Heterogeneity of
            <i>mprF</i>
            Sequences in Methicillin-Resistant Staphylococcus aureus Clinical Isolates: Role in Cross-Resistance between Daptomycin and Host Defense Antimicrobial Peptides

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Staphylococcus aureus is the second leading cause of bacteremia worldwide and the most important pathogen in endovascular infections (1, 2). S. aureus has a particular propensity for developing multidrug resistance (e.g., methicillin-resistant S. aureus [MRSA] and vancomycin-intermediate S. aureus [VISA]), and serious infections with such strains result in enhanced attributable mortality (3).Since its FDA approval in 2003, daptomycin (DAP) has been utilized in many clinical settings, especially for recalcitrant MRSA infections, such as endocarditis (4-6). However, there have been a number of recently reported cases of clinical S. aureus strains that have evolved in vitro DAP resistance in the context of failing DAP treatment regimens, especially in endovascular syndromes. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation [7][8][9][10][11][12].) One of the major features of DAP-resistant (DAP r ) S. aureus strains is the acquisition of gain-in-function mutations in a relatively restricted cadre of genes, especially in the mprF locus (13-18). The MprF protein is responsible for the synthesis and translocation (flipping) of the unique positively charged phospholipid (PL) lysyl-phosphotidylglycerol (L-PG) from the inner-to-outer cell membrane (CM) leaflet (18)(19)(20). Increases in L-PG synthesis and flipping usually result in augmented positive surface charge; many investigators have speculated that this event leads to a charge-repulsive milieu for cationic molecules, such as host defense peptides (HDPs) and calcium-complexed DAP (13,(19)(20)(21)(22)(23). Over the past several years, a number of laboratories, including ours, have linked the presence of singlenucleotide polymorphisms (SNPs) within the mprF locus to the DAP r phenotype (13,14, 21,(24)(25)(26). These SNPs have occurred throughout the mprF open reading frame (ORF), although there are clearly hot spots within this locus for those SNPs linked to DAP resistance (13,14,19, 23). Investigations of mprF SNPs associated with DAP resistance in S. aureus have principally emerged from studies that used only a few or individual isogenic DAP-susceptible (DAP s ) and DAP r strain pairs. To address this limitation, in the current study, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs using 22 DAP s /DAP r isogenic clinical MRSA strain pairs, (ii) the relation-

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confidence: 99%

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Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides

Bayer

Mishra

Chen

et al. 2015

Self Cite

“…MprF is a bifunctional membrane protein responsible for adding lysine to phosphatidylglycerol (PG) and for flipping the positively charged lysyl-PG product to the outer leaflet of the cytoplasmic membrane (7). The SNPs identified in mprF in DAP-NS isolates are located in specific regions of the mprF gene and appear to be gain-in-function mutations hypothesized to decrease susceptibility to Ca 2ϩ -complexed daptomycin by a charge repulsive mechanism (2,8). Studies of the transcriptomic response of S. aureus to daptomycin revealed that daptomycin exposure, in addition to inducing genes involved in cell membrane depolarization, also induced the cell wall stress regulon, indicating that daptomycin directly or indirectly interferes with cell wall synthesis (9).…”

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confidence: 99%

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Bæk

Thøgersen

Mogenssen

et al. 2015

dDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A 477 D) that decreased susceptibility not only to daptomycin but also to vancomycin, ␤-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S 295 L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits. Staphylococcus aureus is a commensal bacterium that can cause a variety of both localized and more invasive infections. Due to its profound ability to acquire resistance to clinically relevant antibiotics, S. aureus remains a major clinical challenge worldwide (1). The most challenging antimicrobial resistance issue in S. aureus has been the evolution and dissemination of methicillinresistant S. aureus (MRSA) strains first in hospitals and later in the general community (1). MRSA infections are typically treated with last-line antibiotics, such as vancomycin and, more recently, daptomycin (DAP), a cyclic lipopeptide derived from Streptomyces roseosporus with bactericidal activity against a broad range of Gram-positive bacteria (2, 3). The mechanism of action of daptomycin has not been fully elucidated, but it is generally accepted that daptomycin inserts into the cytoplasmic membrane of Grampositive bacteria via a calcium-dependent pathway, leading to potassium efflux, membrane depolarization, and, eventually, cell death (2).The development of daptomycin nonsusceptibility (DAP-NS) in S. aureus seems to be a relatively rare phenomenon not involving horizontal gene transfer but, instead, taking place by a stepwise acqu...

“…The nonsusceptibility among these isolates appears to be mediated at least partially through charge repulsion, with resistant strains possessing a more positive surface charge, repelling DAP (19). Indeed, in vitro data suggest that there is a strong correlation between DAP nonsusceptibility and resistance to other positively charged, endogenous antimicrobial proteins (20)(21)(22).There is a need to find appropriate measures to prevent DAP nonsusceptibility and treat such organisms.…”

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β-Lactams Enhance Daptomycin Activity against Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium in In Vitro Pharmacokinetic/Pharmacodynamic Models

Smith

Barber

Raut

et al. 2015

Enterococcus faecalis and Enterococcus faecium are frequently resistant to vancomycin and ␤-lactams. In enterococcal infections with reduced glycopeptide susceptibility, combination therapy is often administered. Our objective was to conduct pharmacokinetic/pharmacodynamic (PK/PD) models to evaluate ␤-lactam synergy with daptomycin (DAP) against resistant enterococci. One E. faecalis strain (R6981) and two E. faecium strains (R6370 and 8019) were evaluated. DAP MICs were obtained. All strains were evaluated for response to LL37, an antimicrobial peptide, in the presence and absence of ceftaroline (CPT), ertapenem (ERT), and ampicillin (AMP). After 96 h, in vitro models were run simulating 10 mg DAP/kg body weight/day, 600 mg CPT every 8 h (q8h), 2 g AMP q4h, and 1 g ERT q24h, both alone and in combination against all strains. DAP MICs were 2, 4, and 4 g/ml for strains R6981, R6370, and 8019, respectively. PK/PD models demonstrated bactericidal activity with DAP-CPT, DAP-AMP, and DAP-ERT combinations against strain 8019 (P < 0.001 and log 10 CFU/ml reduction of >2 compared to any single agent). Against strains R6981 and R6370, the DAP-AMP combination demonstrated enhancement against R6370 but not R6981, while the combinations of DAP-CPT and DAP-ERT were bactericidal, demonstrated enhancement, and were statistically superior to all other regimens at 96 h (P < 0.001) against both strains. CPT, ERT, and AMP similarly augmented LL37 killing against strain 8019. In strains R6981 and R6370, CPT and ERT aided LL37 more than AMP (P < 0.001). Compared to DAP alone, combination regimens provide better killing and prevent resistance. Clinical research involving DAP combinations is warranted. Enterococcus faecalis and Enterococcus faecium together account for 12% of hospital-acquired infections in the United States (1). Often, enterococcal infections are caused by multidrug-resistant strains. For example, 0.4 to 5.2% and 70 to 92.6% of E. faecalis and E. faecium strains are resistant to ampicillin, respectively, and vancomycin resistance is present in up to 12.5% of E. faecalis and 79.7% of E. faecium (2-4). Vancomycin-resistant enterococci (VRE) are associated with increased mortality and complicated infections, such as infective endocarditis (5). Treatment of VRE infections can prove problematic, as available treatment options are potentially limited by static activity and/or platelet suppression with long-term use (6-8). Daptomycin (DAP) is a bactericidal lipopeptide often used against resistant enterococci (9). Mechanistically, it binds with calcium to form a cationic moiety that disrupts membrane potential to confer its antimicrobial effects, similar to endogenous, cationic antimicrobial peptides (10, 11). DAP is frequently dosed at 6 mg/kg body weight daily, although recent clinical and in vitro data suggest improved efficacy at higher doses (7,(12)(13)(14). DAP retains excellent in vitro activity against E. faecalis and E. faecium, with MIC 50/90 values of 1/2 and 2/4 g/ml, respectively (15). Reports of DAP-nonsuscept...