Vancomycin-dependent Enterococcus faecalis clinical isolates and revertant mutants

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Vancomycin-dependent Enterococcus faecalis (VdEnterococci) poses a treatment challenge.
• The VanB resistance phenotype involves a two-component regulatory system activating the vanB operon.
• Understanding resistance mechanisms is crucial for effective treatment strategies.

Purpose of the Study:

• To investigate the genetic basis of vancomycin dependence and resistance in clinical Enterococcus faecalis isolates.
• To analyze mutations in the ddl gene and the vanSB-vanRB regulatory system.
• To characterize the peptidoglycan precursor synthesis under different antibiotic conditions.

Main Methods:

• DNA sequencing of ddl, vanSB, and vanRB genes.
• Reporter assays using DD-dipeptidase activity to measure resistance gene expression.
• Analysis of peptidoglycan precursor synthesis.

Main Results:

• Mutations in the ddl gene impaired host D-Ala:D-Ala ligase activity, leading to synthesis of D-Ala-D-Lac precursors.
• Vancomycin-independent revertants showed restored D-Ala:D-Ala ligase activity (ddl mutations) or constitutive resistance (vanSB mutations).
• Mutations in vanSB affected the sensor's autophosphorylation site or ATP binding domain, leading to constitutive or antibiotic-dependent resistance.

Conclusions:

• The ddl gene mutations are responsible for vancomycin dependence in these isolates.
• Rapid in vitro emergence of vancomycin-independent revertants indicates potential treatment limitations.
• Interruption of vancomycin therapy alone may be insufficient for curing VdEnterococci infections.