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

Abstract

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.

FIG. 1

Chemical structure and antibacterial activities of EGCg against MRSA, MSSA, S. epidermidis, E. coli, and S. enttrica serovar Typhimurium. Bacteria (3 × 10⁶ cells) were inoculated into 3 ml of MHB containing different concentrations of EGCg. The cells were then incubated stationarily at 37°C for 24 h. Growth of bacteria was determined by OD at 600 nm. ○, MRSA F-74; ●, MSSA ATCC 25923; ▵, S. epidermidis ATCC 1228; ▴, E. coli ATCC 25922; ◊, S. enterica serovar Typhimurium TAS 2121.

FIG. 2

Synergistic anti-MRSA (A) and anti-MSSA (B) effects and indifferent anti-E. coli (C) effect between EGCg and β-lactams. MRSA F-74 and MSSA ATCC 25923 were cultured in MHB containing EGCg and oxacillin. E. coli ATCC 25922 was cultured in MHB containing EGCg and ampicillin. Symbols in panel B apply also to panel A.

FIG. 3

Reduction of tolerance of MRSA and MSSA to high ionic strength and low osmotic pressure in the presence of EGCg. (A) MRSA F-74 cells (3 × 10⁶) were cultured in 3 ml of MHB containing different concentrations of EGCg and NaCl at 37°C for 24 h. Growth of bacteria was determined by OD at 600 nm. (B) F-74 cells (10⁴/ml) were incubated in water with various concentrations of EGCg at 37°C for 0, 4, 8, 12, and 24 h. Viable cell numbers were then determined by culturing the cells on MH agar plates for additional 48 h.

FIG. 4

Direct binding of EGCg with peptidoglycan (PG) of the cell wall. PG from S. aureus was added to MHB containing EGCg only (A) or EGCg plus oxacillin (B). The same amount of LPS was used as a control. MRSA F-74 cells were inoculated and cultured at 37°C for 24 h. (A) ○, PG alone; ●, EGCg (50 μg/ml) plus PG; ▵, LPS alone; ▴, EGCg (50 μg/ml) plus LPS. (B) ○, oxacillin alone; ▵, plus EGCg (25 μg/ml); ●, plus EGCg (25 μg/ml) and PG (16 μg/ml); ▴, plus EGCg (25 μg/ml) and PG (32 μg/ml); □, plus EGCg (25 μg/ml) and LPS (32 μg/ml).

FIG. 5

Effects of EGCg in combination with dl-cycloserine (A), minocycline (B), or ofloxacin (C) on MRSA F-74 growth. Symbols in panel C apply to all panels.

FIG. 6

RT-PCR analysis of PBP2′ mRNA expression. Lane M, 100-bp DNA ladder (molecular weight marker); lane 1, MRSA F-74 not treated with EGCg; lane 2, F-74 treated with EGCg (25 μg/ml); lane 3, F-74 treated with oxacillin (4 μg/ml); lane 4, F-74 treated with EGCg (25 μg/ml) and oxacillin (4 μg/ml); lane 5, MSSA FDA 209p as a control. Arrowheads: expected size (528 bp) of S. aureus-specific 16S rRNA (top) and expected size (313 bp) of PBP2′ gene (bottom).