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The Streptococcus mitis -oralis subgroup of the viridans group streptococci (VGS) are the most common cause of infective endocarditis (IE) in many parts of the world. These organisms are frequently resistant in vitro to standard β-lactams (e.g., penicillin; ceftriaxone [CRO]), and have the notable capacity for rapidly developing high-level and durable daptomycin resistance (DAP-R) during exposures in vitro , ex vivo , and in vivo .

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most β-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed ‘NaHCO 3 -responsiveness’, is a property whereby strains are susceptible in vitro to many β-lactams in the presence of NaHCO 3 .

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their

Increased usage of daptomycin (DAP) for methicillin-resistant Staphylococcus aureus (MRSA) infections has led to emergence of DAP-resistant (DAP-R) strains, resulting in treatment failures. DAP-fosfomycin (Fosfo) combinations are synergistically active against MRSA, although the mechanism(s) of this interaction are not fully understood. The current study explores four unique, but likely interrelated activities of DAP-Fosfo combinations: i) synergistic killing; ii) prevention of evolution of DAP-R; iii) resensitization of already DAP-R subpopulations to a DAP-susceptible (DAP-S) phenotype; and iv) perturbations of specific cell envelope phenotypes known to correlate with DAP-R in MRSA. Using an isogenic DAP-S (CB1483) / DAP-R (CB185) clinical MRSA strain-pair, we demonstrated that DAP + Fosfo combinations: i) enhanced killing of both strains in vitro and ex vivo; ii) increased target tissue clearances of the DAP-R strain in an in vivo model of experimental infective endocarditis (IE); iii) prevented emergence of DAP-R in the DAP-S parental strain both in vitro and ex vivo; and iv) resensitized the DAP-R strain to a DAP-S phenotype ex vivo. Phenotypically, following exposure to sub-MIC Fosfo, the DAP-S/ DAP-R strain-pair exhibited distinct modifications in: i) net positive surface charge (p

Increased usage of daptomycin (DAP) for methicillin-resistant Staphylococcus aureus (MRSA) infections has led to emergence of DAP-resistant (DAP-R) strains, resulting in treatment failures. DAP-fosfomycin (Fosfo) combinations are synergistically active against MRSA, although the mechanism(s) of this interaction is not fully understood. The current study explored four unique but likely interrelated activities of DAP-Fosfo combinations: (i) synergistic killing, (ii) prevention of evolution of DAP-R, (iii) resensitization of already DAP-R subpopulations to a DAP-susceptible (DAP-S) phenotype, and (iv) perturbations of specific cell envelope phenotypes known to correlate with DAP-R in MRSA. Using an isogenic DAP-S (CB1483)/DAP-R (CB185) clinical MRSA strain pair, we demonstrated that combinations of DAP plus Fosfo (DAP+Fosfo) (i) enhanced killing of both strains in vitro and ex vivo, (ii) increased target tissue clearances of the DAP-R strain in an in vivo model of experimental infective endocarditis (IE), (iii) prevented emergence of DAP-R in the DAP-S parental strain both in vitro and ex vivo, and (iv) resensitized the DAP-R strain to a DAP-S phenotype ex vivo. Phenotypically, following exposure to sub-MIC Fosfo, the DAP-S/DAP-R strain pair exhibited distinct modifications in (i) net positive surface charge (P

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious clinical threat due to innate virulence properties, high infection rates and the ability to develop resistance to multiple antibiotics, including the lipopeptide, daptomycin (DAP). Acquisition of DAP resistance (DAP-R) in MRSA has been linked with several characteristic alterations in the cell envelope. Clinical treatment of DAP-R MRSA infections has generally involved DAP plus β-lactam combinations, although definable synergy of such combinations varies in a strain-dependent, as well as a β-lactam-dependent manner. We investigated distinct β-lactam-induced cell envelope adaptations of nine clinically-derived DAP-susceptible (DAP-S)/DAP-R strain-pairs following in vitro exposure to a panel of six standard β-lactams (nafcillin, meropenem, cloxacillin, ceftriaxone, cefaclor, or cefoxitin) which differ in their PBP-targeting profiles. In general, in both DAP-S and DAP-R strains, exposure to these β-lactams led to: i) decreased positive surface charge; ii) decreased CM fluidity; iii) increased content and delocalization of anionic phospholipids (i.e., cardiolipin), with the delocalization more pronounced in DAP-R strains; and iv) increased DAP binding in DAP-S (but not DAP-R) strains. Collectively, these results suggest β-lactam-induced alterations in at least three major cell envelope phenotypes (surface charge, membrane fluidity and cardiolipin content) could underlie improved DAP activity, not mediated solely by an increase in DAP binding. This work was presented in part at the 30th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) Meeting, April 18-21, 2020; Poster #P3403 Note - For ease of presentation, we will utilize the terminology, "DAP-R", instead of "DAP-nonsusceptibility".

Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam susceptibility in vitro, ex vivo, and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Here, we investigate the impact of NaHCO3 on factors required for PBP2a functionality., Certain methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit β-lactam susceptibility in vitro, ex vivo, and in vivo in the presence of NaHCO3 (NaHCO3-responsive MRSA). Here, we investigate the impact of NaHCO3 on factors required for PBP2a functionality. Prototype NaHCO3-responsive and -nonresponsive MRSA strains (as defined in vitro) were assessed for the impact of NaHCO3 on the expression of genes involved in PBP2a production-maturation pathways (mecA, blaZ, pbp4, vraSR, prsA, sigB, and floA), membrane PBP2a and PrsA protein content, and membrane carotenoid content. Following NaHCO3 exposure in NaHCO3-responsive (versus nonresponsive) MRSA, there was significantly reduced expression of (i) mecA and blaZ, (ii) the vraSR-prsA gene axis, and (iii) pbp4. Carotenoid production was reduced while floA expression was increased by NaHCO3 exposure in all MRSA strains. This work underscores the distinct regulatory impact of NaHCO3 on a cadre of genes encoding factors required for the maintenance of the MRSA phenotype through PBP2a functionality and maturation.

Staphylococcus argenteus infection was initially described in Aboriginal patients in the Northern Territories of Australia as a predominant cause of skin infections and is rare outside Southeast Asia. A first well-characterized case of S. argenteus infection has now been described in the United States, involving a recurrent hemodialysis catheter infection, in which unstable daptomycin resistance evolved during daptomycin therapy. The unique colonial pigmentation of S. argenteus isolates in strains otherwise identified as Staphylococcus aureus is noteworthy.

Daptomycin-nonsusceptible (DAP-NS) Staphylococcus aureus often exhibits gain-in-function mutations in the mprF gene (involved in positive surface charge maintenance). Standard β-lactams, although relatively inactive against methicillin-resistant S. aureus (MRSA), may prevent the emergence of mprF mutations and DAP-NS. We determined if β-lactams might also impact DAP-NS isolates already possessing an mprF mutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory β-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates ("seesaw" effect). Prolonged (28-d) β-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexisting mprF polymorphisms, accumulation of additional mprF mutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both mprF-dependent phenotypes). β-lactams that either promiscuously or specifically target PBP-1 have significant DAP "resensitizing" effects against DAP-NS S. aureus strains. This may relate to the acquisition of multiple mprF single nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant therapeutic challenges related to its frequency in clinical infections, innate virulence properties, and propensity for multiantibiotic resistance. MRSA is among the most common causes of endovascular infections, including infective endocarditis (IE). Our objective was to employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE. TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (versus daptomycin alone). Primary intravegetation outcomes were maximum size, weights at sacrifice, and MRSA counts at infection baseline versus after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment day 1). A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase plus daptomycin versus the daptomycin alone therapy groups (P = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower than those of the daptomycin alone group (P < 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (P = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower than those of untreated controls (P < 0.0001) and the daptomycin alone group (P < 0.0001). This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size, and weight. Moreover, TTE appears to be an efficient noninvasive tool to assess therapeutic efficacies in experimental MRSA IE.

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) increases β-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains (“NaHCO3 responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO3-responsive or NaHCO3-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO3. Intra-SEV MRSA killing was determined over 72-h exposures. In both “responsive” strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO3-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.

Higher vancomycin MICs have been associated with more complicated courses and higher mortality rates in patients with Staphylococcus aureus bacteremia and infective endocarditis (IE). The aim of this study was to investigate whether the strains belonging to the cohort of 93 patients from a previously published study in which patients with strains with vancomycin MICs of ≥1.5 μg/ml presented higher mortality rates and systemic emboli than patients with strains with vancomycin MICs of

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO(3)) increases β-lactam susceptibility of selected methicillin-resistant Staphylococcus aureus (MRSA) strains (“NaHCO(3) responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO(3)-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro NaHCO(3)-responsive or NaHCO(3)-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO(3). Intra-SEV MRSA killing was determined over 72-h exposures. In both “responsive” strains, supplementation with 25 mM or 44 mM NaHCO(3) significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither in vitro-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO(3) concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO(3)), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO(3)-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO(3) supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO(3)-supplemented media in vitro.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of β-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-β-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

Bacteriophage-derived lysins are cell-wall-hydrolytic enzymes that represent a potential new class of antibacterial therapeutics in development to address burgeoning antimicrobial resistance. CF-301, the lead compound in this class, is in clinical development as an adjunctive treatment to potentially improve clinical cure rates of Staphylococcus aureus bacteremia and infective endocarditis (IE) when used in addition to antibiotics. In order to profile the activity of CF-301 in a clinically relevant milieu, we assessed its in vitro activity in human blood versus in a conventional testing medium (cation-adjusted Mueller-Hinton broth [caMHB]). CF-301 exhibited substantially greater potency (32 to ≥100-fold) in human blood versus caMHB in three standard microbiologic testing formats (e.g., broth dilution MICs, checkerboard synergy, and time-kill assays). We demonstrated that CF-301 acted synergistically with two key human blood factors, human serum lysozyme (HuLYZ) and human serum albumin (HSA), which normally have no nascent antistaphylococcal activity, against a prototypic methicillin-resistant S. aureus (MRSA) strain (MW2). Similar in vitro enhancement of CF-301 activity was also observed in rabbit, horse, and dog (but not rat or mouse) blood. Two well-established MRSA IE models in rabbit and rat were used to validate these findings in vivo by demonstrating comparable synergistic efficacy with standard-of-care anti-MRSA antibiotics at >100-fold lower lysin doses in the rabbit than in the rat model. The unique properties of CF-301 that enable bactericidal potentiation of antimicrobial activity via activation of “latent” host factors in human blood may have important therapeutic implications for durable improvements in clinical outcomes of serious antibiotic-resistant staphylococcal infections.

Optimal treatment options remain unknown for infective endocarditis (IE) caused by penicillin-resistant (PEN-R) viridans group streptococcal (VGS) strains. The aims of this study were to report two cases of highly PEN-R VGS IE, perform a literature review, and evaluate various antibiotic combinations in vitro and in vivo. The following combinations were tested by time-kill studies and in the rabbit experimental endocarditis (EE) model: PEN-gentamicin, ceftriaxone-gentamicin, vancomycin-gentamicin, daptomycin-gentamicin, and daptomycin-ampicillin. Case 1 was caused by Streptococcus parasanguinis (PEN MIC, 4 μg/ml) and was treated with vancomycin plus cardiac surgery. Case 2 was caused by Streptococcus mitis (PEN MIC, 8 μg/ml) and was treated with 4 weeks of vancomycin plus gentamicin, followed by 2 weeks of vancomycin alone. Both patients were alive and relapse-free after ≥6 months follow-up. For the in vitro studies, except for daptomycin-ampicillin, all combinations demonstrated both synergy and bactericidal activity against the S. parasanguinis isolate. Only PEN-gentamicin, daptomycin-gentamicin, and daptomycin-ampicillin demonstrated both synergy and bactericidal activity against the S. mitis strain. Both strains developed high-level daptomycin resistance (HLDR) during daptomycin in vitro passage. In the EE studies, PEN alone failed to clear S. mitis from vegetations, while ceftriaxone and vancomycin were significantly more effective (P

Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major health care concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as “resistant” to β-lactams, often leading to the use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied., Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major health care concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as “resistant” to β-lactams, often leading to the use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied. We identified two distinct MRSA phenotypes upon AST using standard media, with or without sodium bicarbonate (NaHCO3) supplementation: one highly susceptible to the antistaphylococcal β-lactams oxacillin and cefazolin (NaHCO3 responsive) and one resistant to such agents (NaHCO3 nonresponsive). These phenotypes accurately predicted clearance profiles of MRSA from target tissues in experimental MRSA IE treated with each β-lactam. Mechanistically, NaHCO3 reduced the expression of two key genes involved in the MRSA phenotype, mecA and sarA, leading to decreased production of penicillin-binding protein 2a (that mediates methicillin resistance), in NaHCO3-responsive (but not in NaHCO3-nonresponsive) strains. Moreover, both cefazolin and oxacillin synergistically killed NaHCO3-responsive strains in the presence of the host defense antimicrobial peptide (LL-37) in NaHCO3-supplemented media. These findings suggest that AST of MRSA strains in NaHCO3-containing media may potentially identify infections caused by NaHCO3-responsive strains that are appropriate for β-lactam therapy.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of β-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-β-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log(10) CFU/g amounts within SEVs for both strains (∼6 log(10) CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

The phenotypic expression of methicillin resistance among coagulase-negative staphylococci (CoNS) is heterogeneous regardless of the presence of the mecA gene. The potential discordance between phenotypic and genotypic results has led to the use of vancomycin for the treatment of CoNS infective endocarditis (IE) regardless of methicillin MIC values. In this study, we assessed the outcome of methicillin-susceptible CoNS IE among patients treated with antistaphylococcal β-lactams (ASB) versus vancomycin (VAN) in a multicenter cohort study based on data from the International Collaboration on Endocarditis (ICE) Prospective Cohort Study (PCS) and the ICE-Plus databases. The ICE-PCS database contains prospective data on 5,568 patients with IE collected between 2000 and 2006, while the ICE-Plus database contains prospective data on 2,019 patients with IE collected between 2008 and 2012. The primary endpoint was in-hospital mortality. Secondary endpoints were 6-month mortality and survival time. Of the 7,587 patients in the two databases, there were 280 patients with methicillin-susceptible CoNS IE. Detailed treatment and outcome data were available for 180 patients. Eighty-eight patients received ASB, while 36 were treated with VAN. In-hospital mortality (19.3% versus 11.1%; P = 0.27), 6-month mortality (31.6% versus 25.9%; P = 0.58), and survival time after discharge ( P = 0.26) did not significantly differ between the two cohorts. Cox regression analysis did not show any significant association between ASB use and the survival time (hazard ratio, 1.7; P = 0.22); this result was not affected by adjustment for confounders. This study provides no evidence for a difference in outcome with the use of VAN versus ASB for methicillin-susceptible CoNS IE.

Tedizolid, a novel oxazolidinone, exhibits bacteriostatic activity through inhibition of protein synthesis. The efficacies of tedizolid, linezolid, and vancomycin were compared in a murine catheter-related biofilm infection caused by methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) strains engineered for bioluminescence. We observed significantly improved efficacy in terms of decreased S. aureus densities and bioluminescent signals in the tedizolid-treated group versus the linezolid- and vancomycin-treated groups in the model of infection caused by the MSSA and MRSA strains.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro. All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus . (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP s )/daptomycin-resistant (DAP r ) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP r strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP r strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP r strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP r strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus .

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.

The cytoplasmic membrane of Staphylococcus aureus contains ∼20 mol% of the net cationic lipid lysyl-phosphatidylglycerol (LPG). Elevated fractions of LPG are associated with increased resistance to cationic antibiotics, including the lipopeptide daptomycin (DAP). Although the surface charge of the bacterial cytoplasmic membrane is altered by LPG, surface binding of DAP was found to be only moderately affected in anionic vesicles containing 20 mol% LPG. These results suggest that charge repulsion cannot fully explain LPG-mediated resistance to cationic peptides.

Many serious bacterial infections are antibiotic refractory due to biofilm formation. A key structural component of biofilm is extracellular DNA, which is stabilized by bacterial proteins, including those from the DNABII family. TRL1068 is a high-affinity human monoclonal antibody against a DNABII epitope conserved across both Gram-positive and Gram-negative bacterial species. In the present study, the efficacy of TRL1068 for the disruption of biofilm was demonstrated in vitro in the absence of antibiotics by scanning electron microscopy. The in vivo efficacy of this antibody was investigated in a well-characterized catheter-induced aortic valve infective endocarditis model in rats infected with a methicillin-resistant Staphylococcus aureus (MRSA) strain with the ability to form thick biofilms, obtained from the blood of a patient with persistent clinical infection. Animals were treated with vancomycin alone or in combination with TRL1068. MRSA burdens in cardiac vegetations and within intracardiac catheters, kidneys, spleen, and liver showed significant reductions in the combination arm versus vancomycin alone ( P < 0.001). A trend toward mortality reduction was also observed ( P = 0.09). In parallel, the in vivo efficacy of TRL1068 against a multidrug-resistant clinical Acinetobacter baumannii isolate was explored by using an established mouse model of skin and soft tissue catheter-related biofilm infection. Catheter segments infected with A. baumannii were implanted subcutaneously into mice; animals were treated with imipenem alone or in combination with TRL1068. The combination showed a significant reduction of catheter-adherent bacteria versus the antibiotic alone ( P < 0.001). TRL1068 shows excellent promise as an adjunct to standard-of-care antibiotics for a broad range of difficult-to-treat bacterial infections.

Over the past several years, single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been proposed to be associated with a gain-of-function phenotype in terms of daptomycin (DAP) nonsusceptibility (referred to as daptomycin resistance [DAP-R] herein for ease of presentation) in Staphylococcus aureus . We investigated the frequencies of SNPs within the mprF ORF and the relationships of such SNPs to cross-resistance between DAP and cationic host defense peptides (HDPs). Thirty-five well-characterized, unique DAP-susceptible (DAP-S) and DAP-R methicillin-resistant S. aureus (MRSA) isolates of the clonal complex 5 genotype were used. In addition to mprF SNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i) mprF expression, (ii) membrane phospholipid content, (iii) positive surface charge, (iv) DAP binding, and (v) cell wall thickness profiles. A number of DAP-S strains (MICs of ≤1 μg/ml) exhibited mprF SNPs, occasionally with high-level mprF sequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicled mprF hot spot locations associated with DAP-R in S. aureus . In contrast, all 8 DAP-R isolates demonstrated SNPs within such known mprF hot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhanced mprF expression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array of mprF SNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrent mprF dysregulation, were associated with the DAP-R phenotype.

Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAP r ) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAP r strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic Δ mprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAP s )-DAP r strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the Δ mprF strain with singly point-mutated mprF genes ( mprF S295L or mprF T345A ) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAP r strains, the defined mprF SNPs appear to be causally related to this phenotype.