Ind the peptidoglycan layerAFM Study of Effects between EGCG and Cefotaximeof Gram-negative bacteria because of their outer membrane, it is suggested that the mechanism underlying the synergistic effect of EGCG with the antibiotics against Gram-negative bacteria is different from the mechanism underlying the synergistic effects against Gram-positive bacteria. Atomic force microscopy (AFM) is a very useful tool for visualizing the morphology of bacterial surfaces in nanoscale, and has been used to study the antibacterial effects of antibiotics [15], [16], antimicrobial peptides [17], [18], and others [19], [20]. It has been most recently shown by AFM that EGCG has different modes of antibacterial action against Gram-negative and Grampositive bacteria by direct binding to the peptidoglycan layer or through H2O2 production, respectively [19]. In this study, we used AFM to obtain high-resolution images of morphological Thiazole Orange site changes in ESBL-Escherichia coli induced by a sole treatment of EGCG or cefotaxime at sub-MICs (sub-minimum inhibitory concentrations) or a co-treatment of EGCG and cefotaxime at their respective sub-MICs. To explain the cause of the morphological changes on the bacterial cell surface, Mirin site oxidative stress response in ESBL-EC against the treatments were measured.concentration of an antimicrobial agent at which no visible growth will occur after overnight incubation. The effects of combinations were confirmed by the checkerboard method [22]. Two-fold serial dilutions of cefotaxime were tested in combinations with serial dilutions of EGCG. The results were evaluated by a fractional inhibitory concentration (FIC) index. FIC was calculated as MIC of antibiotics alone or EGCG in combination divided by MIC of antibiotics or EGCG alone, and the FIC index was obtained by adding the FICs. FIC indices were interpreted as follows: #0.5, synergy; .0.5 to 1, addition; and .1, indifference.Time-kill StudiesTime-related effects of EGCG, cefotaxime and their combinations were determined by measuring cultures’ CFUs. E. coli suspensions (OD600 = 4) were 100 times diluted with MHB media containing different concentrations of 1655472 cefotaxime or EGCG (or both). The number of surviving bacteria was counted after 0, 1, 2, 4, 6, 8, 10, 12, 14, 16 and 18 h incubated at 37uC.Scanning Electron Microscopy (SEM) Analysis Materials and Methods Bacterial Strain and Growth ConditionAn ESBL-EC strain (BAA-198) [21] was obtained from ATCC. The strain was grown overnight with aeration in a round glass tube containing Mueller ?Hinton Broth (MHB; not cationadjusted; Becton Dickinson) at 37uC. The overnight culture was 100 times diluted with MHB to a final volumes of 5 ml and continued growing with aeration at 37uC till reaching stationary phase determined from optical density at 600 nm (OD600 = 4). Then those cultures with OD600 value of 4 were 100 times diluted with MHB and grown with various concentrations of EGCG (Sigma-Aldrich, St. Louis, MO), cefotaxime (Beta-Lactam antibiotics; Sigma-Aldrich) or their combinations either for MIC determination or time-kill studies. Bacterial growth was calculated by colony-forming unit (CFU) count. CFUs were measured by counting colonies after plating 20 ml of each culture on MHB plates and incubating the plates overnight. Bacterial suspensions were pre- and post-fixed in glutaraldehyde solution and then added to glass cover slips. The glass cover slips were dehydrated in a series of ethanol concentrations (30?5 ) for 15 min followe.Ind the peptidoglycan layerAFM Study of Effects between EGCG and Cefotaximeof Gram-negative bacteria because of their outer membrane, it is suggested that the mechanism underlying the synergistic effect of EGCG with the antibiotics against Gram-negative bacteria is different from the mechanism underlying the synergistic effects against Gram-positive bacteria. Atomic force microscopy (AFM) is a very useful tool for visualizing the morphology of bacterial surfaces in nanoscale, and has been used to study the antibacterial effects of antibiotics [15], [16], antimicrobial peptides [17], [18], and others [19], [20]. It has been most recently shown by AFM that EGCG has different modes of antibacterial action against Gram-negative and Grampositive bacteria by direct binding to the peptidoglycan layer or through H2O2 production, respectively [19]. In this study, we used AFM to obtain high-resolution images of morphological changes in ESBL-Escherichia coli induced by a sole treatment of EGCG or cefotaxime at sub-MICs (sub-minimum inhibitory concentrations) or a co-treatment of EGCG and cefotaxime at their respective sub-MICs. To explain the cause of the morphological changes on the bacterial cell surface, oxidative stress response in ESBL-EC against the treatments were measured.concentration of an antimicrobial agent at which no visible growth will occur after overnight incubation. The effects of combinations were confirmed by the checkerboard method [22]. Two-fold serial dilutions of cefotaxime were tested in combinations with serial dilutions of EGCG. The results were evaluated by a fractional inhibitory concentration (FIC) index. FIC was calculated as MIC of antibiotics alone or EGCG in combination divided by MIC of antibiotics or EGCG alone, and the FIC index was obtained by adding the FICs. FIC indices were interpreted as follows: #0.5, synergy; .0.5 to 1, addition; and .1, indifference.Time-kill StudiesTime-related effects of EGCG, cefotaxime and their combinations were determined by measuring cultures’ CFUs. E. coli suspensions (OD600 = 4) were 100 times diluted with MHB media containing different concentrations of 1655472 cefotaxime or EGCG (or both). The number of surviving bacteria was counted after 0, 1, 2, 4, 6, 8, 10, 12, 14, 16 and 18 h incubated at 37uC.Scanning Electron Microscopy (SEM) Analysis Materials and Methods Bacterial Strain and Growth ConditionAn ESBL-EC strain (BAA-198) [21] was obtained from ATCC. The strain was grown overnight with aeration in a round glass tube containing Mueller ?Hinton Broth (MHB; not cationadjusted; Becton Dickinson) at 37uC. The overnight culture was 100 times diluted with MHB to a final volumes of 5 ml and continued growing with aeration at 37uC till reaching stationary phase determined from optical density at 600 nm (OD600 = 4). Then those cultures with OD600 value of 4 were 100 times diluted with MHB and grown with various concentrations of EGCG (Sigma-Aldrich, St. Louis, MO), cefotaxime (Beta-Lactam antibiotics; Sigma-Aldrich) or their combinations either for MIC determination or time-kill studies. Bacterial growth was calculated by colony-forming unit (CFU) count. CFUs were measured by counting colonies after plating 20 ml of each culture on MHB plates and incubating the plates overnight. Bacterial suspensions were pre- and post-fixed in glutaraldehyde solution and then added to glass cover slips. The glass cover slips were dehydrated in a series of ethanol concentrations (30?5 ) for 15 min followe.
