EGCG against Staphylococci
Staphylococcus aureus is among the most common pathogens to cause community- and hospital-acquired infections. In Europe, S. aureus is the second most common causative microorganism for bacteraemia and is one of the leading causes of sepsis worldwide (Biedenbach et al., 2004). Methicillin-resistant S. aureus (MRSA) is a type of Staphylococci that is resistant to certain antibiotics called β-lactams. Infections with MRSA are more difficult to treat and are therefore associated with a higher mortality rate than those caused by methicillin-susceptible S. aureus (Cosgrove et al., 2003). The methicillin resistance in S. aureus is primarily mediated by the mecA gene, which codes for the modified penicillin-binding protein 2a (PBP2a). PBP2a is located in the bacterial cell wall and has low binding affinity for β-lactams.
The activity of EGCG as single agent and in combination with β-lactams has been assessed in multiple studies. Initially, in vitro data from a study performed over two decades ago indicated that tea extracts, at concentrations found in ordinarily brewed tea, inhibited the growth of MRSA (Toda et al., 1989). Subsequently the biological activity of green tea components including EGCG against S. aureus was investigated (Ikigai et al., 1993). It was reported that the minimum inhibitory concentration (MIC) values of EGCG were below 100 μg mL−1. Initial experiments suggested that negatively charged EGCG exerts its anti-bactericidal activity by binding to the positively charged lipids of the bacterial cell membrane, causing damage to the lipid layer. Subsequently, the interaction of catechins including EGCG with lipid bilayers has been studied in more detail (Kumazawa et al., 2004; Uekusa et al., 2007; Kajiya et al., 2008; Kamihira et al., 2008; Sirk et al., 2008; Cui et al., 2012).
The mechanism of action of EGCG against Staphylococci was further investigated by Yam and co-workers who demonstrated that tea extracts can reverse the phenotypic methicillin resistance in MRSA (Yam et al., 1998). Tea extracts at 25 μg mL−1 were able to inhibit the production of PBP2 by >90% in a constitutively PBP2 producing S. aureus strain. In addition, the production of β-lactamases was inhibited. In contrast, to the study from Yam et al., suppression of PBP2 could not be detected by Zhao et al. either by PBP2 mRNA expression using quantitative PCR or by PBP2 production using latex agglutination (Zhao et al., 2002).
The combination of tea extracts with β-lactams (methicillin, benzylpenicillin) was mostly demonstrated to have a synergistic antibacterial effect. These results were mainly confirmed by Zhao and colleagues who showed that 25 μg mL−1 EGCG was able to reverse the high-level resistance of MRSA to all types of β-lactams, including benzylpenicillin, oxacillin, methicillin, ampicillin and cephalexin (Zhao et al., 2001b); fractional inhibitory concentration indices (FICI) of the β-lactams tested alone against 25 MRSA isolates were low (0.126–0.625), indicating that EGCG has a synergistic effect. In additional studies, the combination of EGCG with ampicillin/sulbactam or carbapenems was also shown to exert a synergistic antibacterial effect and MICs were reduced to the susceptibility breakpoint (Hu et al., 2001; 2002; Stapleton et al., 2004). Furthermore, 12.5 μg mL−1 EGCG in combination with penicillin revealed a synergistic effect in 100% of the 21 MRSA strains tested (Zhao et al., 2002). As previously reported, the production of penicillinase from penicillin-resistant S. aureus was also inhibited by EGCG in a dose-dependent manner.
In addition to EGCG, ECG was also able to reverse β-lactam resistance in clinical MRSA isolates (Stapleton et al., 2004); the gallate moiety of EGC was shown to be essential for oxacillin-modulating activity, as both (-)-epicatechin and (-)-epicatechin-3-cyclohexylcarboxylate were unable to reverse resistance.
Results from Shimamura and co-workers indicated that EGCG binds directly or indirectly to the peptidoglycan of the bacterial cell wall and inhibits the penicillinase activity, protecting penicillin from inactivation (Zhao et al., 2002). Further to its effects when combined with β-lactams, the interactions of EGCG with non-β-lactam antibiotics have been evaluated against MRSA (Hu et al., 2002). The combination of EGCG with antibiotic inhibitors of protein or nucleic acid synthesis was found to be additive or no difference (FICI, 0.5–4.0). In contrast, EGCG tended to have an antagonistic effect on the actions of glycopeptide antibiotics (vancomycin, teicoplanin). These in vitro data indicate that the choice of antibiotic in any potential combination therapy consisting of EGCG plus antibiotic against Staphylococci is critical to achieve a bactericidal effect.
Two studies from Italy have provided further insights into the effects of EGCG on Staphylococci (Sudano Roccaro et al., 2004; Blanco et al., 2005). Blanco et al. showed that 50 μg mL−1 EGCG was able to reverse tetracycline resistance and appeared to improve the MICs of tetracycline in susceptible staphylococcal isolates. In strains in which tetracycline resistance was due to increased expression of a tetracycline efflux pump protein (Tet(K) ), EGCG inhibited the pump activity, which resulted in an increased intracellular retention of tetracycline.
Sudano Roccaro et al. (2004) demonstrated that EGCG was able to decrease slime production and inhibit biofilm formation by ocular S. aureus and S. epidermidis isolates (Sudano Roccaro et al., 2004). These results indicate that in addition to binding to lipid layers and peptidoglycan, EGCG interferes with extracellular polymeric material (glycocalyx).
In experiments using Bagg albino (BALB/c) mice and human PBMCs another interesting antibacterial effect of EGCG was demonstrated, polyphenon, consisting of 50% EGCG, neutralized staphylococcal enterotoxin B in a dose- and incubation time-dependent manner by binding to enterotoxin B molecules (Hisano et al., 2003). Further work is needed to determine the effects of EGCG against different enterotoxins, and whether EGCG has neutralization properties against other staphylococcal superantigens such as toxic shock syndrome toxin.
Taken together, these results indicate that there are multiple mechanisms by which ECGC exerts antibacterial effects against Staphylococci, including bactericidal activity, synergism in combination with other antibiotics, anti-biofilm activity and inhibition of β-lactamase production or neutralization of released toxins. However, not all effects of EGCG against Staphylococci are beneficial. A recent study demonstrated that short exposure of Staphylococcus strains to sublethal doses of EGCG can lead to cross-resistance against antibiotics targeting the bacterial cell wall (vancomycin, oxacillin, ampicillin) (Bikels-Goshen et al., 2010). All EGCG-adapted strains were also more heat tolerant. The reason for this phenomenon was studied by transmission electron microscopy analysis, which revealed that bacterial cells in cultures exposed to EGCG showed pseudo-multicellular appearance and had a more than a twofold increase in the cell wall thickness. In summary, the results of this study indicate that EGCG may also contribute to the development and enhancement of bacterial resistance mechanisms. Animal studies are needed to explore whether these observations are reproducible in vivo.