Separation and Quantification of Murein and Precursors from Enterobacter cloacae after Treatment with Trimethoprim and Sulphadiazine

Abstract

The intracellular concentrations of the soluble murein precursors UDP-Mur-NAc-pentapeptide in the cytoplasm, the membrane-bound lipid precursor disaccharide pentapeptide and the muropeptides of Enterobacter cloacae cultures treated with trimethoprim (12.5 micrograms mL-1) and sulphadiazine (250 micrograms mL-1) were determined by using capillary zone electrophoresis analysis. In the presence of trimethoprim, UDP-Mur-NAc-pentapeptide as well as disaccharide pentapeptide accumulated. In the case of sulphadiazine-treated cells, the concentration of UDP-Mur-NAc-pentapeptide roughly paralleled the control cells but sulphadiazine caused a slow incremental accumulation of disaccharide pentapeptide. The muropeptide composition of the murein indicated that the differences between the peptidoglycans produced by the control cells and the cells grown in the presence of either trimethoprim or sulphadiazine alone or in combination were quite marked. The results suggest that the enhanced activity of trimethoprim plus sulphadiazine against E. cloacae is caused by an additional effect on the inhibition of the bacterial peptidoglycan biosynthesis and that this additional effect is a fundamental part of the antibacterial action of the antimetabolites. This effect leads to changes of cell morphology and resultant changes in bacterial cell permeability.

Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support

“…This is consistent with reports that trimethoprim interferes with the supply of cell-wall precursors in Ent. cloacae (Richards & Xing, 1994;Richards et al, 1995). A wide range of responses was observed in what must (in most cases) be the product of a single cell, or pair of cells, generating diversity during the course of the assay in the presence of trimethoprim above the minimal inhibitory amount.…”

“…Antifolate drugs structurally related to trimethoprim have similar effects on Escherichia coli (Nickerson & Webb, 1956). Trimethoprim also affects cell morphology and the synthesis of cell wall precursors in Enterobacter cloacae (Richards & Xing, 1994), and cell wall formation in Enterococcus faecalis (Richards et al, 1995). It has been suggested that trimethoprim exerts its effect on cell morphology by inhibiting DNA replication (Richards & Xing, 1994).…”

“…Trimethoprim also affects cell morphology and the synthesis of cell wall precursors in Enterobacter cloacae (Richards & Xing, 1994), and cell wall formation in Enterococcus faecalis (Richards et al, 1995). It has been suggested that trimethoprim exerts its effect on cell morphology by inhibiting DNA replication (Richards & Xing, 1994). Most aspects of the occurrence, mechanism and clinical significance of trimethoprim-mediated filamentation are unknown.…”

Metabolic connections between folate and peptidoglycan pathways inPseudomonas aeruginosainform rational design of a dual-action inhibitor

“…Their synergistic mechanism is now known to arise from early folate pathway inhibition by a substrate accumulation feedback loop 61 , however data from the group that originally proposed increased compound uptake showed a striking effect of folate inhibition on the abundance of lipid II in E. cloacae 15 . Thus, while the initial model of mutual of potentiation was incorrect, the data supporting that model still revealed relevant effects of folate inhibition on the cell wall, which we further characterized here.…”

“…Antifolate-induced morphological changes extend beyond filamentation, as cell-wall deficient cells can also arise following folate inhibition 14 . Further linking the folate and cell wall pathways, Richards and Xing reported accumulation of Lipid II in Enterobacter cloacae following TMP and sulfadiazine treatment, suggesting a blockage in PG assembly post-precursor biosynthesis 15 . A screen for Acinetobacter baumannii mutants that were significantly less fit during growth with TMP-SUL uncovered mutants with disruptions in peptidoglycan recycling 16 .…”