Engineering of the start condensation domain with improved N-decanoyl catalytic activity for daptomycin biosynthesis.

Daptomycin, a lipopeptide comprising an N-decanoyl fatty acyl chain and a peptide core, is used clinically as an antimicrobial agent. The start condensation domain (dptC1) is an enzyme that catalyzes the lipoinitiation step of the daptomycin synthesis. In this study, we integrated enzymology, protein engineering, and computer simulation to study the substrate selectivity of the start condensation domain (dptC1) and to screen mutants with improved activity for decanoyl loading. Through molecular docking and computer simulation, the fatty acyl substrate channel and the protein-protein interaction interface of dptC1 are analyzed. Key residues at the protein-protein interface between dptC1 and the acyl carrier were mutated, and a single-point mutant showed more than three-folds improved catalytic efficiency of the target n-decanoyl substrate in comparing with the wild type. Moreover, molecular dynamics simulations suggested that mutants with increased catalytic activity may correlated with a more "open" and contracted substrate binding channel. Our work provides a new perspective for the elucidation of lipopeptide natural products biosynthesis, and also provides new resources to enrich its diversity and optimize the production of important components.

An induced mutation of ABC-transporter component VraF(K84E) contributes to vancomycin resistance and virulence in Staphylococcus aureus strain MW2.

Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.

Dual release of daptomycin and BMP-2 from a composite of ß-TCP ceramic and ADA gelatin.

BACKGROUND: Antibiotic-containing carrier systems are one option that offers the advantage of releasing active ingredients over a longer period of time. In vitro sustained drug release from a carrier system consisting of microporous ß-TCP ceramic and alginate has been reported in previous works. Alginate dialdehyde (ADA) gelatin gel showed both better mechanical properties when loaded into a ß-TCP ceramic and higher biodegradability than pure alginate. METHODS: Dual release of daptomycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21, and 28 by HPLC and ELISA. After release, the microbial efficacy of the daptomycin was verified and the biocompatibility of the composite was tested in cell culture. RESULTS: Daptomycin and the model compound FITC protein A (n = 30) were released from the composite over 28 days. A Daptomycin release above the minimum inhibitory concentration (MIC) by day 9 and a burst release of 71.7 ± 5.9% were observed in the loaded ceramics. Low concentrations of BMP-2 were released from the loaded ceramics over 28 days.

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

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-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.

Population pharmacokinetics of daptomycin in critically ill patients receiving extracorporeal membrane oxygenation.

BACKGROUND: Daptomycin is widely used in critically ill patients for Gram-positive bacterial infections. Extracorporeal membrane oxygenation (ECMO) is increasingly used in this population and can potentially alter the pharmacokinetic (PK) behaviour of antibiotics. However, the effect of ECMO has not been evaluated in daptomycin. Our study aims to explore the effect of ECMO on daptomycin in critically ill patients through population pharmacokinetic (PopPK) analysis and to determine optimal dosage regimens based on both efficacy and safety considerations. METHODS: A prospective, open-label PK study was carried out in critically ill patients with or without ECMO. The total concentration of daptomycin was determined by UPLC-MS/MS. NONMEM was used for PopPK analysis and Monte Carlo simulations. RESULTS: Two hundred and ninety-three plasma samples were collected from 36 critically ill patients, 24 of whom received ECMO support. A two-compartment model with first-order elimination can best describe the PK of daptomycin. Creatinine clearance (CLCR) significantly affects the clearance of daptomycin while ECMO has no significant effect on the PK parameters. Monte Carlo simulations showed that, when the MICs for bacteria are  ≥1 mg/L, the currently recommended dosage regimen is insufficient for critically ill patients with CLCR > 30 mL/min. Our simulations suggest 10 mg/kg for patients with CLCR between 30 and 90 mL/min, and 12 mg/kg for patients with CLCR higher than 90 mL/min. CONCLUSIONS: This is the first PopPK model of daptomycin in ECMO patients. Optimal dosage regimens considering efficacy, safety, and pathogens were provided for critical patients based on pharmacokinetic-pharmacodynamic analysis.

Elucidating Daptomycin's Antibacterial Efficacy: Insights into the Tripartite Complex with Lipid II and Phospholipids in Bacterial Septum Membrane.

This study elucidated the mechanism of formation of a tripartite complex containing daptomycin (Dap), lipid II, and phospholipid phosphatidylglycerol in the bacterial septum membrane, which was previously reported as the cause of the antibacterial action of Dap against gram-positive bacteria via molecular dynamics and enhanced sampling methods. Others have suggested that this transient complex ushers in the inhibition of cell wall synthesis by obstructing the downstream polymerization and cross-linking processes involving lipid II, which is absent in the presence of cardiolipin lipid in the membrane. In this work, we observed that the complex was stabilized by Ca2+-mediated electrostatic interactions between Dap and lipid head groups, hydrophobic interaction, hydrogen bonds, and salt bridges between the lipopeptide and lipids and was associated with Dap concentration-dependent membrane depolarization, thinning of the bilayer, and increased lipid tail disorder. Residues Orn6 and Kyn13, along with the DXDG motif, made simultaneous contact with constituent lipids, hence playing a crucial role in the formation of the complex. Incorporating cardiolipin into the membrane model led to its competitively displacing lipid II away from the Dap, reducing the lifetime of the complex and the nonexistence of lipid tail disorder and membrane depolarization. No evidence of water permeation inside the membrane hydrophobic interior was noted in all of the systems studied. Additionally, it was shown that using hydrophobic contacts between Dap and lipids as collective variables for enhanced sampling gave rise to a free energy barrier for the translocation of the lipopeptide. A better understanding of Dap's antibacterial mechanism, as studied through this work, will help develop lipopeptide-based antibiotics for rising Dap-resistant bacteria.

Regulatory interactions between daptomycin- and bacitracin-responsive pathways coordinate the cell envelope antibiotic resistance response of Enterococcus faecalis.

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

Daptomycin Exposure Prediction With a Limited Sampling Strategy.

BACKGROUND: Daptomycin is a cyclic lipopeptide antibiotic used to treat serious infectious endocarditis caused by Staphylococcus aureus . The pharmacodynamic parameter correlating best with efficacy is the ratio of the estimated area under the concentration (AUC 0-24 )-time curve to the minimum inhibitory concentration. The aim of the study is to develop a limited sampling strategy to estimate AUC 0-24 using a reduced number of samples. METHODS: Sixty-eight daptomycin AUC 0-24 values were calculated for 50 White patients who underwent treatment for at least 5 consecutive days. Plasma concentrations were detected using a validated high-performance liquid chromatography-tandem mass spectrometry analytical method, with daptomycin-d5 as an internal standard. Multiple regression was used to evaluate the ability of 2 concentration-time points to predict the AUC 0-24 calculated from the entire pharmacokinetic profile. Prediction bias was calculated as the mean prediction error, whereas prediction precision was estimated as the mean absolute prediction error. The development and validation datasets comprised 40 and 10 randomly selected patients, respectively. RESULTS: The AUC 0-24 (mg*h/L) was best estimated using the daptomycin trough concentration and plasma concentrations detected 2 hours after dosing. We calculated a mean prediction error of 1.6 (95% confidence interval, -10.7 to 10.9) and a mean absolute prediction error of 11.8 (95% confidence interval, 5.3-18.3), with 73% of prediction errors within ±15%. CONCLUSIONS: An equation was developed to estimate daptomycin exposure (AUC 0-24 ), offering clinical applicability and utility in generating personalized dosing regimens, especially for individuals at high risk of treatment failure or delayed response.

Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

A machine learning approach to predict daptomycin exposure from two concentrations based on Monte Carlo simulations.

Daptomycin is a concentration-dependent lipopeptide antibiotic for which exposure/effect relationships have been shown. Machine learning (ML) algorithms, developed to predict the individual exposure to drugs, have shown very good performances in comparison to maximum a posteriori Bayesian estimation (MAP-BE). The aim of this work was to predict the area under the blood concentration curve (AUC) of daptomycin from two samples and a few covariates using XGBoost ML algorithm trained on Monte Carlo simulations. Five thousand one hundred fifty patients were simulated from two literature population pharmacokinetics models. Data from the first model were split into a training set (75%) and a testing set (25%). Four ML algorithms were built to learn AUC based on daptomycin blood concentration samples at pre-dose and 1 h post-dose. The XGBoost model (best ML algorithm) with the lowest root mean square error (RMSE) in a 10-fold cross-validation experiment was evaluated in both the test set and the simulations from the second population pharmacokinetic model (validation). The ML model based on the two concentrations, the differences between these concentrations, and five other covariates (sex, weight, daptomycin dose, creatinine clearance, and body temperature) yielded very good AUC estimation in the test (relative bias/RMSE = 0.43/7.69%) and validation sets (relative bias/RMSE = 4.61/6.63%). The XGBoost ML model developed allowed accurate estimation of daptomycin AUC using C0, C1h, and a few covariates and could be used for exposure estimation and dose adjustment. This ML approach can facilitate the conduct of future therapeutic drug monitoring (TDM) studies.

Whole-genome sequencing identifies MprF mutations in a genetically diverse population of daptomycin non-susceptible Staphylococcus aureus in Australia.

OBJECTIVES: Daptomycin is one of the few last-line antimicrobials available for the treatment of multidrug-resistant Staphylococcus aureus infections. An increasing number of daptomycin non-susceptible S. aureus infections has been reported worldwide, including Australia. Resistance to daptomycin is multifactorial and involves chromosomal mutations in genes encoding proteins involved in cell membrane and cell wall synthesis. METHODS: In this study, we performed broth microdilution (BMD) to determine the daptomycin minimum inhibitory concentration (MIC) of 66 clinical isolates of S. aureus previously reported as daptomycin non-susceptible by the VITEKⓇ 2. We used whole-genome sequencing to characterise the isolates and screened the genomes for mutations associated with daptomycin non-susceptibility. RESULTS: Only 56 of the 66 isolates had a daptomycin MIC >1 mg/L by BMD. Although the 66 isolates were polyclonal, ST22 was the predominant sequence type and one-third of the isolates were multidrug resistant. Daptomycin non-susceptibility was primarily associated with MprF mutations-at least one MprF mutation was identified in the 66 isolates. Twelve previously reported MprF mutations associated with daptomycin non-susceptibility were identified in 83% of the isolates. Novel MprF mutations identified included P314A, P314F, P314T, S337T, L341V, F349del, and T423R. CONCLUSIONS: Daptomycin non-susceptible S. aureus causing infections in Australia are polyclonal and harbour MprF mutation(s). The identification of multidrug-resistant daptomycin non-susceptible S. aureus is a public health concern.

Daptomycin-induced eosinophilic pneumonia.

ABSTRACT: Daptomycin-induced eosinophilic pneumonia (DIEP) is a rare complication of daptomycin use. Manifestations most commonly include fever, hypoxia, dyspnea, cough, eosinophilia, and lung changes on radiographs and CT. Patients typically have had recent daptomycin exposure and develop fever, dyspnea, infiltrates on chest radiograph, more than 25% eosinophils on bronchoalveolar lavage, and improvement of symptoms after withdrawal of daptomycin. Treatment includes discontinuation of daptomycin, corticosteroids, and supportive measures such as supplemental oxygen. Clinicians should have a high index of suspicion for DIEP in patients who develop new onset of pulmonary and systemic signs and symptoms after initiation of daptomycin.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Daptomycin Dose Optimization in Pediatric Staphylococcus aureus Bacteremia: A Pharmacokinetic/Pharmacodynamic Investigation.

Daptomycin is an antibiotic with Gram-positive activity, including methicillin-resistant Staphylococcus aureus, for which optimal pediatric dosing is unknown. This study aimed to evaluate daptomycin exposures achieved with package label dosing and to identify dosing regimens necessary to enhance efficacy and minimize toxicity in children with S. aureus bacteremia. Monte Carlo simulations were performed to determine probability of target attainment (PTA) for six pediatric age cohorts. Area under the curve to minimum inhibitory concentration ratio (AUC0-24:MIC) ≥666 was used to determine the PTA for efficacy (PTAE). Minimum concentration (Cmin) ≥24.3 mg/L determined the PTA for toxicity (PTAT). Acceptable dosing regimens were those which achieved the combined target of ≥90% PTAE and ≤5% PTAT. Package label dosing of daptomycin yielded insufficient efficacy with only 26.3% PTAE in children 13-24 months, 39.5% PTAE in children 2-6 years, 30.1% PTAE in children 7-11 years, and 50.1% PTAE in adolescents ≥12 years. To achieve the combined efficacy and safety target, doses of 18-24 mg/kg in children 3-12 months, 20-24 mg/kg in children 13-24 months, 19-24 mg/kg in children 2-6 years, 17-19 mg/kg in children 7-11 years, and 10-14 mg/kg in adolescents ≥12 years are necessary. Package label dosing resulted in suboptimal exposure for the majority of pediatric patients in all age groups evaluated. If targeting validated efficacy and safety endpoints, daily daptomycin doses of at least 20 mg/kg in children ≤6 years, 17 mg/kg in children 7-11 years, and 10 mg/kg in adolescents ≥12 years are necessary. Clinical studies evaluating these higher doses are needed.

Getting to the heart of the matter-are two agents really better than one for the treatment of staphylococcal infective endocarditis?

The recently published European Society of Cardiology guidelines for infective endocarditis management recommends daptomycin combination therapy for the treatment of staphylococcal endocarditis in severe penicillin allergy, rather than daptomycin monotherapy. We discuss the evidence base behind this recommendation, highlighting concerns regarding the lack of robust clinical studies, increased cost and logistical considerations, and adverse effects of combination therapy. Although further studies are required to elucidate the role of combination vs monotherapy in these patients, we propose a pragmatic management approach to reduce the risk of adverse antimicrobial side effects and limit costs, while aiming to maintain treatment efficacy.

Comparative effectiveness of daptomycin versus vancomycin among patients with methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections: A systematic literature review and meta-analysis.

BACKGROUND: In the treatment of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSIs), vancomycin stands as the prevalent therapeutic agent. Daptomycin remains an alternative antibiotic to treat MRSA BSIs in cases where vancomycin proves ineffective. However, studies have conflicted on whether daptomycin is more effective than vancomycin among patients with MRSA BSI. OBJECTIVE: To compare the effectiveness of daptomycin and vancomycin for the prevention of mortality among adult patients with MRSA BSI. METHODS: Systematic searches of databases were performed, including Embase, PubMed, Web of Science, and Cochrane Library. The Newcastle Ottawa Scale (NOS) and Revised Cochrane risk-of-bias tool for randomized trials (RoB 2) were used to assess the quality of individual observational and randomized control studies, respectively. Pooled odd ratios were calculated using random effects models. RESULTS: Twenty studies were included based on a priori set inclusion and exclusion criteria. Daptomycin treatment was associated with non-significant lower mortality odds, compared to vancomycin treatment (OR = 0.81; 95% CI, 0.62, 1.06). Sub-analyses based on the time patients were switched from another anti-MRSA treatment to daptomycin demonstrated that switching to daptomycin within 3 or 5 days was significantly associated with 55% and 45% decreased odds of all-cause mortality, respectively. However, switching to daptomycin any time after five days of treatment was not significantly associated with lower odds of mortality. Stratified analysis based on vancomycin minimum inhibitory concentration (MIC) revealed that daptomycin treatment among patients infected with MRSA strains with MIC≥1 mg/L was significantly associated with 40% lower odds of mortality compared to vancomycin treatment. CONCLUSION: Compared with vancomycin, an early switch from vancomycin to daptomycin was significantly associated with lower odds of mortality. In contrast, switching to daptomycin at any time only showed a trend towards reduced mortality, with a non-significant association. Therefore, the efficacy of early daptomycin use over vancomycin against mortality among MRSA BSIs patients may add evidence to the existing literature in support of switching to daptomycin early over remaining on vancomycin. More randomized and prospective studies are needed to assess this association.

Gausemycin Antibiotic Family Acts via Ca2+-Dependent Membrane Targeting.

We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.

Removal of common antimicrobial agents by sustained low-efficiency dialysis.

Adequate dosing of antimicrobials is paramount for treating infections in critically ill patients undergoing kidney replacement therapy; however, little is known about antimicrobial removal by sustained low-efficiency dialysis (SLED). The objective was to quantify the removal of cefepime, daptomycin, meropenem, piperacillin-tazobactam, and vancomycin in patients undergoing SLED. Adult patients ≥18 years with acute kidney injury (AKI) or end-stage kidney disease receiving one of the select antimicrobials and requiring SLED were included. Blood and dialysate flow rates were maintained at 250 and 100 mL/min, respectively. Simultaneous arterial and venous blood samples for the analysis of antibiotic concentrations were collected hourly for 8 hours during SLED (on-SLED). Arterial samples were collected every 2 hours for up to 6 hours while not receiving SLED (off-SLED) for the calculation of SLED clearance, half-life (t1/2) on-SLED and off-SLED, and the fraction of removal by SLED (fD). Twenty-one patients completed the study: 52% male, mean age (±SD) 53 ± 13 years, and mean weight of 98 ± 30 kg. Eighty-six percent had AKI, and 4 patients were receiving cefepime, 3 daptomycin, 10 meropenem, 6 piperacillin-tazobactam, and 13 vancomycin. The average SLED time was 7.3 ± 1.1 hours, and the mean ultrafiltration rate was 95 ± 52 mL/hour (range 10-211). The t1/2 on-SLED was substantially lower than the off-SLED t1/2 for all antimicrobials, and the SLED fD varied between 44% and 77%. An 8-hour SLED session led to significant elimination of most antimicrobials evaluated. If SLED is performed, modification of the dosing regimen is warranted to avoid subtherapeutic concentrations.

Synergistic bactericidal effects of phage-enhanced antibiotic therapy against MRSA biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) causes biofilm-related medical device infections. Phage-antibiotic combinations offer potential therapy due to proven in vitro antibiofilm efficacy. We evaluated phage-antibiotic synergy against biofilms using modified checkerboard and 24-h time-kill assays. Humanized-simulated daptomycin (DAP) (10, 8, and 6 mg/kg q24h) and ceftaroline (CPT) (600 mg q12h) were combined with Intesti13, Sb-1, and Romulus phages (tMOI 1, q12h). Assays were conducted in 168-h biofilm reactor models against DAP non-susceptible (DNS) vancomycin intermediate S. aureus (VISA) MRSA D712 and DAP-susceptible MRSA 8014. Synergistic activity and bactericidal activity were defined as ≥2log10 CFU/mL reduction from antibiotic-only regimens and ≥3log10 CFU/mL decrease from baseline at 24 h. Differences were analyzed by one-way analysis of variance with Tukey's post hoc test (P ≤ 0.05 is considered significant). Surviving bacteria were examined for antibiotic minimum biofilm inhibitory concentration (MBIC) changes and phage susceptibility. In 168-h biofilm models, humanized DAP 10 mg/kg + CPT, combined with a 2-phage cocktail (Intesti13 + Sb-1) against D712, and a 3-phage cocktail (Intesti13 + Sb-1 + Romulus) against 8014, demonstrated synergistic bactericidal activity. At 168 h, bacteria were minimally detectable [2log10 CFU/cm2 (-Δ4.23 and -Δ4.42 log10 CFU/cm2; both P < 0.001)]. Antibiotic MBIC remained unchanged compared to baseline across various time points. None of the tested bacteria at 168 h exhibited complete phage resistance. This study reveals bactericidal efficacy of DAP + CPT with 2-phage and 3-phage cocktails against DNS VISA and MRSA isolates (D712 and 8014) in biofilm models, maintaining susceptibility. Further research is needed for diverse strains and durations, aligning with infection care. IMPORTANCE: The prevalence of biofilm-associated medical device infections caused by methicillin-resistant Staphylococcus aureus (MRSA) presents a pressing medical challenge. The latest research demonstrates the potential of phage-antibiotic combinations (PACs) as a promising solution, notably in vitro antibiofilm efficacy. By adopting modified checkerboard and 24-h time-kill assays, the study investigated the synergistic action of phages combined with humanized-simulated doses of daptomycin (DAP) and ceftaroline (CPT). The results were promising: a combination of DAP, CPT, and either a 2-phage or 3-phage cocktail effectively exhibited bactericidal activity against both DAP non-susceptible vancomycin intermediate S. aureus MRSA and DAP-susceptible MRSA strains within 168-h biofilm models. Moreover, post-treatment evaluations revealed no discernible rise in antibiotic resistance or complete phage resistance. This pioneering work suggests the potential of PACs in addressing MRSA biofilm infections, setting the stage for further expansive research tailored to diverse bacterial strains and treatment durations.