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.

Therapeutics for Vancomycin-Resistant Enterococcal Bloodstream Infections.

Vancomycin-resistant enterococci (VRE) are common causes of bloodstream infections (BSIs) with high morbidity and mortality rates. They are pathogens of global concern with a limited treatment pipeline. Significant challenges exist in the management of VRE BSI, including drug dosing, the emergence of resistance, and the optimal treatment for persistent bacteremia and infective endocarditis. Therapeutic drug monitoring (TDM) for antimicrobial therapy is evolving for VRE-active agents; however, there are significant gaps in the literature for predicting antimicrobial efficacy for VRE BSIs. To date, TDM has the greatest evidence for predicting drug toxicity for the three main VRE-active antimicrobial agents daptomycin, linezolid, and teicoplanin. This article presents an overview of the treatment options for VRE BSIs, the role of antimicrobial dose optimization through TDM in supporting clinical infection management, and challenges and perspectives for the future.

Membrane Lipids Augment Cell Envelope Stress Signaling via the MadRS System to Defend Against Antimicrobial Peptides and Antibiotics in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Phage-antibiotic synergy against daptomycin-nonsusceptible MRSA in an ex vivo simulated endocardial pharmacokinetic/pharmacodynamic model.

Phage-antibiotic combinations (PAC) offer a potential solution for treating refractory daptomycin-nonsusceptible (DNS) methicillin-resistant Staphylococcus aureus (MRSA) infections. We examined PAC activity against two well-characterized DNS MRSA strains (C4 and C37) in vitro and ex vivo. PACs comprising daptomycin (DAP) ± ceftaroline (CPT) and a two-phage cocktail (Intesti13 + Sb-1) were evaluated for phage-antibiotic synergy (PAS) against high MRSA inoculum (109 CFU/mL) using (i) modified checkerboards (CB), (ii) 24-h time-kill assays (TKA), and (iii) 168-h ex vivo simulated endocardial vegetation (SEV) models. PAS was defined as a fractional inhibitory concentration ≤0.5 in CB minimum inhibitory concentration (MIC) or a ≥2 log10 CFU/mL reduction compared to the next best regimen in time-kill assays and SEV models. Significant differences between regimens were assessed by analysis of variance with Tukey's post hoc modification (α = 0.05). CB assays revealed PAS with Intesti13 + Sb-1 + DAP ± CPT. In 24-h time-kill assays against C4, Intesti13 + Sb-1 + DAP ± CPT demonstrated synergistic activity (-Δ7.21 and -Δ7.39 log10 CFU/mL, respectively) (P < 0.05 each). Against C37, Intesti13 + Sb-1 + CPT ± DAP was equally effective (-Δ7.14 log10 CFU/mL each) and not significantly different from DAP + Intesti13 + Sb-1 (-Δ6.65 log10 CFU/mL). In 168-h SEV models against C4 and C37, DAP ± CPT + the phage cocktail exerted synergistic activities, significantly reducing bio-burdens to the detection limit [2 log10 CFU/g (-Δ7.07 and -Δ7.11 log10 CFU/g, respectively)] (P < 0.001). At 168 h, both models maintained stable MICs, and no treatment-emergent phage resistance occurred with DAP or DAP + CPT regimens. The two-phage cocktail demonstrated synergistic activity against two DNS MRSA isolates in combination with DAP + CPT in vitro and ex vivo. Further in vivo PAC investigations are needed.

The Spx stress regulator confers high-level ß-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA has acquired resistance to next-generation ß-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators, which, through poorly described mechanisms, enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased ß-lactam MICs up to 16-fold and transformed MRSA cells with low levels of resistance to being homogenously highly resistant to ß-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPR interference (CRISPRi) to knock down spx transcription, we unambiguously linked hyper-resistance to the accumulation of Spx. Spx was previously proposed to be essential; however, our data suggest that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in ß-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane, including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox-sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum ß-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

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.

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.

Daptomycin eosinophilic pneumonia, a systematic review of the literature and case series.

PURPOSE: Daptomycin-induced eosinophilic pneumonia (DIEP) is a rare yet severe adverse event that requires rapid recognition and management. Diagnosing a definite case is challenging and involves meeting the American Thoracic Society (ATS) criteria, although alternative criteria have been suggested. This study aims to conduct a systematic review of literature and includes a case series. METHODS: Six cases of DIEP identified at Perugia Hospital, Perugia, Italy have been described. A systematic review was carried out adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement guidelines. RESULTS: a total of 74 cases of DIEP were analysed. Using ATS clinical criteria, 15 were classified as definite (20.3%), 54 as probable (73.0%), and 5 as possible (6.8%). Phillips criteria and the Lyon Algorithm identified 43/74 (58.2%) and 64/67 (95.5%) cases as definite, respectively. Bronchoalveolar lavage (BAL) was performed in 43 cases, revealing an average eosinophil count of 28.6% (SD 24.4). Radiological findings highlighted recurring features like bilateral opacities (68.1%), ground-glass opacities (41.7%), patchy infiltrates (30.6%), and peripheral predominance (19.4%). Upon suspicion, daptomycin was discontinued; 20 cases required no additional treatment, 38 received corticosteroids, and 12 received both corticosteroids and antibiotics. Recovery rates were high across all treatment types (≥ 73.7%). Most reports described rapid improvement post-withdrawal (within 96 h). CONCLUSIONS: DIEP is a rare, fast-progressing condition where early diagnosis and prompt treatment are vital. Diagnosis relies on clinical, laboratory, and radiological evaluations. Stopping daptomycin is essential, with corticosteroids often necessary. Further research is needed to enhance diagnostic accuracy for this disease.

Daptomycin Plus Oxacillin for Persistent Methicillin-Susceptible Staphylococcus aureus Bacteremia.

BACKGROUND: The preferred antibiotic salvage regimen for persistent methicillin-susceptible Staphylococcus aureus bacteremia (MSSAB) is unclear. Ertapenem with cefazolin or an antistaphylococcal penicillin has been primarily described, but identifying alternative carbapenem-sparing options may support antibiotic stewardship efforts and decrease the risk of antibiotic-associated Clostridioides difficile infection. OBJECTIVE: We sought to evaluate the effectiveness and safety of daptomycin plus oxacillin (D/O) for persistent MSSAB. METHODS: This was a single-center, retrospective cohort of patients with persistent MSSAB who received D/O between January 1, 2014, and January 1, 2023. Adult patients were included if they had blood cultures positive for MSSA ≥72 hours and received D/O combination for ≥48 hours. Patients were excluded if they were pregnant, incarcerated, or received another antibiotic considered to have excellent activity against MSSA. The primary outcome was time to MSSA bacteremia clearance post-daptomycin initiation. Secondary outcomes included microbiological cure, hospital length of stay, 90-day all-cause mortality, MSSA bacteremia-related mortality, 90-day readmission for MSSAB, and incidence of antibiotic-associated adverse effects. Time to MSSAB clearance post-D/O initiation was plotted using Kaplan-Meier estimation. RESULTS: Seven unique patient encounters were identified including 4 with endocarditis. Despite a median MSSA bacteremia duration of 7.8 days, median clearance was 2 days post-daptomycin initiation. All achieved microbiological cure, and no adverse effects were reported. Ninety-day all-cause mortality, MSSAB-related mortality, and 90-day readmission for MSSAB occurred in 28.6%, 14.3%, and 14.3% of patients, respectively. CONCLUSIONS AND RELEVANCE: D/O was an effective, well-tolerated salvage regimen in this cohort and may represent a carbapenem-sparing option for persistent MSSAB.

Perioperative daptomycin for prophylaxis of vancomycin-resistant Enterococcus infection in colonized liver transplant recipients.

BACKGROUND: Infection with vancomycin-resistant Enterococcus (VRE) in liver transplant recipients (LTR) has been associated with extended hospital stays, increased readmission rates, graft failure, and death. A tailored perioperative surgical prophylaxis regimen targeting VRE may reduce postoperative infections in VRE-colonized patients. This study investigated the outcomes of perioperative daptomycin in this patient population. METHODS: This retrospective, single-center cohort study included LTR ≥ 18 years old who were VRE-colonized from June 2018 to November 2022. VRE colonization was identified by a VRE rectal swab screen or a positive VRE culture prior to transplant. Two groups were analyzed: daptomycin versus no daptomycin. All LTR received perioperative piperacillin-tazobactam for 24 h. If VRE-colonized, one dose of daptomycin (6 mg/kg) was given pre- and postoperatively. Demographics, clinical characteristics, risk factors for VRE infection, and daptomycin dose were collected. The primary outcome was VRE infection at 14 days and 90 days post-transplant. RESULTS: There were 36 VRE-colonized LTR; 19 received daptomycin and 17 did not. Baseline characteristics and risk factors for VRE infection were similar between groups. More VRE infections occurred in the no daptomycin group within 14 days post-transplant (24% vs. 0%, p = .04), but at 90 days posttransplant there was no significant difference (29% vs. 16%, p = .43). The average daptomycin dose was 7.1 mg/kg. CONCLUSION: Perioperative daptomycin reduced the rate of VRE infections in VRE-colonized LTR within 14 days posttransplant but not at 90 days. Future studies should evaluate if higher doses and/or longer duration of perioperative daptomycin can reduce VRE infections beyond 14 days post-transplant.

Daptomycin Exposure as a Risk Factor for Daptomycin-Induced Eosinophilic Pneumonia and Muscular Toxicity.

BACKGROUND: High-dose daptomycin is increasingly used in patients with bone and joint infection (BJI). This raises concerns about a higher risk of adverse events (AEs), including daptomycin-induced eosinophilic pneumonia (DIEP) and myotoxicity. We aimed to examine pharmacokinetic and other potential determinants of DIEP and myotoxicity in patients with BJI receiving daptomycin. METHODS: All patients receiving daptomycin for BJI were identified in a prospective cohort study. Cases were matched at a 1:3 ratio, with controls randomly selected from the same cohort. Bayesian estimation of the daptomycin daily area under the concentration-time curve over 24 hours (AUC24h) was performed with the Monolix software based on therapeutic drug monitoring (TDM) data. Demographic and biological data were also collected. Risk factors of AEs were analyzed using Cox proportional hazards model. RESULTS: From 1130 patients followed over 7 years, 9 with DIEP, 26 with myotoxicity, and 106 controls were included in the final analysis. Daptomycin AUC24h, C-reactive protein, and serum protein levels were associated with the risk of AEs. The adjusted hazard ratio of DIEP or myotoxicity was 3.1 (95% confidence interval [CI], 1.48-6.5; P < .001) for daptomycin AUC24h > 939 mg/h/L, 9.8 (95% CI, 3.94-24.5; P < .001) for C-reactive protein > 21.6 mg/L, and 2.4 (95% CI, 1.02-5.65; P = .04) for serum protein <72 g/L. CONCLUSIONS: We identified common determinants of DIEP and myotoxicity in patients with BJI. Because the risk of AEs was associated with daptomycin exposure, daptomycin TDM and model-informed precision dosing may help optimize the efficacy and safety of daptomycin treatment in this setting. A target AUC24h range of 666 to 939 mg/h/L is suggested.

Combinations of Daptomycin plus Ceftriaxone, but Not Ascending Daptomycin Dose-Regimens, Are Effective in Experimental Endocarditis Caused by Streptococcus mitis-oralis Strains: Target Tissue Clearances and Prevention of Emergence of Daptomycin-Resistance.

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 ß-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. In this study, we used 2 prototypic DAP-susceptible (DAP-S) S. mitis-oralis strains (351; and SF100), which both evolved stable, high-level DAP-R in vitro within 1 to 3 days of DAP passage (5 to 20 µg/mL DAP). Of note, the combination of DAP + CRO prevented this rapid emergence of DAP-R in both strains during in vitro passage. The experimental rabbit IE model was then employed to quantify both the clearance of these strains from multiple target tissues, as well as the emergence of DAP-R in vivo under the following treatment conditions: (i) ascending DAP-alone dose-strategies encompassing human standard-dose and high-dose-regimens; and (ii) combinations of DAP + CRO on these same metrics. Ascending DAP-alone dose-regimens (4 to 18 mg/kg/d) were relatively ineffective at either reducing target organ bioburdens or preventing emergence of DAP-R in vivo. In contrast, the combination of DAP (4 or 8 mg/kg/d) + CRO was effective at clearing both strains from multiple target tissues (often with sterilization of bio-burdens in such organs), as well as preventing the emergence of DAP-R. In patients with serious S. mitis-oralis infections such as IE, especially caused by strains exhibiting intrinsic ß-lactam resistance, initial therapy with combinations of DAP + CRO may be warranted.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

Eradication of Staphylococcus epidermidis within Biofilms: Comparison of Systemic versus Supratherapeutic Concentrations of Antibiotics.

Biofilm-forming bacterial infections result in clinical failure, recurring infections, and high health care costs. The antibiotic concentrations needed to eradicate biofilm require further research. We aimed to model an in vitro prosthetic joint infection (PJI) to elucidate the activity of traditional systemic concentrations versus supratherapeutic concentrations to eradicate a Staphylococcus epidermidis biofilm PJI. We evaluated S. epidermidis high-biofilm-forming (ATCC 35984) and low-biofilm-forming (ATCC 12228) isolates in an in vitro pharmacodynamic biofilm reactor model with chromium cobalt coupons to simulate prosthetic joint infection. Vancomycin, daptomycin, levofloxacin, and minocycline were used alone and combined with rifampin to evaluate the effect of biofilm eradication. We simulated three exposures: (i) humanized systemic dosing alone, (ii) supratherapeutic doses (1,000× MIC), and (iii) and dosing in combination with rifampin. Resistance development was monitored throughout the study. Simulated humanized systemic doses of a lipoglycopeptide (daptomycin), a fluoroquinolone (levofloxacin), a tetracycline (minocycline), and a glycopeptide (vancomycin) alone failed to eradicate a formed S. epidermidis biofilm. Supratherapeutic doses of vancomycin (2,000 µg/mL) and minocycline (15 µg/mL) with or without rifampin (15 µg/mL) failed to eradicate biofilms. However, a levofloxacin supratherapeutic dose (125 µg/mL) with rifampin eradicated the high-biofilm-producing isolate by 48 h. Interestingly, supratherapeutic-dose exposures of daptomycin (500 µg/mL) alone eradicated high- and low-biofilm-forming isolates in established biofilms. The concentrations needed to eradicate biofilms on foreign materials are not obtained with systemic dosing regimens. The failure of systemic dosing regimens to eradicate biofilms validates clinical findings with recurring infections. The addition of rifampin to supratherapeutic dosing regimens does not result in synergy. Supratherapeutic daptomycin dosing may be effective at the site of action to eradicate biofilms. Further studies are needed.

Profiling daptomycin resistance among diverse methicillin-resistant Staphylococcus aureus lineages in China.

Daptomycin (DAP) is effective against methicillin-resistant Staphylococcus aureus (MRSA). However, reduced susceptibility to DAP in MRSA may lead to treatment failures. We aim to determine the distribution of DAP minimum inhibitory concentrations (MICs) and DAP heteroresistance (hDAP) among MRSA lineages in China. A total of 472 clinical MRSA isolates collected from 2015 to 2017 in China were examined for DAP susceptibility. All isolates (n = 472) were found to be DAP susceptible, but 35.17% (166/472) of them exhibited a high DAP MIC (MIC >0.5 µg/mL). The high DAP MIC group contained a larger proportion of isolates with a higher vancomycin or teicoplanin MIC (>1.5 µg/mL) than the low DAP MIC group (19.3% vs 7.8%, P < 0.001; 22.3% vs 8.2%, P < 0.001). We compared the clonal complex (CC) distributions and clinical characteristics in MRSA isolates stratified by DAP MIC. CC5 isolates were less susceptible to DAP (MIC50 = 1 µg/mL) than CC59 isolates (MIC50 = 0.5 µg/mL, P < 0.001). Population analysis profiling revealed that 5 of 10 ST5 and ST59 DAP-susceptible MRSA isolates investigated exhibited hDAP. The results also showed that CC5 MRSA with an agrA mutation (I238K) had a higher DAP MIC than those with a wild-type agrA (P < 0.001). The agrA-I238K mutation was found to be associated with agr dysfunction as indicated by the loss of δ-hemolysin production. In addition, agr/psmα defectiveness was associated with hDAP in MRSA. Whole-genome sequencing analysis revealed mutations in mprF and walR/walK in DAP-resistant subpopulations, and most DAP-resistant subpopulations (6/8, 75%) were stable. Our study suggests that the increased DAP resistance and hDAP in MRSA may threaten the effectiveness against MRSA infections.

Physiologically based pharmacokinetic modelling to inform combination dosing regimens of ceftaroline and daptomycin in special populations.

AIMS: The combination of daptomycin and ceftaroline used as salvage therapy is associated with higher survival and decreased clinical failure in complicated methicillin-resistant Staphylococcus aureus (MRSA) infections that are resistant to standard MRSA treatment. This study aimed to evaluate dosing regimens for coadministration of daptomycin and ceftaroline in special populations including paediatrics, renally impaired (RI), obese and geriatrics that generate sufficient coverage against daptomycin-resistant MRSA. METHODS: Physiologically based pharmacokinetic models were developed from pharmacokinetic studies of healthy adults, geriatric, paediatric, obese and RI patients. The predicted profiles were used to evaluate joint probability of target attainment (PTA), as well as tissue-to-plasma ratios. RESULTS: The adult dosing regimens of 6 mg/kg every (q)24h or q48h daptomycin and 300-600 mg q12h ceftaroline fosamil by RI categories achieved ≥90% joint PTA when the minimum inhibitory concentrations in the combination are at or below 1 and 4 µg/mL against MRSA. In paediatrics, wherein there is no recommended daptomycin dosing regimen for S. aureus bacteraemia, ≥90% joint PTA is achieved when the minimum inhibitory concentrations in the combination are up to 0.5 and 2 µg/mL for standard paediatric dosing regimens of 7 mg/kg q24h daptomycin and 12 mg/kg q8h ceftaroline fosamil. Model predicted tissue-to-plasma ratios of 0.3 and 0.7 in the skin and lung, respectively, for ceftaroline and 0.8 in the skin for daptomycin. CONCLUSION: Our work illustrates how physiologically based pharmacokinetic modelling can inform appropriate dosing of adult and paediatric patients and thereby enable prediction of target attainment in the patients during multitherapies.

Comparison of the efficacy and safety of standard- and high-dose daptomycin: A systematic review and meta-analysis.

AIMS: Standard doses of daptomycin at 4 and 6 mg/kg were used for the treatment of skin and soft tissue for infections and bacteraemia, respectively. However, increased doses of daptomycin are recommended for complicated infections by Gram-positive organisms. METHODS: A systematic review was conducted using 4 databases. We compared treatment success between standard-dose (SD, 4-6 mg/kg) and high-dose (HD, >6 mg/kg) daptomycin in patients with all-cause bacteraemia, complicated bacteraemia, infective endocarditis, osteomyelitis and foreign body/prosthetic infection as the primary outcome. We also compared the success between SD and HD2 (≥8 mg/kg) daptomycin treatments in patients with these diseases as the secondary outcome. The incidence of creatine phosphokinase (CPK) elevation was evaluated as safety. RESULTS: In patients with complicated bacteraemia and infective endocarditis, the treatment success was significantly lower in the SD group than in the HD group (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76 and OR 0.50, 95% CI 0.30-0.82) and HD2 group (OR 0.38, 95% CI 0.21-0.69 and OR 0.30, 95% CI 0.15-0.60), respectively. A significant difference was demonstrated only in the HD2 group in patients with bacteraemia, including simple infection. SD did not decrease the success rate for the treatment of osteomyelitis and foreign body/prosthetic infection. The incidence of elevated CPK was significantly lower in SD group than in HD group. CONCLUSION: SD daptomycin was associated with significantly lower treatment success than HD in patients with complicated bacteraemia/infective endocarditis. The CPK elevation should be considered in patients treated with high daptomycin doses.

Practical Mouse Model to Investigate Therapeutics for Staphylococcusaureus Contaminated Surgical Mesh Implants.

INTRODUCTION: The use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus-infected surgical mesh and provide results from antibiotic and immunotherapeutic testing. MATERIALS AND METHODS: Implantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 "don't eat me" molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points. RESULTS: A single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection. CONCLUSIONS: This fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.

Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

AIMS: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. METHODS AND RESULTS: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l). CONCLUSIONS: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.