AFM Probing the Mechanism of Synergistic Effects of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate (EGCG) with Cefotaxime against Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli

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Abstract

Background

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics.

Methods and Findings

The mechanism of synergistic activity of a combination of (−)-epigallocatechin-3-gallate (EGCG) and β-lactam antibiotics cefotaxime was studied on Extended-spectrum β-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs.

Conclusions

These results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.

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Most cited references 25

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A common mechanism of cellular death induced by bactericidal antibiotics.

Michael A Kohanski, Daniel Dwyer, Boris Hayete … (2007)

Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.

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Morphological plasticity as a bacterial survival strategy.

Sheryl Justice, David Hunstad, Lynette Cegelski … (2008)

Bacteria have evolved complex systems to maintain consistent cell morphologies. Nevertheless, in certain circumstances, bacteria alter this highly regulated process to transform into filamentous organisms. Accumulating evidence attributes important biological roles to filamentation in stressful environments, including, but not limited to, sites of interaction between pathogenic bacteria and their hosts. Filamentation could represent an intended response to specific environmental cues that promote survival amidst the threats of consumption and killing.

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Mechanism of synergy between epigallocatechin gallate and beta-lactams against methicillin-resistant Staphylococcus aureus.

Hong-Chuan Zhao, T Shimamura, S Okubo … (2001)

Compared to MICs (more than 800 microg/ml) of (-)-epigallocatechin gallate (EGCg) against Escherchia coli, MICs of EGCg against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) were 100 microg/ml or less. Furthermore, less than 25 microg EGCg per ml obviously reversed the high level resistance of MRSA to all types of tested beta-lactams, including benzylpenicillin, oxacillin, methicillin, ampicillin, and cephalexin. EGCg also induced a supersusceptibility to beta-lactams in MSSA which does not express mecA, encoding penicillin-binding protein 2' (PBP2'). The fractional inhibitory concentration (FIC) indices of the tested beta-lactams against 25 isolates of MRSA were from 0.126 to 0.625 in combination with 6.25, 12.5 or 25 microg of EGCg per ml. However, no synergism was observed between EGCg and ampicillin against E. coli. EGCg largely reduced the tolerance of MRSA and MSSA to high ionic strength and low osmotic pressure in their external atmosphere, indicating damage of the cell wall. Unlike dextran and lipopolysaccharide, peptidoglycan from S. aureus blocked both the antibacterial activity of EGCg and the synergism between EGCg and oxacillin, suggesting a direct binding of EGCg with peptidoglycan on the cell wall. EGCg showed a synergistic effect with DL-cycloserine (an inhibitor of cell wall synthesis unrelated to PBP2') but additive or indifferent effect with inhibitors of protein and nuclear acid synthesis. EGCg did not suppress either PBP2' mRNA expression or PBP2' production, as confirmed by reverse transcription-PCR and a semiquantitative PBP2' latex agglutination assay, indicating an irrelevance between the synergy and PBP2' production. In summary, both EGCg and beta-lactams directly or indirectly attack the same site, peptidoglycan on the cell wall. EGCg synergizes the activity of beta-lactams against MRSA owing to interference with the integrity of the cell wall through direct binding to peptidoglycan.

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Author and article information

Contributors

Vladimir N. Uversky: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2012

Publication date (Electronic): 13 November 2012

Volume: 7

Issue: 11

Electronic Location Identifier: e48880

Affiliations

[1 ]Department of Chemistry and Nano Sciences, Ewha Womans University, Seoul, Korea

[2 ]Division of Environmental Science and Ecological Engineering, Korea University, Seoul, Korea

[3 ]Mechanobiology Institute, National University of Singapore, Singapore, Singapore

[4 ]Department of Medicine, College of Medicine, Chung-Ang University, Seoul, Korea

University of South Florida College of Medicine, United States of America

Author notes

* E-mail: nanopark@ 123456ewha.ac.kr

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: YC SP. Performed the experiments: YC SHK HK JY. Analyzed the data: YC KK WP SP. Contributed reagents/materials/analysis tools: KK WP SP. Wrote the paper: KK WP SP.

Article

Publisher ID: PONE-D-12-25401

DOI: 10.1371/journal.pone.0048880

PMC ID: 3496731

PubMed ID: 23152812

SO-VID: f28295f2-2d42-4aac-aea3-67354be4d18d

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 22 August 2012

Date accepted : 1 October 2012

Page count

Pages: 6

Funding

This work was equally supported by grants (SRC Program: 2012-0000647, #20110021114, #2012-0001138) from Korean National Research Foundation as well as a grant (Code #PJ007492) from Korean Rural Development Administration. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AFM Probing the Mechanism of Synergistic Effects of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate (EGCG) with Cefotaxime against Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli

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Abstract

Background

Methods and Findings

Conclusions

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PLOS Climate

Most cited references 25

A common mechanism of cellular death induced by bactericidal antibiotics.

Morphological plasticity as a bacterial survival strategy.

Mechanism of synergy between epigallocatechin gallate and beta-lactams against methicillin-resistant Staphylococcus aureus.

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