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Synergistic anti-bacterial and proteomic effects of epigallocatechin gallate on clinical isolates of imipenem-resistant Klebsiella pneumoniae.



Imipenem-resistant Klebsiella pneumoniae (IRKP) were used to explore the synergistic anti-bacterial and proteomic effects of imipenem alone or in combination with epigallocatechin gallate (EGCg). The minimal inhibitory concentrations (MICs) of EGCG for 12 clinically isolated IRKP strains ranged from 300 to 650 [micro]g/ml. Each of the 12 IRKP strains experienced a 4- to 64-fold reduction in the MIC of imipenem upon co-incubation with 0.25 x MIC level of EGCg. The time-kill method was used on the 12 IRKP clinical isolates to evaluate the bactericidal activities of imipenem alone or with EGCg. Compared to imipenem alonet EGCg with imipenem demonstrated enhanced bactericidal activity. Two-dimensional polyacry-lamide gel electrophoresis identified eight down-regulated and four up-regulated proteins in the IRKP strain upon exposure to 1 x MIC of EGCg. Analysis of the outer membrane protein profiles of IRKP cultures treated with EGCg revealed unique changes in outer membrane proteins. In addition, scanning electron microscopic analysis demonstrated the presence of cells with wrinkled surfaces containing perforations and irregular rod-shaped forms after treatment with EGCg or imipenem. These studies demonstrate that EGCg can synergize the bacterial activity of imipenem and differentially stimulate the expression of various proteins in IRKP.

[c] 2011 Elsevier GmbH. All rights reserved.


Keywords: Epigallocatechin gallate Klebsiella pneumonia Imipenem resistance Anti-bacterial activity


Gram-negative pathogens such as Escherichia coll Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii are commonly associated with intra-abdominal sepsis and pneumonia in critical patients (Doumith et ah, 2009; Jung et al., 2004). Imipenem is recommended as a first-line therapy for multidrug-resistant gram-negative bacteria; however, resistance to imipenem has recently emerged (Ardanuy et al., 1998; Cao et al., 2000; Gulmez et al., 2008; Normann et al., 2009). The mechanism of imipenem resistance involves the production of specific carbapenemases as well as the loss of porin proteins (Domenech-Sanchez et al., 1999; Hesna et al., 2002; Yang et al., 2009). Imipenem resistance caused by the alteration in Hpopolysaccharide (LPS) levels has also been reported in Enterobacter aerogenes (Bornet et al., 2000; Leying et al., 1991). A significant increase in the prevalence of imipenem-resistant K. pneumoniae (IRKP) has been observed in common human pathogens. The rapid emergence of IRKP has limited the availability of anti-bacterial treatment options. The combination with a plant-derived anti-bacterial drug is the only alternative for the time being.

Tea polyphenols (TPP) have been widely reported to have antioxidant and anti-cancer effects as well as cardiovascular health benefits in human (Hara-Kudo et al., 2005; Hemaiswarya et al., 2008; Navarro-Martinez et al., 2006; Osterburg et al., 2009). Notably, TPP are effective anti-microbial compounds against a variety of pathogenic and antibiotic-resistant microorganisms (Zhao et al, 2003; Cho et al., 2008). A recent review discussed how EGCg, a main constituent of green tea catechins, is capable of acting synergistically with various [beta]-lactams against methicillin-resistant Staphylococcus aureus (Hemaiswarya et al., 2008). In the present study, we examined the synergistic anti-bacterial effects and the proteomic changes caused by EGCg and imipenem against IRKP clinical isolates.

Materials and methods

Bacterial strains and culture conditions

Twelve clinical isolates of K. pneumoniae were obtained from Soonchunhyang University hospital (Cheonan, Korea). The K. pneumoniae strain ATCC 700603 was used as an imipenem-susceptible control strain. Muller-Hinton broth (MHB) or Luria-Bertani broth (LB) was used for minimal inhibitory concentration (MIC) tests, and bacterial cultures were maintained at 37 [degrees] C for 18-20 h.

EGCg and antibiotics

EGCg (>90%) and imipenem were purchased from Merck Sharp and Dohme Corp (Rahway, NJ, USA). Imipenem was dissolved in 10mM phosphate buffer (pH 7.0). An e-test imipenem strip was purchased from AB Biodisk (Solna, Sweden).

Determination of MIC and FIC of imipenem and EGCg

The MICs of imipenem, EGCg and combined imipenem-EGCg for the K. pneumoniae clinical isolates were determined by the agar dilution method using MH agar. MH agar plates were also used for imipenem susceptibility tests. MIC was defined as the lowest drug concentration that inhibited bacterial growth. To measure the synergistic effect of EGCg and imipenem against IRKP strains, fractional inhibitory concentrations (FICs) were measured according to the following formula: (MIC of imipenem plus EGCg/MIC of imipenem alone) + (MIC of EGCg plus imipenem/MIC of EGCg alone). FIC indices were interpreted as synergistic when values were [less than equal to] 0.5, as additive or indifferent when values were > 0.5-4.0 and as antagonistic when values were >4.0.

Time-kill assays

The bactericidal activity was determined in duplicate using the time-kill method according to CLSI guidelines (1999). Imipenem-susceptible K. pneumoniae (ISKP) and IRKP were harvested, washed three times with phosphate buffer and then suspended in the same buffer to a final concentration of 5 x [10.sup.6] cfu/ml in 4 ml of MHB. Both imipenem and EGCg were used at different concentrations in the experiments. Viable cells were counted every 6h for 0-24 h. The time-kill assays were carried out in triplicate.

Preparation of outer membrane proteins

Outer membrane proteins were prepared by the sodium N-lauroyl sarcosinate-insoluble method (Domenech-Sanchez et al., 1999). Cell pellets were suspended 1 ml of 10 mM HEPES buffer (pH 7.4) at 4 [degrees] C and then sonicated with a Sonic Dismembrator (Fisher Scientific Co., USA). The unbroken cells and debris were removed by centrifugation at 15,000 x g for 2 min at 4 [degrees] C. The supernatant was transferred to a microcentrifuge tube, and total cell membranes were precipitated from the supernatant fluid by centrifugation at 15,000 x g for 30 min at 4 C. Total cell membrane pellets were then resuspended in 0.2 ml of 10 mM HEPES buffer (pH 7.4). The cytoplasmic membranes were solubilized by the addition of 0.2 ml of 2% sodium N-lauroyl sarcosinate in 10 mM HEPES buffer and then incubated at 25 [degrees] C for 30 min. After the supernatant fluid was removed by centrifugation, the outer membrane was washed with 0.5ml of lOmM HEPES buffer. The outer membranes were suspended in 50 [micro]of 10 mM HEPES buffer (pH 7.4).

Two-dimensional gel electrophoresis (2-DE)

Cultures of IRKP were grown overnight in MH medium in the presence or absence of 0.5 x MIC EGCg were centrifuged at 8500xg for 30min at 4[degrees]C to remove intact cells. Proteins in the culture supernatants were precipitated by the addition of 100% trichloroacetic acid to a final concentration of 10%. After overnight incubation at 4 [degrees] C, the precipitate was centrifuged at 10,000xg for 30min at 4 [degrees] C and then washed with ice-cold 100% acetone. Two-dimensional gel electrophoresis was performed according to previously described methods (Cho et al., 2007; Kurupati et al., 2006). Isoelectric focusing (IEF) was performed using the Ettan [TM] IPC phor System (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Proteins were dissolved in 350 [micro]l of rehydration buffer [8 M urea, 2% CHAPS (w/v) and 0.5% IPG buffer pH 3-10]. Isoelectric focusing was performed in seven steps: 300 V for 30 min, 500 V for 30 min, 1000 V for 1 h, 3000 V for 1.5 h, 5000 V 1.5 h, gradient 8000 V for 2 h and finally, 8000 V for 1 h. The second dimension was run on a 12% SDS polyacrylamide gel using a PROTEAN II xi electrophoresis kit (BIO-RAD, Hercules, CA, USA).

In-gel digestion and MALDI-TOF/MS

Protein spots were excised from silver-stained 2-DE gels, and then the peptides were digested with trypsin according to previously described methods (Cho et al., 2007; Perkins et al., 1999). Digested peptides were re-dissolved using 0.1% trifluoroacetic acid (TFA). To reduce the chemical background noise for MALDI-TOF, sample peptides were desalted using Zip-tip Gig pipette tips (Millipore, Bedford, MA, USA). Peptides were eluted onto a MALDI plate using a CHCA matrix solution (10 mg/ml CHCA in 0.5% TFA/50% acetonitrile, 1:1). All mass spectra were acquired in reflection mode using a 4700 Proteomics Analyzer (Applied Biosystems, Framing-ham, MA, USA). Afterwards, proteins were identified from MALDI fingerprint data using MASCOT ( against the NCBI database (Kurupati et al., 2006; Perkins et al., 1999).

SEM analysis ofK. pneumoniae exposed to EGCG and imipenem

Colonies of K. pneumoniae species that were grown on LB agar plates for 24 h were excised into small agar blocks of 0.5 cm3. Each of the colony containing agar blocks were exposed to EGCg and imipenem in phosphate buffer for 2 h at 37 C. Subsequently, the colonies were pre-and post-fixed and then dehydrated in an ethanol series of increasing concentration (30-95%) for 15min followed by 100% ethanol for 20min. The dehydrated cells were immersed in hexamethyldisilazane (Electron Microscopy Sciences, Ft. Washington, PA) for 1 h and then air-dried. The cells were coated with gold and examined by scanning electron microscopy (Joel Ltd., Japan).

Results and discussion

MICs of imipenem and EGCg against isolates and synergistic effect

Isolates were highly resistant to imipenem with the majority of the isolates having an MIC >8 [micro]g/ml. The MICs of imipenem and EGCg for the clinical isolates of K. pneumoniae are summarized in Table 1. The MICs of EGCg exposed to the IRKP isolates were ranged from 300 to 650 [micro]g/ml. When imipenem was combined with EGCg, a synergistic effect was observed at a concentration less than one-fourth of that of the MIC of EGCg alone. The MICs for imipenem were decreased up to 64-fold for all IRKP strains. These results demonstrate that EGCg was synergistic with imipenem for each of the 12 IRKP strains tested. This finding was consistent with previous reports that a special synergy effect can occur when antibiotics are combined with an agent that antagonizes bacterial resistance mechanisms (Wagner and Ulrich-Merzenich, 2009).
Table 1
The MICsand FIC indices of imipenem in combination with EGCg exposed to
12 IRKP isolates.

Strain  MIC([mu]/ml)                                    FIC Index
no                                                         (b)

            EGCg      Alone  With 0.25 x  With 0.5 x
                               MIC EGCg      MIC EGCg

1                450     32          0.5       0.125      0.265
2                350     32            1        0.25      0.281
3                650     32            4         0.5      0.375
4                300      8          0.5       <0.06      0.265
5                550     16            2       0.125      0.375
6                350     16          0.5       0.125      0.281
7                550     32            1        0.06      0.281
8                500      8            1       <0.06      0.375
9                350     16          0.5       0.125      0.281
10               600     32            2       <0.06      0.312
ii               6S0      8          0 S        0 06      0 312
12               500     16            1       <0.06      0.312
ATCC             300      1         0.06      ND (a)      0.310

(a) ND, not determined,
(b) FIC indices were interpreted as synergistic when values were
[less than equal to] 0.5, as additive or indifferent when values
were > 0.5-4.0.

Time-kill assays

The bactericidal effects of imipenem with EGCg on ISKP and IRKP were evaluated using time-kill curves (Fig. 1). In the absence of imipenem or EGCg, both ISKP and IRKP grew well. However, at 0.5 x MIC of imipenem, ISKP was more sensitive than IRKP, whereas IRKP was more sensitive than ISKP at 0.5 x MIC of EGCg. The decrease in viable counts of ISKP and IRKP was rapid with 0.5 x MIC of EGCg when compared to 0.25 x MIC of EGCg. However, the strongest growth inhibition was observed with the combination of 0.25 x MICofimipenemand0.5 x MIC of EGCg against IRKP; almost all of the IRKP cells were killed within 24 h of incubation. Similar experiments were performed for all of the IRKP clinical isolates. The results show that co-incubation of sub-MIC levels of EGCg and imipenem synergistically increased imipenem's killing activity towards all of the K. pneumoniae strains tested.


Expression of outer membrane proteins

The outer membrane of Gram-negative bacteria is composed of a bilayer containing lipopolysaccharide and outer membrane proteins (OMPs). OMPs are important to the permeability of antimicrobial agents (Kurupati et al. 2006). In K. pneumoniae, OmpK36 and OmpK35 are major porins implicated in antibiotic resistance. SDS-PAGE analysis of the OMPs of the ISKP and IRKP strains (Fig. 2) revealed an absence of OmpK36 and OmpK35 in IRKP. The OMPs were confirmed to be porins, which can allow imipenem to penetrate the K. pneumoniae cells. Expression of the 371kDa OMP in all ISKP and IRKP strains was increased when exposed to 0.5 x MIC of EGCg. This OMP was identified as OmpA by MALDI-TOF/MS. Llobet et al. (2009) reported that OmpA confers resistance to antimicrobial peptides. On the other hand, the expression of the 26 kDa OMP, OmpF, was increased in IRKP by EGCg, whereas the expression of the 17 kDa OMP, Omp 17 K, was decreased in ISKP and IRKP by EGCg. These results are similar to those reported by Climent et al. (1997), who found that overproduction of OmpK l 7 protein in E. coli causes significantly decreased OmpA and OrnpF production.



Changes in total protein in IRKP exposed to EGCg

As shown in Fig. 3, 0.5 x MIC of EGCg by itself did not produce bactericidal effects, but this concentration of EGCg significantly reduced the lethal imipenem concentration. To examine the effect of 0.5 x MIC of EGCg on IRKP, the strain was incubated in MH medium overnight in the presence or absence of 0.5 x MIC of EGCg (300[micro]g/ml EGCg). IRKP cells were subjected to isoelectric focusing (at a pH ranging from 4 to 7) and 2-DE to determine the ability of EGCg to either induce or suppress protein expression. Twelve proteins were successfully identified and characterized by MALDI-TOF/MS as shown in Table 2.
Table 2
Protein expression profiling of imipenem-resistant K. pneumoniae
exposed to EGCg.

Spot no.             Identified protein      Accession    Score EGCg
                                              number       change (a)

Response to stress, cellular processes:
1                    Molecular chaperone     ABR75475.1   123
                     DnaK                                 [up arrow]

2                    Chaperonin GroEL        ABR79862.1   85
                                                          [up arrow]

9                    Alkyl hydroperoxide     ABR76100.1   119
                     reductase,  AhpC                     [up arrow]

12                   Superoxide dismutase.   ABR77425.1   63
                     SodB                                 [up arrow]
                                                          [up arrow]

Energy metabolism

7                    Glyceraldehyde-3-       ABR76630.1   95
                     phosphate                            [down arrow]

8                    Pyruvate kinase         ABR77561.1   58
                                                          [down arrow]
Biosynthesis, biosynthesis of cofactor,
protein synthesis

3                    Elongation factor Tu    ABR79111.1   106
                                                          [down arrow]

4                    Acetyl-coA carboxylase  ABR80246.1   82
                                                          [down arrow]

6                    Molybdenum cofactor     ABR76246.1   69
                     biosynthesis protein A               [down arrow]

10                   50S ribosomal           ABR79948.1   75
                     protein L9                           [down arrow]
Cell envelope

5                    Outer membrane protein  ABR75926.1   72
                                                          [down arrow]
                                                          [down arrow]

DNA metabolism

11                   Single stranded DNA     ABR79801.1   76
                     binding protein                      [down arrow]

(a.) [up arrow] [up arrow], strong up regulation by EGCg; [up arrow],
weak up regulation by EGCg. [down arrow] [down arrow], strong down
regulation by EGCg; [down arrow], weak down regulation by EGCg.

Four proteins were up-regulated upon exposure to EGCg. The molecular chaperones Dnak and GroEL, which are important in the stress response and survival of K. pneumoniae, were significantly increased in IRKP after exposure to EGCg. Increased expression in the detoxification associated proteins, alkyl hydroperoxide reductase (AhpC) and superoxide dismutase (SodB) was also observed.

On the other hand, eight proteins expressed under normal physiological conditions were not expressed or markedly decreased upon exposure to EGCg. These down-regulated proteins were identified as energy metabolism proteins (e.g., glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase), biosynthesis proteins (e.g., elongation factor Tu, acetyl-coA carboxylase, molybdenum cofactor biosynthesis protein A, 50S ribosomal protein L9), a cell envelope protein (e.g., outer membrane protein) and a DNA metabolism protein (e.g., single-stranded DNA binding protein). All of these proteins were dramatically decreased in IRKP after exposure to EGCg. These results are reminiscent of those reported by Cho et al. (2007) who found that sub-MIC levels of TPP reduce the expression of various proteins in E. coli. While the synergistic mechanism of EGCg in the enhanced killing of IRKP by imipenem is still unclear, these data demonstrate that sub-MIC exposure to EGCg dramatically affected the expression of several important IRKP proteins.

Effects of EGCg and imipenem stress on cell morphology

The morphological changes of the K. pneumoniae species exposed to 0.25 x MIC of imipenem, 0.5 x MIC of EGCg or 0.5 x MIC of EGCg plus 0.5 x MIC of imipenem were examined. The scanning electron micrograph results revealed that normal cells grown on MH medium in the absence of EGCg and imipenem were of typical rod shape with smooth surface (Fig. 4A). However, cells treated with 1 x MIC of imipenem and 1 x MIC of EGCg or 0.5 x MIC of EGCg with imipenem for 2 h showed several destructive openings on their cell envelopes as well as a preponderance of irregular rod-shaped forms with wrinkled surfaces (Fig. 4B-D). Several reports have shown that high concentrations of various chemicals are toxic to cells because of their ability to cause disruption of membrane components (Chang etal., 2004; Choetal., 2007). Ikigaietal. (1993) reported that EGCg damages bacterial membranes and that the bactericidal effect of EGCg is attributed to membrane perturbation.


In the present study, the combination of EGCg and imipenem showed synergistic antibacterial effects against IRKP and 1SKP. Furthermore, bacterial cell exposure to EGCg resulted in proteomic changes related to survival. These findings suggest that EGCg extracted from green tea acts as a killing agent and a stressor to pathogenic bacteria. Consequently, the combination of EGCg with chemical antibiotics has been shown to be an effective alternative treatment strategy against pathogenic and antibiotic-resistant bacteria such as IRKP.


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Yun-Seok Cho (a), Jay Jooroung Oh (b), Kye-Heon Oh (a), *

(a.) Department of Biotechnology, Soonchunhyang University, P.O. Box 97, Asan, Chung-Norn 336-600, Republic of Korea

(b.) Program of Microbiology, Indiana University, Bloomington, IN 47402, USA

* Corresponding author. Tel.:+82 41 530 1353; fax: +82 41 530 1350. E-moil address: (K.-H. Oh).

doi: 10.1016/j.phymed.2011.03.012

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Title Annotation:ELSEVIER
Author:Cho, Yun-Seok; Oh, Jay jooyoung; Oh, Kye-Heon
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Aug 15, 2011
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