Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus

Abstract

Background: Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.

Methods: Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.

Results: On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.

Conclusion: Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.

Figure 1. Percentage of daptomycin-exposed pairs (n = 12) with a mutation in each functional gene category.

All daptomycin-nonsusceptible isolates had at least one mutation in a gene coding for phospholipid biosynthesis, including mprF, cls2 or pgsA.

Figure 2. Phospholipid biosynthesis genes are integral to the development of reduced susceptibility to daptomycin in S. aureus.

(A) The predicted amino acid changes associated with the 11 SNPs identified within mprF. (B) The five mutations identified in cls2 were mapped to four positions in the protein, all within the two N-terminal transmembrane domains. (C) Six mutations were identified in pgsA and were mapped to four positions in the protein, three of which were in transmembrane domains. The ‘A’ numbers correspond to the daptomycin-nonsusceptible isolates, and the arrows point to the position of the amino acid change.

Figure 3. Schematic of our working hypothesis for the functional effect of the observed mutations.

mprF mutations lead to an increase in lysinylation of phosphotidylglycerol (PG) to form L-PG, and an increase in translocation of this positively charged L-PG to the outer leaflet of the membrane, leading to electrorepulsion of daptomycin. In isolation, or in concert with mprF mutations, mutations in cls2 may lead to altered membrane charge or effect binding of daptomycin to the membrane. Finally, PgsA is important in the initial step of phospholipid biosynthesis, converting CDP-diacylglycerol (CDP-DG) into PG.

Figure 4. Transmission electron microscopy of two clinical pairs showing a thickening of the cell wall in the daptomycin-nonsusceptible isolates.

(A) and (B) represent A8819 (daptomycin-susceptible) and A8817 (daptomycin-nonsusceptible), respectively. (C) and (D) represent A8796 (daptomycin-susceptible) and A8799 (daptomycin-nonsusceptible), respectively. P<0.001 for both.