Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediate Levels In Resistant Enterococcus faecium

Abstract

Vancomycin is a natural glycopeptide antibiotic and is used for the treatment of multi-drug resistant Gram positive bacteria. However, resistance to vancomycin has emerged. Vancomycin resistance in VanA-type Enterococcus faecium is due to a vancomycin resistance cassette comprised of vanH, vanX, vanY, vanZ, vanR and vanS genes. These genes encode for an alternative peptidoglycan biosynthesis pathway in which D-Ala-D-Ala is replaced with D-Ala-D-Lac. Vancomycin binds with a lower affinity to D-Ala-D-Lac as it does to the nomal dipeptide. In this study, several of the unique UDP-linked intermediates from VanA-type vancomycin resistant Enterococcus faecium (VRE) were preparatively purified from vancomycin treated cultures and used to optimize their LC-MS/MS quantification. Cytoplasmic pool levels were then measured in VRE before and after vancomycin exposure. VRE in the absence of vancomycin had low but readily detectible basal levels of VanA pathway intermediates. After a 15 min vancomycin exposure, normal pathway intermediates were only modestly affected, whereas alternative pathway intermediates increased substantially. Alternative pathway intermediates then decreased back toward unexposed levels after longer vancomycin exposure. A vancomycin concentration profile demonstrated a midpoint of vancomycin effect between 1 and 4 μg/mL. Time course experiments revealed a complex response to vancomycin exposure, with most normal pathway intermediates responding rapidly (minutes) to vancomycin exposure, and alternative pathway intermediates increasing for 15-20 min and then decreasing partially back toward normal levels. qRT-PCR study also demonstrated that mRNA levels of VanA pathway genes also took about 15 min to reach their maximal level of induction.