Synergistic activity and mechanism of action of ceftazidime and apigenin combination against ceftazidime-resistant Enterobacter cloacae

The purpose of this investigation was to examine the antibacterial and synergistic effect of naturally occurring flavonoids, apigenin, quercetin, naringenin and ceftazidime when use singly and in combination against ceftazidime-resistant Enterobacter cloacae strains by minimum inhibitory concentration (MIC), checkerboard and viable count methods. The mode of actions were also studied by electronmicoscopy, enzyme assay, outer and inner membrane permeabilisation. The results showed that these strains were positive in the ESBL–ampC genes combination by multiplex PCR. These findings were confirmed by MICs that these strains were resistant to ceftazidime, cefepime and flomoxef at >1024, 16–24, >256 μg/ml respectively, while susceptible to imipenem at 1–2 μg/ml. The synergistic activity was observed at ceftazidime plus either apigenin or naringenin combinations with FIC indixes between <0.01 and <0.27 against these strains, whereas ceftazidime plus clavulanic acid or quercetin did not exhibit synergy. The modulation of ceftazidime-resistance by apigenin or narigenin significantly enhanced the activities of ceftazidime. The 5,7-OH group of A ring and one 4′-OH group of the B ring in apigenin and naringenin are important for synergistic activity. Viable counts showed that the killing of ceftazidime-resistant E. cloacae DMST 21394 (CREC) cells by 3 μg/ml ceftazidime was potentiated by 3 μg/ml apigenin to low levels (103 cfu/ml) over 6 h. Electronmicroscopy clearly showed that ceftazidime 3 μg/ml in combination with 3 μg/ml of apigenin also caused marked morphological damage of cell wall, cell shape and plasma membrane of this strain. Enzymes assays indicated that apigenin showed marked inhibitory activity against penicillinase type IV from E. cloacae. The results for outer membrane (OM) permeabilization in both nitrocefin (NCF) assay and crystal violet uptake showed that the combination of ceftazidime plus apigenin significantly altered OM permeabilisation of CREC compared to control or single treatment of these agents. Both o-nitrophenyl-β-d-galactoside (ONPG) uptake and release of UV-absorbing material concentrations results exhibited that ceftazidime and apigenin combination damaged CREC cytoplasmic membrane (CM) and caused subsequent leakage of intracellular constituents. From the results, it can be concluded that apigenin and naringenin have the synergistic effect with ceftazidime to reverse bacterial resistance to this cephalosporin against CREC. This activity may be involved three mechanisms of action by apigenin. The first is on the peptidoglycan synthesis inhibition. The second mechanism is inhibition the activity of certain β-lactamase enzymes. The third mode of action is alteration of OM and CM permeabilization. Apigenin and naringenin have a sufficient margin of safety for therapeutic use. For this reason, apigenin and naringenin offer for the development of a valuable adjunct to ceftazidime against CREC, which currently almost cephalosporins resistance.