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International Journal of Antimicrobial Agents 36 (2010) 129131

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

journa l homepage: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

ntimicrobial activity of the green tea polyphenol ()-epigallocatechin-3-gallateEGCG) against clinical isolates of Stenotrophomonas maltophilia

icola C. Gordona, David W. Warehama,b,

Division of Infection, Barts & The London NHS Trust, London, UKCentre for Immunology and Infection, Blizard Institute of Cell and Molecular Science, Barts & The London School of Medicine and Dentistry,ueen Mary University of London, London, UK

r t i c l e i n f o

rticle history:eceived 5 March 2010ccepted 17 March 2010

eywords:tenotrophomonas maltophiliaGCG

a b s t r a c t

Stenotrophomonas maltophilia is increasingly recognised as an important nosocomial pathogen. Treat-ment options are limiteddue to intrinsic resistance tomany antibiotics aswell as concerns over toxicity ofthe mainstay of treatment, co-trimoxazole. Epigallocatechin-3-gallate (EGCG), the major catechin foundin green tea, has been shown to have antimicrobial effects against a number of bacterial pathogens. Weevaluated the in vitro activity of this compoundagainst 40 clinical isolates of S.maltophilia.MIC50/90 values(minimal inhibitory concentrations for 50% and 90% of the organisms, respectively) were 256mg/L whendetermined by agar dilution and 512mg/L by brothmicrodilution. MBC values (minimal bactericidal50/90concentrations for 50% and 90% of the organisms, respectively) were 512mg/L. In timekill assays, thebactericidal activity of EGCGwas analysed by viable colony counts as well as a colorimetric assay for bac-terial reduction of XTT. EGCGwas slowly bactericidal at 4MIC,with a 2.5 log reduction in viable bacteriaat 24h. EGCGhas promising in vitro antimicrobial activity against S.maltophilia. Although themechanism

furthts arelsevieof action is not yet clear,other antimicrobial agen

2010 E

. Introduction

Stenotrophomonas maltophilia is a non-fermentative Gram-egative bacillus that has gained increasing importance as anmportant nosocomial pathogen in recent years. It is a recognisedause of skin and soft-tissue, respiratory, bloodstream and pros-hetic device infections, especially in immunocompromised andritically ill patients [1]. The organism has also been implicated in aumber of outbreaks secondary to environmental sources [24].lthough S. maltophilia is considered to have relatively limitedathogenic potential, when infection does occur treatment is com-licated by intrinsic resistance to a wide range of antimicrobials5] and methodological difficulties in performing susceptibilityesting [1]. At present, the mainstay of treatment is trimetho-rim/sulfamethoxazole (co-trimoxazole), an antibiotic whose useor other infections has declined due to problems with toxicity

nd intolerance. Surveillance studies are few, but the emergence ofesistance to co-trimoxazolehas been reported, inpartmediatedbyhe acquisition of genes encoding sulphonamide resistance (sul1/2)hich reside onmobile genetic elements [6]. Alternative treatment

Corresponding author. Present address: Centre for Immunology and Infection,lizard Institute of Cell and Molecular Science, 4 Newark Street, Whitechapel,ondon 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).

924-8579/$ see front matter 2010 Elsevier B.V. and the International Society of Chemoi:10.1016/j.ijantimicag.2010.03.025er studies to evaluate its clinical potential and role in combination withwarranted.r B.V. and the International Society of Chemotherapy. All rights reserved.

options are limited to ticarcillin/clavulanic acid, minocycline, thenewer fluoroquinolones (moxifloxacin) and possibly tigecycline[7]. As with all multidrug-resistant (MDR) Gram-negative bacte-ria, there is an urgent need to identify new agents with promisingactivity that can be exploited and developed further.

Epigallocatechin-3-gallate (EGCG) is the major polyphenolfound in the leaves of Camellia sinensis (tea), present in par-ticularly high concentrations in green (unfermented) tea. It hasbeen reported to have a variety of health benefits, includinganticancer and antimicrobial properties [8,9]. Significant antimi-crobial activity in vitro has been demonstrated against a varietyof Gram-positive, Gram-negative and fungal pathogens, includingmeticillin-resistant Staphylococcus aureus (MRSA) and MDR Acine-tobacter baumannii [10]. Consequently, it is a possible candidate fordevelopment as a treatment for infections caused by other MDRorganisms. In this study, we evaluated the antimicrobial activity ofEGCG against a collection of S. maltophilia clinical isolates.

2. Methods2.1. Characterisation of bacterial isolates

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

otherapy. All rights reserved.

http://www.sciencedirect.com/science/journal/09248579http://www.elsevier.com/locate/ijantimicagmailto:d.w.wareham@qmul.ac.ukdx.doi.org/10.1016/j.ijantimicag.2010.03.025

1 ournal of Antimicrobial Agents 36 (2010) 129131

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Fig. 1. Effect of 200g of ()-epigallocatechin-3-gallate (EGCG) applied to a blankfilter paper disk on an IsoSensitest agar plate inoculated with Stenotrophomonasmaltophilia ATCC 10258.

Fig. 2. Timekill curves showing the effect of ()-epigallocatechin-3-gallate (EGCG)30 N.C. Gordon, D.W. Wareham / International J

trains involved in respiratory, wound and bloodstream infec-ions. Isolates were identified by biochemical profiling using API0E (bioMrieux, France) and the MicroScan WalkAway systemSiemens Healthcare Diagnostics, Deerfield, IL). Susceptibility test-ng to routine antimicrobials was performed by the British Societyor Antimicrobial Chemotherapy (BSAC) disk diffusion method11] and with the Negative Combo 42 panel on the MicroScan.tenotrophomonas maltophilia ATCC 10258 was used as a represen-ative type strain.

.2. In vitro susceptibility testing of EGCG

Stock solutions (10000mg/L) of EGCG (Sigma-Aldrich, St Louis,O) were prepared in dimethyl sulphoxide (DMSO) and diluted in

terile distilled water. A series of IsoSensitest agar plates (Oxoid,asingstoke, UK) supplemented with EGCG at 0256mg/L wasrepared to enable minimal inhibitory concentrations (MICs) toe determined by agar dilution. Plates were inoculated with 104

olony-forming units (CFU)/mL of S. maltophilia (1L of a 1 in 10ilution of a 0.5McFarland suspension) prepared in sterile distilledater. Broth microtitre dilution tests were performed in 96-welllates using IsoSensitest broth supplemented with 0512mg/LGCG and an inoculum of 105 CFU/mL. All susceptibility tests wereead after 18h incubation at 30 C in air. Minimal bactericidal con-entrations (MBCs)were determined by transferring 10L aliquotsrom wells without visible signs of growth in the broth microtitreilution plates into freshmediawithout EGCG and incubating themor a further 18h.

.3. Timekill assays

The killing kinetics of EGCG at 1, 2 and 4 MIC were deter-ined against S. maltophilia ATCC 10258. Viable bacterial countsere performed after 0, 2.5, 5, 10 and 24h incubation by plat-

ng serial 10-fold dilutions of broth cultures onto IsoSensitest agarnd incubating for 24h. A timekill assay was also performedsing bacterial 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-etrazolium-5-carboxanilide (XTT) metabolism as a marker of celliability [12]. At each time point, cells were harvested from 1mLliquots of culture, washedwith 1mL of phosphate-buffered salinePBS) and mixed with 1mL of 0.5mg/mL XTT (Sigma) in PBS sup-lemented with 50Mmenadione. Following incubation for 2h at7 C, XTT reductionwas quantified colorimetrically by absorbancet 492nm measured on a VersaMaxTM tuneable microplate readerMolecular Devices Limited, Surrey, UK).

. Results

.1. Characterisation of isolates

Of the 40 isolates collected, 19were from respiratory samples, 8rom bloodstream infections, 7 from catheter tips, 3 from wounds,from drain fluids and 1 from an ileal biopsy. One isolate appearedusceptible to trimethoprim by disk diffusion (>20mm zone ofnhibition surrounding a 2.5g trimethoprim disk), whilst theemaining 39 isolates gave no zone of inhibition. Six isolates werelso identified as resistant to co-trimoxazole using the MicroScanalkAway system, and this was confirmed by disk diffusion sus-

eptibility testing. Stenotrophomonas maltophilia ATCC 10258 wasesistant to trimethoprim and sensitive to co-trimoxazole..2. Susceptibility to EGCG

In agar diffusion assays, zones of inhibition of up to 25mmere observed around blank filter paper disks containing EGCGolution (Fig. 1). By agar dilution, EGCG inhibited growth of allat concentrations of 0, 256, 512 and 1024mg/L (0, 1, 2 and 4 MIC) onStenotrophomonasmaltophiliaATCC10258by the plate colony count technique.MIC,minimal inhibitory concentration; CFU, colony-forming units.

strains at a concentration of 256mg/L. One isolate was inhibited at128mg/L. Using brothmicrotitre dilution, 2 isolates were inhibitedat 128mg/L, 3 isolates at 256mg/L and 34 isolates at 512mg/L; theMIC was >512mg/L for a single isolate. The MIC50/90 values (MICfor 50% and 90% of the organisms, respectively) were 256mg/L byagar dilution and 512mg/L by broth microtitre dilution. The MBCwas 128mg/L for 2 isolates, 256mg/L for 3 isolates and 512mg/Lfor the remaining 35 isolates. EGCG MICs of the clinical strainswere similar to those of S. maltophilia ATCC 10258 and there wasno correlation between the MIC of EGCG and resistance to co-trimoxazole.

3.3. Timekill assays

Timekill curves generated by viable plate counts and XTTreduction are shown in Figs. 2 and 3, respectively. EGCG appearedto be slowly bactericidal at 4 MIC, with an approximate 2.5 logreduction in CFU after 24h incubation.

4. DiscussionThe antimicrobial activity of EGCG has been demonstrated pre-viously against a variety of organisms, including Mycobacteriumtuberculosis [13], pathogenic yeasts [14] and most recently A. bau-mannii [10]. Our data confirm that S. maltophilia is also susceptible

N.C. Gordon, D.W. Wareham / International Journa

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

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

o EGCG, with the MIC90 being considerably lower than for A. bau-annii (256mg/L vs. 625mg/L).The precise mechanism of action of EGCG remains unclear as

t appears to have a variety of inhibitory effects on bacteria. It haseen shown to cause membrane disruption both in Gram-negativend Gram-positive organisms [15] and it can inhibit bacterial DNAyrase, preventing DNA supercoiling and leading to bacterial celleath [16]. Overall, the MICs for Gram-negative organisms areigher than those for Gram-positive organisms, an effect that maye due to differences in cell wall lipopolysaccharide charge [15].t has also been proposed that EGCG has antifolate activity similaro that of trimethoprim, which may be responsible for its activ-ty against S. maltophilia [17]. As no difference was observed inheMICof EGCGagainst trimethoprim-resistant or co-trimoxazole-esistant S. maltophilia and S. maltophilia ATCC 10258, we suspecthat additional mechanisms are involved. This is supported by theffect of EGCG on A. baumannii, an organism intrinsically resistanto trimethoprim.

Although EGCG reaches detectable levels in serum followingral administration of green tea powder [18], concern has beenaised about the potential for toxicity when administered at highose [19]. It may also be difficult to achieve adequate serum con-entrations for treatment of Gram-negative infections in its currentorm. However, it is unlikely that there would be sufficient absorp-ion to give rise to toxicity if applied topically. Green tea extractas been used safely on skin to protect against ultraviolet damage20], suggesting that EGCG canbeused topicallywithout significantpithelial injury. However, further data are required regarding itsffect at a higher concentration. EGCG may also help to accelerateound healing owing to antioxidant and free-radical scavengingroperties [21], making it an ideal candidate for topical applicationo infected wounds and burns. Alternatively, there may be poten-ial for development as a surface disinfectant or antibacterial handub. A further possibility might be inhaled therapy for the treat-ent of lower respiratory tract infections, particularly in patientst high risk of multiresistant infections such as individuals withystic fibrosis or ventilated patients.

Of particular interest is the effect of EGCG on susceptibility tother antibiotics. In S. aureus, significant synergy with -lactams

gainstMRSA has been observed. This effect is blocked by the addi-ion of peptidoglycan, suggesting that there may be direct bindingf EGCG to peptidoglycan residues within the cell wall leading toonformational changes that increase susceptibility to -lactams22]. Alterations to cell wall teichoic acids by the related compound

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l 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/antimicrobialcombinations against multiresistant Gram-negative pathogens istherefore another avenue in need of further investigation.

Funding: No funding sources.Competing interests: None declared.Ethical approval: Not required.

References

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17] Navarro-Martnez MD, Navarro-Pern E, Cabezas-Herrera J, Ruiz-Gmez J,Garca-Cnovas F, Rodrguez-Lpez JN. Antifolate activity of epigallocatechingallate against Stenotrophomonas maltophilia. Antimicrob Agents Chemother2005;49:291420.

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21] Kim H, Kawazoe T, Han DW, Matsumara K, Tsutsumi S, Hyon SH. Enhancedwoundhealingbyanepigallocatechin-incorporatedcollagensponge indiabeticmice. Wound Repair Regen 2008;16:71420.22] ZhaoWH, AsanoN, Hu ZQ, Shimamura T. Restoration of antibacterial activity of-lactams by epigallocatechin gallate against -lactamase-producing speciesdepending on location of -lactamase. J Pharm Pharmacol 2003;55:73540.

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Antimicrobial activity of the green tea polyphenol ()-epigallocatechin-3-gallate (EGCG) against clinical isolates of Sten...IntroductionMethodsCharacterisation of bacterial isolatesIn vitro susceptibility testing of EGCGTimekill assays

ResultsCharacterisation of isolatesSusceptibility to EGCGTimekill assays

DiscussionReferences