Effect of a Point Mutation in mprF on Susceptibility to Daptomycin, Vancomycin, and Oxacillin in an MRSA Clinical Strain.

Feng-Jui Chen,I-Wen Huang,Chen-Hsiang Lee,Chung-Shi Yang,Tsai-Ling Lauderdale,Pei-Chi Hsu,Yu-Chieh Hsu

doi:10.3389/fmicb.2018.01086

Abstract

We previously reported the sequential recovery of daptomycin-nonsusceptible MRSA clinical isolates with an L431F substitution in the MprF protein. The aim of the present study is to determine the effect of this mutation by replacing the mprF gene on the chromosome of a daptomycin-susceptible progenitor strain, CGK5, to obtain CGK5mut having the L431F MprF mutation. Compared to CGK5, the daptomycin and vancomycin MICs of CGK5mut increased from 0.5 to 3 μg/ml and from 1.5 to 3 μg/ml, respectively; however, its oxacillin MIC decreased from 128 to 1 μg/ml in medium without added 2% NaCl. The expression levels of vraSR and several other cell-wall synthesis-related genes were significantly increased in CGK5mut, and the mutant also had significantly reduced negative cell membrane charge, thicker cell wall, and longer doubling time. These features were abolished in the reverse mutant carrying F431L MprF, confirming the pleiotropic effects of the L431F MprF mutation. We believe that this is the first work that shows a single MprF missense mutation can lead to not only changes in the cell membrane but also increased expression of vraSR and subsequently increased resistance to daptomycin and vancomycin while simultaneously conferring increased susceptibility to oxacillin in an isogenic MRSA strain.

Highlights

Daptomycin, a cyclic lipopeptide antibiotic, is one of the last-line agents for the treatment of certain severe multidrug-resistant Staphylococcus aureus infections, including those caused by methicillinresistant S. aureus (MRSA) (Enoch et al, 2007)
The daptomycin MIC of CGK5mut was 6-fold higher (3 μg/ml) compared to that of the wild-type parental strain CGK5 (0.5 μg/ml), a level of increase similar to what we previously reported for DAP-NS clinical isolates (Lee et al, 2010)
We found that CGK5mut grows slower than both CGK5 and CGK5mutR in Mueller Hinton II broth (MHB) (Figure 2)

Summary

Introduction

Daptomycin, a cyclic lipopeptide antibiotic, is one of the last-line agents for the treatment of certain severe multidrug-resistant Staphylococcus aureus infections, including those caused by methicillinresistant S. aureus (MRSA) (Enoch et al, 2007). Effect of a Mutation in mprF on Susceptibility mprF, yycG (walK), vraSR, tagA, and dltABCD (Friedman et al, 2006; Baltz, 2009; Bertsche et al, 2011; Song et al, 2013). These loci are all part of the cell wall stimulon in S. aureus and include genes encoding proteins involved in the production of membrane phospholipids. This results in increased amounts of LPG relative to phosphatidylglycerol (PG) on the outer leaflet of the cytoplasmic membrane and an accompanying reduction in cell membrane negative charge (Baltz, 2009; Rubio et al, 2012)

Objectives

Methods

Results

Discussion

Conclusion