Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost

Abstract

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Keywords: MRSA; biological cost; daptomycin; in vivo.

FIGURE 1

Timeline of the infection and treatments related to the isolation of the clinical SA6819, SA6820, and SA6850 strains. (d: day, CIP: ciprofloxacin, DAP: daptomycin, LNZ: linezolid, RIF: rifampicin, SXT: trimethoprim/sulfamethoxazole, and VAN: vancomycin).

FIGURE 2

(A) Competition experiment between the clinical SA6820 (DAP-R) and the clinical recurrent SA6850 (DAP-S) strains. The DAP-R sub-population of SA6820 is indicated with (•), the whole population (SA6820 + SA6850) growing on BHI agar without antibiotic is indicated with (x). (B) Single culture of SA6820 showed that in absence of competition with a susceptible strain, the resistance is stable during 10 days.

FIGURE 3

(A) Progressive reversion of DAP resistance during extended in vitro single culture of the clinical DAP-R strain SA6820 by daily serial passages in drug-free BHI broth. Every 5 days, enumeration of the DAP resistant population was performed by plating the culture and its serial dilutions on BHI agar supplemented with calcium (CaCl₂, 50 mg/L) and containing or not DAP 2 mg/L resulting in (SA6820-revertant). The DAP-R sub-population of SA6820 is indicated with (•), the whole population (SA6820 + SA6820-revertant) growing on BHI agar without antibiotic is indicated with (x). (B) Competition experiment between the clinical strain SA6820 (DAP-R) and its in vitro revertant SA6820-revertant (DAP-S). The DAP-R sub-population of SA6820 is indicated with (•), the whole population growing on BHI agar without antibiotic is indicated with (x).

FIGURE 4

Phylogenetic tree based on the SNP comparisons versus N315 showing the evolution of the different isolates: the strains SA6819, SA6820, SA6820-revertant, and SA6850 are shown in bold. All strains represented are from the ST5 MLST type.

FIGURE 5

(A) Competitive cultures performed between SA6820ΔmprF and SA6820 showed increased fitness of SA6820ΔmprF over SA6820 (DAP-R). DAP-R sub-population of SA6820 is indicated with (•) and the whole population growing on BHI agar (SA6820 + SA6820ΔmprF) is indicated with (x). (B) Competition experiment between the two DAP-S strains: SA6850 (ciprofloxacin marker □) and SA6820ΔmprF (chloramphenicol marker Δ).

FIGURE 6

(A) Competition experiment between the clinical strains CB-5014 (DAP-R) and CB-5013 (DAP-S) and (B) CB-1634 (DAP-R) and CB-1631 (DAP-S) in comparison with the single culture of their corresponding DAP-R resistant counterparts (C,D). The DAP-R sub-population of each experiment is indicated with (•), and is represented by either CB5014 (C) or CB1634 (D) grown with DAP; the whole population of CB5014 (C) + CB1634 (D) growing on BHI agar without antibiotic is indicated with (x).