In vitro efficacy of ceftriaxone/sulbactam against Escherichia coli isolates producing CTX-M-15 extended-spectrum beta-lactamase.

on the antibacterial activity of commonly used and therapeutically relevant b-lactam antibiotics. The effect of glutathione (GSH) and ascorbic acid on the susceptibility of E. coli K-12 strain MG1655 to ampicillin and penicillin was investigated. MICs of these antibiotics in the presence and absence of 10 mM of these antioxidants were determined by agar dilution method as outlined by the CLSI (formerly the NCCLS). 3 An inoculum of approximately 10 4 –10 5 cfu (simultaneously determined by plating) per spot (in a volume of 10 mL) was applied to the agar plates with increasing antibiotic concentration. MIC was the lowest concentration of antimicro-bial agent that prevented visible growth after 20 h of incubation at 378C. The experiments were carried out at least twice and the representative results are mentioned here. We observed that the presence of 10 mM GSH made MG1655 cells more susceptible to these b-lactams, because addition of GSH resulted in reduced MICs of ampicillin and penicillin from 8 and 64 mg/L to 4 and 48 mg/L, respectively. However, we found that this effect was specific to GSH, as the presence of ascorbic acid did not make any difference to the antibiotic susceptibility of MG1655. Total intracellular glutathione (GSH in) is a sum of reduced glutathione (GSH) þ oxidized glutathione (GSSG) amounts and bacterial cells can readily take up both GSH and GSSG, resulting in enhanced GSH in. 4 As GSH in along with GSH to GSSG ratio are key regulators of various intracellular redox reactions, we investigated whether this glutathione-mediated phenotype is dependent on its redox status outside the cell. For this purpose the MICs of the above-mentioned b-lactams were determined in the presence of 5 mM GSSG. The results showed that GSSG also decreased the MICs of the antibiotics to the same extent as observed with GSH, implying that glutathione-mediated augmentation of b-lactam susceptibility is independent of its redox status outside the cell. Gamma glutamyl-transepeptidase (ggt, EC 2.3.2.2)-mediated cleavage of glutathione is required for the uptake of extracellular glutathione by bacterial cells. 5 In addition, as ggt catalyses the transfer of the gamma glutamyl group of glutathione and related gamma-glutamyl amides to other amino acids and peptides (transpeptidation), it is likely to be involved in the transpeptida-tion step of bacterial cell wall synthesis. Considering that b-lactams act at the transpeptidation step of cell wall synthesis, glutathione-mediated augmentation of b-lactam antibacterial activity could be due to the …