Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Antibiotic resistance – why is the problem so difficult to solve?

Antibiotic resistance has been increasing along with antibiotic use. At the same time, the supply of new drugs to replace those rendered inefficient by the development has been dwindling, leading to concerns that we may soon lack efficient means to treat bacterial infections. Though the problem has received considerable interest, there are no indications that the situation is about to change. The present review maintains that this is because the two objectives - preserving the efficiency of existing drugs and increasing the supply of new ones - are partly opposing. Hence, creating an incentive structure compatible with both of them is not easy. Nevertheless, it is suggested that levying a fee on the use of antibiotics, and earmarking the proceeds from this fee for subsidizing development of new antibiotics, would be an important step towards increasing incentives for a better antibiotic stewardship while preserving incentives to develop new substances