EGCG (Sigma-Aldrich, St Louis,
MO) were prepared in dimethyl sulphoxide (DMSO) and diluted in
sterile distilled water. A series of IsoSensitest agar plates (Oxoid,
Basingstoke, UK) supplemented with EGCG at 0256 mg/L was
prepared to enable minimal inhibitory concentrations (MICs) to
be determined by agar dilution. Plates were inoculated with 104
colony-forming units (CFU)/mL of S. maltophilia (1 L of a 1 in 10
dilution of a 0.5 McFarland suspension) prepared in sterile distilled
water. Broth microtitre dilution tests were performed in 96-well
plates using IsoSensitest broth supplemented with 0512 mg/L
EGCG and an inoculum of 105 CFU/mL. All susceptibility tests were
read after 18 h incubation at 30 C in air. Minimal bactericidal concentrations (MBCs) were determined by transferring 10 L aliquots
from wells without visible signs of growth in the broth microtitre
dilution plates into fresh media without EGCG and incubating them
for a further 18 h.

Fig. 1. Effect of 200 g of ()-epigallocatechin-3-gallate (EGCG) applied to a blank

lter paper disk on an IsoSensitest agar plate inoculated with Stenotrophomonas
maltophilia ATCC 10258.

2.3. Timekill assays

The killing kinetics of EGCG at 1, 2 and 4 MIC were determined against S. maltophilia ATCC 10258. Viable bacterial counts
were performed after 0, 2.5, 5, 10 and 24 h incubation by plating serial 10-fold dilutions of broth cultures onto IsoSensitest agar
and incubating for 24 h. A timekill assay was also performed
using bacterial 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2Htetrazolium-5-carboxanilide (XTT) metabolism as a marker of cell
viability [12]. At each time point, cells were harvested from 1 mL
aliquots of culture, washed with 1 mL of phosphate-buffered saline
(PBS) and mixed with 1 mL of 0.5 mg/mL XTT (Sigma) in PBS supplemented with 50 M menadione. Following incubation for 2 h at
37 C, XTT reduction was quantied colorimetrically by absorbance
at 492 nm measured on a VersaMaxTM tuneable microplate reader
(Molecular Devices Limited, Surrey, UK).
3. Results
3.1. Characterisation of isolates
Of the 40 isolates collected, 19 were from respiratory samples, 8
from bloodstream infections, 7 from catheter tips, 3 from wounds,
2 from drain uids and 1 from an ileal biopsy. One isolate appeared
susceptible to trimethoprim by disk diffusion (>20 mm zone of
inhibition surrounding a 2.5 g trimethoprim disk), whilst the
remaining 39 isolates gave no zone of inhibition. Six isolates were
also identied as resistant to co-trimoxazole using the MicroScan
WalkAway system, and this was conrmed by disk diffusion susceptibility testing. Stenotrophomonas maltophilia ATCC 10258 was
resistant to trimethoprim and sensitive to co-trimoxazole.

Fig. 2. Timekill curves showing the effect of ()-epigallocatechin-3-gallate (EGCG)

at concentrations of 0, 256, 512 and 1024 mg/L (0, 1, 2 and 4 MIC) on
Stenotrophomonas maltophilia ATCC 10258 by the plate colony count technique. MIC,
minimal inhibitory concentration; CFU, colony-forming units.

strains at a concentration of 256 mg/L. One isolate was inhibited at

128 mg/L. Using broth microtitre dilution, 2 isolates were inhibited
at 128 mg/L, 3 isolates at 256 mg/L and 34 isolates at 512 mg/L; the
MIC was >512 mg/L for a single isolate. The MIC50/90 values (MIC
for 50% and 90% of the organisms, respectively) were 256 mg/L by
agar dilution and 512 mg/L by broth microtitre dilution. The MBC
was 128 mg/L for 2 isolates, 256 mg/L for 3 isolates and 512 mg/L
for the remaining 35 isolates. EGCG MICs of the clinical strains
were similar to those of S. maltophilia ATCC 10258 and there was
no correlation between the MIC of EGCG and resistance to cotrimoxazole.
3.3. Timekill assays
Timekill curves generated by viable plate counts and XTT
reduction are shown in Figs. 2 and 3, respectively. EGCG appeared
to be slowly bactericidal at 4 MIC, with an approximate 2.5 log
reduction in CFU after 24 h incubation.
4. Discussion

3.2. Susceptibility to EGCG

In agar diffusion assays, zones of inhibition of up to 25 mm
were observed around blank lter paper disks containing EGCG
solution (Fig. 1). By agar dilution, EGCG inhibited growth of all

The antimicrobial activity of EGCG has been demonstrated previously against a variety of organisms, including Mycobacterium
tuberculosis [13], pathogenic yeasts [14] and most recently A. baumannii [10]. Our data conrm that S. maltophilia is also susceptible

N.C. Gordon, D.W. Wareham / International Journal of Antimicrobial Agents 36 (2010) 129131

131

()-epicatechin gallate also lead to conformational changes [23],

with a reduction in oxacillin MICs for both meticillin-sensitive and
-resistant strains of S. aureus. The effect of EGCG/antimicrobial
combinations against multiresistant Gram-negative pathogens is
therefore another avenue in need of further investigation.
Funding: No funding sources.
Competing interests: None declared.
Ethical approval: Not required.
References

Fig. 3. Timekill curves showing the effect of ()-epigallocatechin-3-gallate (EGCG)

at concentrations of 0, 256, 512 and 1024 mg/L (0, 1, 2 and 4 MIC) on
Stenotrophomonas maltophilia ATCC 10258 by quantication of XTT reduction. MIC,
minimal inhibitory concentration; OD492, optical density at 492 nm.

to EGCG, with the MIC90 being considerably lower than for A. baumannii (256 mg/L vs. 625 mg/L).
The precise mechanism of action of EGCG remains unclear as
it appears to have a variety of inhibitory effects on bacteria. It has
been shown to cause membrane disruption both in Gram-negative
and Gram-positive organisms [15] and it can inhibit bacterial DNA
gyrase, preventing DNA supercoiling and leading to bacterial cell
death [16]. Overall, the MICs for Gram-negative organisms are
higher than those for Gram-positive organisms, an effect that may
be due to differences in cell wall lipopolysaccharide charge [15].
It has also been proposed that EGCG has antifolate activity similar
to that of trimethoprim, which may be responsible for its activity against S. maltophilia [17]. As no difference was observed in
the MIC of EGCG against trimethoprim-resistant or co-trimoxazoleresistant S. maltophilia and S. maltophilia ATCC 10258, we suspect
that additional mechanisms are involved. This is supported by the
effect of EGCG on A. baumannii, an organism intrinsically resistant
to trimethoprim.
Although EGCG reaches detectable levels in serum following
oral administration of green tea powder [18], concern has been
raised about the potential for toxicity when administered at high
dose [19]. It may also be difcult to achieve adequate serum concentrations for treatment of Gram-negative infections in its current
form. However, it is unlikely that there would be sufcient absorption to give rise to toxicity if applied topically. Green tea extract
has been used safely on skin to protect against ultraviolet damage
[20], suggesting that EGCG can be used topically without signicant
epithelial injury. However, further data are required regarding its
effect at a higher concentration. EGCG may also help to accelerate
wound healing owing to antioxidant and free-radical scavenging
properties [21], making it an ideal candidate for topical application
to infected wounds and burns. Alternatively, there may be potential for development as a surface disinfectant or antibacterial hand
rub. A further possibility might be inhaled therapy for the treatment of lower respiratory tract infections, particularly in patients
at high risk of multiresistant infections such as individuals with
cystic brosis or ventilated patients.
Of particular interest is the effect of EGCG on susceptibility to
other antibiotics. In S. aureus, signicant synergy with -lactams
against MRSA has been observed. This effect is blocked by the addition of peptidoglycan, suggesting that there may be direct binding
of EGCG to peptidoglycan residues within the cell wall leading to
conformational changes that increase susceptibility to -lactams
[22]. Alterations to cell wall teichoic acids by the related compound

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