Comparative genome sequencing of an isogenic pair of USA800 clinical methicillin-resistant Staphylococcus aureus isolates obtained before and after daptomycin treatment failure

Abstract

We describe here a clinical daptomycin treatment failure in a patient with recurrent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in whom daptomycin was administered after a failed empirical treatment course with vancomycin and piperacillin-tazobactam. We had the opportunity to compare the genome sequences of an isogenic pair of daptomycin-susceptible and -resistant MRSA isolates obtained before and after initiation of daptomycin therapy, respectively. The genotype of both isolates was USA800, ST5, SCCmec type IV, agr type II. There was no increase in cell wall thickness in the daptomycin-resistant strain despite having decreased susceptibility to both vancomycin and daptomycin. By comparing the genome sequences by pyrosequencing, we identified a polymorphism (S337L) in the tenth transmembrane segment of the multiple peptide resistance factor, MprF, encoding lysyl phosphatidylglycerol transferase. This enzyme has been shown previously to promote repulsion of daptomycin at the cell surface by addition of positively charged lysine to phosphatidylglycerol. Also, the hlb open reading frame (ORF) encoding the β-toxin was interrupted by a prophage in the daptomycin-susceptible strain; this phage was missing in the daptomycin-resistant isolate and the hlb ORF was restored. Loss of the phage in the resistant isolate also resulted in loss of the virulence factor genes clpP, scn, and sak. This is the first study to use pyrosequencing to compare the genomes of a daptomycin-susceptible/resistant MRSA isolate pair obtained during failed daptomycin therapy in humans.

Fig. 1.

Patient's antibiotic treatment course.

Fig. 2.

MIC determination and growth of Dap^s (DapS) and Dap^r (DapR) strains in various concentrations of vancomycin and daptomycin. Numbers appearing in the legends refer to the amounts (in mg/liter) of antibiotic in which the strains were incubated.

Fig. 3.

Cell wall thickness comparisons of Dap^s (S) and Dap^r (R) strains. (A) Cell wall thickness measurements of peripheral and cross walls. (B) Transmission electron microscopy of Dap^s (top) and Dap^r (bottom) strains.

Fig. 4.

Dap resistance-associated polymorphisms in the translated mprF gene product, lysyl phosphatidylglycerol transferase (LPGT). Bars 1 to 14 refer to transmembrane domains in which mutations were found. Superscript letters indicate reference sources as follows: a, the present study; b, Yang et al. (65); c, Mishra et al. (48); d, Friedman et al. (29); e, Julian et al. (40); f, Murthy et al. (52); and g, Pillai et al. (56a).