Daptomycin Resistant Staphylococcus aureus Clinical Strain With Novel Non-synonymous Mutations in the mprF and vraS Genes: A New Insight Into Daptomycin Resistance.

Artur J Sabat,Hajo Grundmann,Monica Monaco,Viktoria Akkerboom,Alexander W Friedrich,Giulia Errico,Maria Del Grosso,Annalisa Pantosti,Marco Tinelli

doi:10.3389/fmicb.2018.02705

Abstract

Objectives: Daptomycin (DAP) resistance in Staphylococcus aureus is uncommon but there are increasing reports of the emergence of resistance during DAP therapy. Most clinical DAP-resistant S. aureus isolates investigated carried mutations in the mprF gene. The aim of this study was to identify mutations between a clinical pair of methicillin-susceptible S. aureus (MSSA) isolates (DAP-susceptible and DAP-resistant). Additionally, the activity of genes previously associated with DAP resistance was assessed.Materials and Methods: Two MSSA isolates from patient with left-sided endocarditis were analyzed by whole genome sequencing (WGS) and reverse transcription-quantitative real-time PCR (RT-qPCR). The first isolate, DAP-susceptible, was obtained before initiation of treatment and the second isolate, DAP-resistant, was recovered after 4 weeks of DAP therapy.Results: Comparison of complete genomes of DAP-susceptible and its DAP-resistant variant identified two non-synonymous and one synonymous mutations. The non-synonymous mutations consisted of a S829L substitution in mprF and a T331I substitution in vraS. The RT-qPCR experiments revealed an increased expression of vraS, dltA, mprF, and sceD genes in DAP-resistant variant. Strikingly, the expression of dltA and mprF genes was significantly downregulated by DAP.Conclusion: The mprF and vraS genes were previously associated with DAP resistance, however, none of the mutations described in this study had been previously identified and linked to DAP resistance. Moreover, we provide a new insight into the DAP action on S. aureus, in which the expression of key genes in DAP resistance is decreased by the antibiotic.

Highlights

Daptomycin (DAP) is an alternative to vancomycin for invasive methicillin-resistant Staphylococcus aureus (MRSA) infections or for serious methicillin-susceptible S. aureus (MSSA) infections in patients who are allergic to beta-lactams
MprF and vraS genes were previously associated with DAP resistance, none of the single-nucleotide polymorphisms (SNPs) that we identified had been previously described and linked to DAP resistance
Our study revealed a novel amino acid substitution in C-terminal catalytic synthase domain of the MprF protein at amino acid residue 829 (S829L), which was found in a clinical isolate with the DAP-resistant phenotype after prolonged treatment of a patient with endocarditis initially infected with a fully susceptible strain

Summary

Introduction

Daptomycin (DAP) is an alternative to vancomycin for invasive methicillin-resistant Staphylococcus aureus (MRSA) infections or for serious methicillin-susceptible S. aureus (MSSA) infections in patients who are allergic to beta-lactams. The amino acid substitutions in the MprF protein identified in the strains showing resistance to DAP lead to altered cell membrane phospholipid profiles. It results in a cell membrane positive charge increase and changes in cell membrane fluidity (Mishra et al, 2009). Data from numerous studies have suggested that charge repulsion arising from the mprF- and dlt-mediated enhancement of positive surface charge is mainly responsible for DAP-resistant phenotype among S. aureus strains (Jones et al, 2008; Patel et al, 2011; Peleg et al, 2012; Gasch et al, 2013; Yang et al, 2013; Cafiso et al, 2014; Kang et al, 2017; Ma et al, 2018). The emergence of mutations in these regulatory genes have been associated with DAP resistance in S. aureus (Friedman et al, 2006; Mehta et al, 2012)

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