Activity Of Daptomycin Research Articles

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to β-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Daptomycin Liposomes Exhibit Enhanced Activity against Staphylococci Biofilms Compared to Free Drug.

The purpose of the present study was to investigate the anti-staphylococcal activity of liposomal daptomycin against four biofilm-producing S. aureus and S. epidermidis clinical strains, three of which are methicillin-resistant. Neutral and negatively charged daptomycin-loaded liposomes were prepared using three methods, namely, thin-film hydration (TFH), a dehydration-rehydration vesicle (DRV) method, and microfluidic mixing (MM); moreover, they were characterized for drug encapsulation (EE%), size distribution, zeta-potential, vesicle stability, drug release, and drug integrity. Interestingly, whilst drug loading in THF and DRV nanosized (by extrusion) vesicles was around 30-35, very low loading (~4%) was possible in MM vesicles, requiring further explanatory investigations. Liposomal encapsulation protected daptomycin from degradation and preserved its bioactivity. Biofilm mass (crystal violet, CV), biofilm viability (MTT), and growth curve (GC) assays evaluated the antimicrobial activity of neutral and negatively charged daptomycin-liposomes towards planktonic bacteria and biofilms. Neutral liposomes exhibited dramatically enhanced inhibition of bacterial growth (compared to the free drug) for all species studied, while negatively charged liposomes were totally inactive. Biofilm prevention and treatment studies revealed high antibiofilm activity of liposomal daptomycin. Neutral liposomes were more active for prevention and negative charge ones for treating established biofilms. Planktonic bacteria as well as the matured biofilms of low daptomycin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) strains were almost completely eradicated by liposomal-daptomycin, indicating the need for their further exploration as antimicrobial therapeutics.

Thiazolopyrimidinone Derivative H5-23 Enhances Daptomycin Activity against Linezolid-Resistant Enterococcus faecalis by Disrupting the Cell Membrane.

The increasing emergence and dissemination of multidrug-resistant (MDR) Gram-positive pathogens pose a serious threat to global public health. Previous reports have demonstrated that the compound H5-23, which has a thiazolopyrimidinone core structure, exhibited antibacterial activity against Staphylococcus epidermidis in vitro. However, the antibacterial activity in vivo and mechanism of action of H5-23 against MDR bacteria have not been fully studied. In this study, we report that H5-23 has wide-spectrum antibacterial activity against Gram-positive bacteria. When combined with daptomycin (DAP), H5-23 demonstrates enhanced antimicrobial activity, effectively killing both planktonic and persister cells, as well as eradicating biofilm formation by linezolid-resistant Enterococcus faecalis. The development of resistance shows that H5-23 has a low propensity to induce antibiotic resistance compared to that of linezolid in vitro. Mechanistic studies reveal that H5-23 increases membrane permeability and disrupts membrane integrity, resulting in increased production of reactive oxygen species (ROS), metabolic perturbations, and ultimately cell death. Additionally, we demonstrate the synergistic antibacterial effect of H5-23 combined with DAP in a murine model. These findings suggest that H5-23 is a promising antimicrobial agent and provides a potential strategy for enhancing the efficacy of DAP in combating multidrug-resistant E. faecalis.

Antimicrobial Activity of Ceragenins against Vancomycin-Susceptible and -Resistant Enterococcus spp.

Ceragenins (CSAs) are a new class of antimicrobial agents designed to mimic the activities of endogenous antimicrobial peptides. In this study, the antibacterial activities of various ceragenins (CSA-13, CSA-44, CSA-90, CSA-131, CSA-138, CSA-142, and CSA-192), linezolid, and daptomycin were assessed against 50 non-repeated Enterococcus spp. (17 of them vancomycin-resistant Enterococcus-VRE) isolated from various clinical specimens. Among the ceragenins evaluated, the MIC50 and MIC90 values of CSA-44 and CSA-192 were the lowest (2 and 4 μg/mL, respectively), and further studies were continued with these two ceragenins. Potential interactions between CSA-44 or CSA-192 and linezolid were tested and synergistic interactions were seen with the CSA-192-linezolid combination against three Enterococcus spp., one of them VRE. The effects of CSA-44 and CSA-192 on the MIC values of vancomycin were also investigated, and the largest MIC change was seen in the vancomycin-CSA-192 combination. The in vivo effects of CSA-44 and CSA-192 were evaluated in a Caenorhabditis elegans model system. Compared to no treatment, increased survival was observed with C. elegans when treated with ceragenins. In conclusion, CSA-44 and CSA-192 appear to be good candidates (alone or in combination) for the treatment of enterococcal infections, including those from VRE.

1824. Combination therapy of daptomycin/colistin for extreme drug resistant Acinetobacter baumannii bacteremia

Abstract Background There are experimental data demonstrating synergistic in-vitro activity of colistin and daptomycin against extremely drug-resistant (XDR) strains of Acinetobacter baumannii.Aim of this study is the description of a case-series of patients with confirmed XDR A. baumannii bloodstream infection (BSI) receiving an antimicrobial regimen including colistin and daptomycin. Methods Prospective study of patients with BSI caused by XDR A. baumannii from May 2021 to February 2022 in a tertiary level hospital. Demographic, clinical and laboratory data were recorded and evaluated from all patients. BacT Alert system (BiomerieuxR) was used for blood cultures and bacterial identification and susceptibility testing was performed using VITEK® 2. Colistin susceptibility testing (dilution method) were interpreted in accordance to Clinical and Laboratory Standards Institute (CLSI) criteria. Results A total of 16 episodes of XDR A. baumannii were recorded in 16 patients (14 males). Median age was 60 (range 38-85) years. Ten patients (56.2%) were hospitalized in medical wards, and 43.8% in surgery wards. One-third of patients had previously been admitted to the Intensive Care Unit, while all patients had an indwelling central vascular catheter. Fourteen of the 16 XDR strains of A. baumannii were also resistant to colistin. Also, 2 of the 16 strains had a MIC of 2μg/ml for tigecycline, while in the remaining 14 the MIC was > 2μg/ml. All isolates had meropenem MIC >16μg/ml. All patients received a combination of colistin -daptomycin -meropenem -tigecycline. Clinical and laboratory improvement was noted in 15 of the 16 patients within the 14 days of treatment. Fifteen out of the 16 patients showed a microbiological response: the mean duration between the start of treatment and the first negative blood culture was 12 days.Finally, no patient experienced a marked increase in CPK due to daptomycin use. Conclusion The outcome of this case series of patients receiving colistin-daptomycin combination as part of a broader synergistic antimicrobial regimen to treat XDR A. baumannii was satisfactory, with no significant adverse reactions. This potential clinical benefit of the colistin/daptomycin combination should be further evaluated in a larger comparative study. Disclosures All Authors: No reported disclosures.

Daptomycin exerts differential immunomodulatory effects on host responses against methicillin-resistant Staphylococcus aureus biofilms

Daptomycin (DAP) is indicated for difficult-to-treat Gram-positive infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). Exposure of S. aureus to subinhibitory antimicrobial concentrations (sub-MICs) has been shown to alter cell morphology and biofilm formation. This study aimed to investigate the influence of DAP biofilm sub-MICs on the damage caused by human polymorphonuclear neutrophils (PMNs) against MRSA biofilms and the potential immunomodulatory activity of DAP on human monocytes (MNCs) exposed to MRSA biofilms. DAP activity against biofilms and the impact of DAP on PMN-induced biofilm damage were evaluated by the XTT reduction assay, whereas pathogen recognition, signal transduction and cytokine modulation of DAP on MNCs in response to MRSA biofilms were assessed by RT-PCR and ELISA methodology. The MIC50 of DAP to MRSA biofilms was 16-32 mg/L. Pre-treatment of MRSA with 1, 2 or 4 mg/L DAP caused a synergistic effect on PMN-mediated biofilm damage, being dependent on the effector-to-target ratio. MNCs responded to MRSA biofilms and DAP through Toll like receptor 2 (TLR2) upregulation and increased NLRP3 inflammasome production. DAP caused 2.5-fold greater TLR2 mRNA levels than those caused by MRSA biofilms. A predominantly inflammatory response was induced by either component, causing the release of significantly increased IFN-γ, TNF-α, IL-8 and IL-6 levels by MNCs exposed to the combination treatment. MRSA biofilms alone or combined with DAP caused low amounts of IL-10 production, but increased IL-1β levels. DAP may condition MNCs towards an inflammatory response through TLR2 engagement and NLRP3 inflammasome activation, possibly controlling biofilm-associated pathogenicity.

A54145 Factor D Is Not Less Susceptible to Inhibition by Lung Surfactant than Daptomycin.

A54145 factor D (A5D) is a cyclic lipopeptide antibiotic that shares several structural and mechanistic features with the clinically important antibiotic daptomycin, such as their requirement for calcium and phosphatidylglycerol (PG) for activity. Studies by others have suggested that daptomycin's activity is strongly inhibited by lung surfactant while A5D's activity is not. This finding has inspired efforts, albeit unsuccessful, to develop an A5D analogue that is highly active in the presence of lung surfactant and can be used for treating community acquired pneumonia (CAP). Here we demonstrate that A5D, like daptomycin, has a strong preference for the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration) of PG. This PG stereoisomer was determined to be the only stereoisomer of PG in lung surfactant. Both antibiotics are completely antagonized by approximately 1-2 mol equiv of 2R,2'S-PG. Studies performed in the presence of lung surfactant revealed that the antagonism of these peptides by surfactant is mainly due to their interaction with PG and that A5D is not significantly less susceptible to inhibition by lung surfactant than daptomycin.

Antibiotic sensitivity analysis of clinical coagulase-negative staphylococci

Background. The increasing role of coagulase-negative staphylococci in the occurrence of staphylococcal infections leads to the need for close attention to them. Special control is required over the sensitivity of bacteria to antibiotics and the spread of methicillin resistance, as a sign of multiple resistance to antibacterial drugs. It is also important to identify the virulence factors of coagulase-negative staphylococci, which determine their behavior in the environment.The aim. To evaluate the sensitivity of strains of coagulase-negative staphylococci to clinically significant antibiotics daptomycin, vancomycin, linezolid and oxacillin and lantibiotic warnerin.Methods. Determination of the minimal inhibitory concentrations of antibacterial compounds for clinical coagulase-negative staphylococci by standard methods of serial dilutions and disc diffusion. Identification of the phenomenon of decreased susceptibility of bacteria to vancomycin by population analysis and concentration gradient. Lipid analysis by thin layer chromatography. Results. High antibacterial activity of vancomycin, daptomycin and linezolid against clinical strains of coagulase-negative staphylococci was shown. The upper limit of the minimum inhibitory concentrations of vancomycin within the sensitive phenotype and the expansion of the ranges of the minimum inhibitory concentrations of daptomycin and warnerin towards an increase in oxacillin-resistant isolates were revealed. The heterogeneous nature of sensitivity to vancomycin of the cultures of the studied strains and the possibility of their rapid enrichment with subpopulations with reduced sensitivity to this antibiotic have been established. The selection of resistance of coagulase-negative staphylococci to vancomycin was accompanied by an increase in the synthesis of lysylphosphatidylglycerol and a decrease in their sensitivity to cationic peptide compounds.Conclusion. The revealed prevalence of the methicillin-resistant phenotype of clinical strains of coagulase-negative staphylococci, along with the presence in the lipid spectrum of the universal factor of resistance to cationic antibacterial compounds, lysylphosphatidylglycerol, entails the need for new methodological solutions for diagnosing infections caused by coagulase-negative staphylococci.

Addition of daptomycin to levofloxacin increased the efficacy of levofloxacin monotherapy against a methicillin-susceptible Staphylococcus aureus strain in experimental meningitis and prevented development of resistance in vitro.

Introduction. Daptomycin and levofloxacin were tested as monotherapies and in combination against the antibiotic-susceptible S. aureus strain MSSA 1112 in a rabbit meningitis model and the effect of the combination on induction of resistance was determined in vitro. Hypothesis/Gap Statement. Treatment combination might be more efficacious than the monotherapies regarding antibacterial efficacy in vivo and development of resistance. Aim. To demonstrate a synergy of the antibiotic combination daptomycin and levofloxacin in experimental meningitis, and also its ability to reduce the emergence of resistance against the antibiotics in vitro. Methodology. Changes of the susceptibility to fluoroquinolones and daptomycin were determined by the measurement of the MIC and mutations were detected by whole genome sequence comparison of the mutants with the parent strain MSSA 1112. Meningitis was induced by intracisternal inoculation of 105 c.f.u. (colony forming unit) of MSSA 1112 and treatment was started 10 h later by injection of daptomycin (15 mg kg−1) and levofloxacin (10 mg kg−1) standard doses. Cerebrospinal fluid (CSF) samples were repeatedly collected during therapy in order to determine killing rates and results of bactericidal activity were expressed in Δlog10c.f.u. ml-1 over 8 h. Results. The combination of daptomycin with levofloxacin was significantly (P<0.001) superior to levofloxacin monotherapy and increased the antibacterial activity of daptomycin. In vitro, MSSA 1112 was cycled over 6 days with either increasing concentrations of levofloxacin or daptomycin or with a combination of levofloxacin with half of the MIC of daptomycin or daptomycin with half of the MIC of levofloxacin leading to mutations in target genes as identified by whole genome sequence analysis. Addition of low concentration of daptomycin (0.25 mg l−1) reduced levofloxacin-induced resistance in vitro. Addition of levofloxacin in low concentration (0.125 mg l−1) did not influence daptomycin-induced resistance. Conclusion. These findings highlight the lack of reciprocal interference of antibiotics in combination with regard to the development of resistance.

Proteomic Correlates of Enhanced Daptomycin Activity following β-Lactam Preconditioning in Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus.

Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus β-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro β-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected β-lactam pretreatments on DAP plus β-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring β-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each β-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without β-lactam preconditioning. Relative proteomic quantitation comparing β-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with β-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to β-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

In-vitro Activity of Tigecycline versus Daptomycin against Clinical Isolates of Enterococcus Species

Introduction: Tigecycline is a potential therapeutic agent for multidrug resistant gram positive and gram negative organisms. The clinical efficacy of synergic combination of daptomycin with betalactam antibiotics have been described against various species of Enterococcus isolates. Aim: To detect the in-vitro activity of tigecycline and daptomycin against Enterococcus species and their antimicrobial activity with Vitek 2 automated system. Materials and Methods: This was a cross-sectional study conducted at Department of Microbiology, Vinayaka Mission’s Kirupananda Variyar (VMKV) Medical College and Hospitals, Salem, Tamil Nadu, India. Isolates of Enterococcus obtained over a period of two years from March 2016 - March 2018 from various clinical samples were identified by standard biochemical method and their antimicrobial susceptibility pattern was determined. Minimum Inhibitory Concentration (MIC) of tigecycline and daptomycin was determined by Vitek 2 automated system. Analysis of data was done by using frequency distribution and percentage. Results: Out of 211 Enterococcus isolates studied, 23 (10.90%) isolates showed decreased susceptibility to daptomycin with MIC of ≥8 µg/mL. All isolates showed 100% susceptibility to tigecycline. Conclusion: The present study showed potent antimicrobial activity of tigecycline against various species of Enterococcus. Decreased susceptibility to daptomycin has been observed among the study isolates. Further clinical investigations are required to know the potential benefits of these agents in therapy.

A RARE CASE OF SUCCESSFUL TREATMENT OF PROSTHETIC MITRAL VALVE INFECTIVE ENDOCARDITIS WITH A NONSURGICAL APPROACH

TOPIC: Cardiovascular Disease TYPE: Medical Student/Resident Case Reports INTRODUCTION: Infective endocarditis (IE) is an infection of the endocardial surface of the valve, native or prosthetic. Primary treatment for IE involving a prosthetic valve is surgical intervention (1, 2) and if left untreated can be fatal. Incidence rate of deaths associated with IE is 4.6/100,000/year (1). We present a case of a 64-year-old male who was successfully treated medically without any surgical intervention for prosthetic mitral valve IE. CASE PRESENTATION: This is a 64-year-old male with known past medical history of aortic valve replacement, tricuspid and mitral valve repair, atrial fibrillation status post cardioversion and ablation, and hypertension who complained of fevers, palpitations, and dyspnea for a day. He was started on vancomycin and piperacillin-tazobactam pending blood cultures. The initial and repeat blood cultures were positive for methicillin sensitive Staphylococcus aureus (MSSA) and the antibiotics were switched to nafcillin. Due to persistent bacteremia, transesophageal echocardiography (TEE) was recommended which showed filamentous echodensity on the mitral annuloplasty ring. Patient had an allergic reaction to nafcillin therefore on discharge antibiotics were switched to Ceftriaxone 2 gm intravenously (IV) and Daptomycin 600 mg IV daily for 6 weeks. TEE was repeated six weeks later, which showed no vegetations or abscesses. DISCUSSION: Cases of IE caused by Staphylococcus aureus are 'almost always surgical diseases,' according to the American Heart Association/American College of Cardiology/American Thoracic Society guidelines for the management of valvular heart disease (2). Since this patient refused surgery after having multiple heart surgeries in the past, the risks/benefits of surgery needed to be carefully evaluated. Daptomycin and Ceftriaxone were used because Daptomycin exerts rapid bactericidal activity against S. aureus, including strains tolerant or resistant to other agents. When used in combination with Daptomycin, Ceftriaxone improves bactericidal activity through synergistic effects. There is evidence that rapid bacterial killing has potential clinical advantages over bacteriostatic therapy in certain infections (3). The combination of beta lactams with Daptomycin also have an effect on enhanced bacterial clearance (3). This case report provides evidence that medical therapy is a viable alternative to surgical treatment. To our knowledge, this is the first report of successful treatment of prosthetic mitral valve infective endocarditis with a non-surgical approach. CONCLUSIONS: With the correct combination of antibiotics and routine follow up, non surgical candidates can be managed medically. While conclusions can be made based on the improvement and clearance of bacteremia of this patient, future studies are needed to strengthen evidence showing the benefit of medical treatment over surgical approaches for prosthetic valve endocarditis. REFERENCE #1: Cresti A, Chiavarelli M, Scalese M, et al. Epidemiological and mortality trends in infective endocarditis, a 17-year population-based prospective study. Cardiovasc Diagn Ther. 2017;7(1):27-35. REFERENCE #2: Bonow RO, Carabello BA, Chatterjee K, et al. 2008 Focused update incorporated into the ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Valvular Heart Disease). Circulation 2008;2013:e523–661. REFERENCE #3: Dhand A, Bayer AS, Pogliano J, et al. Use of antistaphylococcal beta-lactams to increase daptomycin activity in eradicating persistent bacteremia due to methicillin-resistant Staphylococcus aureus: Role of enhanced daptomycin binding.Clin Infect Dis 2011;53:158-163. DISCLOSURES: No relevant relationships by Dilesha Kumanayaka, source=Web Response No relevant relationships by Richard Miller, source=Web Response No relevant relationships by monica mutyala, source=Web Response No relevant relationships by Fayez Shamoon, source=Web Response Speaker/Speaker's Bureau relationship with Gilead Please note: $5001 - $20000 by jihad slim, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with ViiV Please note: $5001 - $20000 by jihad slim, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with Abbvie Please note: $5001 - $20000 by jihad slim, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with merck Please note: $5001 - $20000 by jihad slim, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with Jansen Please note: $5001 - $20000 by jihad slim, source=Web Response, value=Honoraria

Vancomycin and daptomycin stability in heparin or sodium citrate lock solutions.

We investigated physical and chemical stability of daptomycin and vancomycin in heparin or sodium citrate lock solutions. The aim of this study was to find the optimal combination of antimicrobials and additives for lock solutions, which maximized patient safety. Vancomycin and daptomycin were diluted with heparin or sodium citrate to achieve final concentrations of vancomycin-heparin 2.5 mg/mL-833.33 U/mL, vancomycin-citrate 2.5-33.3 mg/mL, daptomycin-heparin 5 mg/mL-800 U/mL, and daptomycin-citrate 5-32 mg/mL and they were stored at room temperature (+25°C), 4°C, -20°C, and 37°C. Physical and chemical stability were analyzed for each antibiotic-anticoagulant combination in all conditions immediately after preparation, at 24, 48, 72 h and at different time points until unstable concentrations were obtained. Daptomycin-sodium citrate microbiological activity was also studied by evaluating two Staphylococcus aureus cultures in a calcium enriched medium with a daptomycin E test, with and without sodium citrate. After incubation at 37°C vancomycin and daptomycin combined with heparin retained at least 90% of the initial concentration over 48 h. Vancomycin-sodium citrate solution stored at 37°C reduced more than 10% of the initial concentration at 24 h. On the other hand, daptomycin-sodium citrate preparation was stable at 37°C for 72 h but the microbiological activity of daptomycin was lower in the presence of sodium citrate. The purpose is to prepare vancomycin and daptomycin lock solutions combined with heparin. They should be changed at 48 h and its stability is over 3 days at 25°C and 7 days at 4°C, which allow Hospital Pharmacy Services to manage their stocks. Daptomycin-sodium citrate combination is more stable for extended periods but its bioactivity has not been demonstrated.

In Vitro Anti-Biofilm Activity of Bacteriophage K (ATCC 19685-B1) and Daptomycin against Staphylococci.

The purpose of the present study was to investigate anti-staphylococcal activity of daptomycin and bacteriophage K, alone or in combination, against biofilm-producers and non-producers S. aureus and S. epidermidis strains, under biofilm forming and cells’ proliferation conditions. Daptomycin and bacteriophage K (ATCC 19685B1), in different concentrations, were tested against 10 Staphylococcus aureus and 10 S. epidermidis, characterized by phenotypes and genotypes. The quantitative microtiter plate (crystal violet, CV), methylthiazoltetrazolium (MTT), and growth curve (GC) assays were performed. No statistically significant difference was found between species, whereas daptomycin alone performed better using medium and high concentrations of the drug and bacteriophage K was more active against strains with higher susceptibility, by CV and MTT assays. Best results were achieved using both agents combined in high concentrations. Bacteriophage K was effective within 3.8 and 2.4 h, depending on the concentration used, by the GC assay. Combination of daptomycin with bacteriophage K was more effective against staphylococci, depending on the concentrations used and strains’ susceptibility. Further studies are needed to evaluate if this approach might be a choice for prevention or therapy of biofilm-associated infections.

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus.

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

An atomic perspective on improving daptomycin's activity

BackgroundRecently, through comprehensive medicinal chemistry efforts, we have found a new daptomycin analogue, termed kynomycin, showing enhanced activity against both methicillin-resistant S. aureus and vancomycin-resistant Enterococcus in vitro and in vivo, with improved pharmacokinetics and lower cytotoxicity than daptomycin. MethodsIn this study we compared the physicochemical properties of kynomycin with those of daptomycin from an atomic perspective by using Nuclear Magnetic Resonance spectroscopy and Molecular Dynamics simulations. Results and conclusionWe observed that kynurenine methylation changes daptomycin's key physicochemical properties; its calcium dependent oligomerization efficiency is improved and the modified kynurenine strengths contacts with the lipid tail and tryptophan residues. In addition, it is observed that, compared to daptomycin, kynomycin tetramer is more stable and binds stronger to calcium. The combined experiments provide key clues for the improved antibacterial activity of kynomycin. General significanceWe expect that this approach will help study the calcium binding and oligomerization features of new calcium dependent peptide antibiotics.

Cell Membrane Adaptations Mediate β-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro.

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

Catalase Protects Biofilm of Staphylococcus aureus against Daptomycin Activity.

Daptomycin is recommended for the treatment of Staphylococcus aureus infections due to its bactericidal activity. However, its mechanism of action is poorly understood. The involvement of reactive oxygen species (ROS) in the bactericidal activity of daptomycin has been proved against planktonic S. aureus, but not against the biofilm of S. aureus. Therefore, we evaluated if ROS contributes to the effect of daptomycin against biofilm of S. aureus. Biofilms of wild type, catalase deficient and daptomycin-resistant S. aureus strains were grown in microtiter-plates. After three days, the biofilms were exposed to daptomycin with or without thiourea in the presence of a ROS indicator. After overnight incubation, the amount of ROS and the percentage of surviving bacteria were determined. The bacterial survival was higher and the amount of ROS was lower in the wild type than in the catalase deficient biofilm, demonstrating a protective effect of catalase against daptomycin. The induction of cytotoxic ROS formation by daptomycin was verified by the addition of thiourea, which reduced the amount of ROS and protected the wild type biofilm against high concentrations of daptomycin. Accordingly, only the highest concentration of daptomycin reduced the bacterial survival and increased the ROS formation in the resistant biofilm. In conclusion, daptomycin induced the production of cytotoxic levels of endogenous ROS in S. aureus biofilm and the presence of catalase protected the biofilm against the lethality of the induced ROS.

In vitro antimicrobial activity of daptomycin alone and in adjunction with either amoxicillin, cefotaxime or rifampicin against the main pathogens responsible for bacterial meningitis in adults

As daptomycin adjunction is currently under clinical evaluation in the multicentre phase II AddaMAP study to improve the prognosis of pneumococcal meningitis, the present work aimed at evaluating the in vitro antimicrobial activity of daptomycin-based combinations against some of the most frequent species responsible for bacterial meningitis. Clinically relevant strains of Streptococcus pneumoniae, Listeria monocytogenes, Haemophilus influenzae and Neisseria meningitidis were obtained from National Reference Centers. The antimicrobial activity of amoxicillin, cefotaxime and rifampicin, either alone or in association with daptomycin, was explored through the determination of minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) as well as time-kill assay (TKA) using the broth microdilution method. All species taken together, the adjunction of daptomycin had no deleterious impact on the antimicrobial activity of amoxicillin, cefotaxime or rifampicin in vitro. Regarding Gram-positive bacteria, FICI and TKA analysis confirmed a global improvement of growth inhibition and bactericidal activity due to the adjunction of daptomycin. The synergistic effect prevailed for L. monocytogenes as demonstrated by FICI mainly <0.5 and a dynamic TKA-based synergy rate >50%. In addition, daptomycin-based associations did not modify the activity of β-lactam antibiotics or rifampicin against Gram-negative bacteria, notably N. meningitidis. These results bring comforting evidence towards the clinical potential of daptomycin adjunction in the treatment of bacterial meningitis, which supports the ongoing AddaMAP clinical trial.

Cloxacillin or fosfomycin plus daptomycin combinations are more active than cloxacillin monotherapy or combined with gentamicin against MSSA in a rabbit model of experimental endocarditis.

In vitro and in vivo activity of daptomycin alone or plus either cloxacillin or fosfomycin compared with cloxacillin alone and cloxacillin plus gentamicin were evaluated in a rabbit model of MSSA experimental endocarditis (EE). Five MSSA strains were used in the in vitro time-kill studies at standard (105-106 cfu/mL) and high (108 cfu/mL) inocula. In the in vivo EE model, the following antibiotic combinations were evaluated: cloxacillin (2 g/4 h) alone or combined with gentamicin (1 mg/kg/8 h) or daptomycin (6 mg/kg once daily); and daptomycin (6 mg/kg/day) alone or combined with fosfomycin (2 g/6 h). At standard and high inocula, daptomycin plus fosfomycin or cloxacillin were bactericidal against 4/5 and 5/5 strains, respectively, while cloxacillin plus gentamicin was bactericidal against 3/5 strains at standard inocula but against none at high inocula. Fosfomycin, cloxacillin, gentamicin and daptomycin MIC/MBCs of the MSSA-678 strain used in the EE model were: 8/64, 0.25/0.5, 0.25/0.5 and 1/8 mg/L, respectively. Adding gentamicin to cloxacillin significantly reduced bacterial density in vegetations compared with cloxacillin monotherapy (P = 0.026). Adding fosfomycin or cloxacillin to daptomycin [10/11 (93%) and 8/11 (73%), respectively] significantly improved the efficacy of daptomycin in sterilizing vegetations [0/11 (0%), P < 0.001 for both combinations] and showed better activity than cloxacillin alone [0/10 (0%), P < 0.001 for both combinations] and cloxacillin plus gentamicin [3/10 (30%), P = 0.086 for cloxacillin plus daptomycin and P = 0.008 for fosfomycin plus daptomycin]. No recovered isolates showed increased daptomycin MIC. The addition of cloxacillin or fosfomycin to daptomycin is synergistic and rapidly bactericidal, showing better activity than cloxacillin plus gentamicin for treating MSSA EE, supporting their clinical use.