This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E. faecalis, S. aureus, and C. striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S. aureus and C. striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

h. Therapeutic enhancement of combinations was defined as >2 log 10 CFU/ml reduction over the most active single agent. The effect of ceftaroline on the membrane charge, cell wall thickness, susceptibility to killing by the human cathelicidin LL37, and daptomycin binding were evaluated. Therapeutic enhancement was observed with daptomycin plus ceftaroline in both strains and vancomycin plus ceftaroline against D592. Ceftaroline exposure enhanced daptomycin-induced depolarization (81.7% versus 72.3%; P ‫؍‬ 0.03) and killing by cathelicidin LL37 (P < 0.01) and reduced cell wall thickness (P < 0.001). Fluorescence-labeled daptomycin was bound over 7-fold more in ceftaroline-exposed cells. Whole-genome sequencing and mutation analysis of these strains indicated that change in daptomycin susceptibility is related to an fmtC (mprF) mutation. The combination of daptomycin plus ceftaroline appears to be potent, with rapid and sustained bactericidal activity against both daptomycinsusceptible and -nonsusceptible strains of MRSA.

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Vancomycin is the standard of care for the treatment of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptible S. aureus (VSSA) using the spectra produced by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis. Delayed initiation of appropriate antibiotic therapy results in increased mortality rates for patients with sepsis (1-6). Staphylococcus aureus is a pathogen frequently isolated from patients with sepsis, with worse clinical outcomes noted for patients with methicillin-resistant S. aureus (MRSA) (7). Vancomycin remains the standard of care for the treatment of invasive infections with MRSA (8, 9). Infections with vancomycin-nonsusceptible isolates are associated with prolonged bacteremia, longer hospital stays, and greater rates of clinical treatment failure than infections with vancomycin-susceptible S. aureus (VSSA) (10, 11).Vancomycin nonsusceptibility falls into two related phenotypes: vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). The VISA phenotype is reliably detected by broth microdilution, and these strains are characterized by a vancomycin MIC of 4 or 8 g/ml. Reliance on susceptibility testing to identify the VISA phenotype therefore delays the identification of these strains and thus the initiation of appropriate antimicrobial therapy. Because only a subpopulation of cells of hVISA strains (Յ10 Ϫ5 to 10 Ϫ6 ) have vancomycin MICs in the intermediate range, hVISA isolates frequently test susceptible to vancomycin (i.e., MICs of Յ2 g/ml) by broth microdilution methods, which typically tes...

ObjectiveTo delineate the prevalence and factors associated with antimicrobial use across six referral hospitals in Tanzania using WHO point prevalence survey (PPS) methodology to inform hospital-specific antimicrobial stewardship programmes.DesignCross-sectional analytical study.SettingSix referral hospitals in Tanzania.ParticipantsPatients irrespective of age and gender (n=948) admitted in the six referral hospital wards before 8:00 hours on each day of the survey were included in December 2019. Using the WHO PPS methodology, data on hospitals, wards, patients, antibiotics, and indications for antibiotics were collected.Outcome measuresWe analysed the prevalence of antibiotic use by referral hospital, ward, indication and patient characteristics as the main outcomes. We also described adherence to the Tanzania Standard Treatment Guidelines (STG) and WHO’s AWaRe categorisation of antibiotics.ResultsApproximately 62.3% of inpatients were prescribed antibiotics, predominantly from the Access group of antibiotics (ceftriaxone, metronidazole or ampicillin–cloxacillin). The overall adherence of antibiotic prescriptions to the Tanzania STG was high (84.0%), with the exception of Sekou Toure Regional Referral Hospital (68.0%) and Maweni Regional Referral Hospital (57.8%). The most common indication for antibiotic prescriptions was community-acquired infections (39.8%). Children less than 2 years of age (OR 1.73, 95% CI 1.02 to 2.92, p=0.039); admission to surgical wards (OR 4.90, 95% CI 2.87 to 8.36, p <0.001); and admission to paediatric wards (OR 3.93, 95% CI 2.16 to 7.15, p <0.001) were associated with increased odds of antibiotic use. Only 2 of 591 patients were prescribed antibiotics based on culture and antimicrobial susceptibility testing results.ConclusionsEmpirical use of antibiotics is common, and the Access group of antibiotics is predominantly prescribed in children less than 2 years and patients admitted to surgical and paediatric wards. Lack of utilisation of antimicrobial susceptibility testing services in these hospitals requires urgent interventions. Routine monitoring of antibiotic use is recommended to be part of antibiotic stewardship programmes in Tanzania.

Objectives: Dalbavancin is a long-acting lipoglycopeptide with activity against gram-positives, including methicillin-resistant Staphylococcus aureus (MRSA). The potential for lipoglycopeptides, with half-lives greater than 1 week, to select for resistance is unknown. Here we explore a case of MRSA central lineassociated bloodstream infection in which dalbavancin and vancomycin non-susceptibility emerged in a urine isolate collected after the patient was treated with vancomycin and dalbavancin sequentially. Methods: Isolates from blood and urine underwent susceptibility testing, and whole genome sequencing (WGS). The blood isolate was subjected to successive passage in vitro in the presence of escalating dalbavancin concentrations and the emergent isolate was subjected to repeat susceptibility testing and WGS. Results: The blood isolate was fully susceptible to vancomycin; however, MICs of the urine isolate to dalbavancin, vancomycin, telavancin, and daptomycin were at least fourfold higher than the bloodderived strain. Both strains were indistinguishable by spa and variable number tandem repeat (VNTR) typing, and WGS revealed only seven variants, indicating clonality. Four variants affected genes, including a 3bp in-frame deletion in yvqF, a gene which has been implicated in glycopeptide resistance. Vancomycin and dalbavancin non-susceptibility emerged in the blood isolate after successive passage in vitro in the presence of dalbavancin, and WGS identified a single non-synonymous variant in yvqF. Conclusions: This is the first case in which VISA has emerged in the context of a dalbavancin-containing regimen. The selection for cross-resistance to vancomycin in vitro by dalbavancin exposure alone is troubling. Clinicians should be aware of the possibility for emergence of dalbavancin non-susceptibility and glycopeptide cross-resistance arising following therapy.

Device-related infections with this pathogen frequently require prolonged parenteral therapy.

Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance ( Enterococcus spp. are among the leading causes of nosocomial infections (1). Vancomycin-resistant Enterococcus faecium has been recognized as an important multidrug-resistant pathogen for which new or improved therapies are urgently needed (2, 3). Daptomycin is a lipopeptide antibiotic with potent in vitro bactericidal activity against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE). Although daptomycin lacks an approved indication from the FDA, it is frequently utilized as an alternative agent for the management of VRE infections. However, the optimal pharmacokinetic/pharmacodynamic (PK/PD) targets for resistance prevention and therapeutic success with daptomycin have not been defined (4). In fact, the susceptibility breakpoint designating resistance in enterococci also remains questionable; however, for ease of presentation, we will use the term resistance to refer to nonsusceptible enterococci with daptomycin MICs of Ͼ4 mg/liter. Most experts agree that the daptomycin doses of 4 to 6 mg/kg of body weight/day used for staphylococcal infections are likely inadequate for VRE infections due to reduced susceptibility observed in enterococci (3-5). Daptomycin MIC 50 and MIC 90 are 2 and 4 mg/liter for E. faecium, and 0.5 and 1 mg/liter for Enterococcus faecalis versus 0.25 and 0.5 mg/liter for staphylococci (6). Reports of daptomycin resistance are now becoming more common, emphasizing the need for improved daptomycin dosing paradigms to help preserve the utility of this drug against these difficult to treat pathogens (7-10). Due to the substantial morbidity and mortality associated with enterococcal infections, it is critical to identify characteristics that correlate with treatment failure and therefore improve antimicrobial optimization and patient outcomes.In order to derive daptomycin exposure targets for resistance prevention, we evaluated simulated daptomycin regimens from 4 to 12 mg/kg/day in a 14-day PK/PD model of simulated endocardial vegetations (SEVs) against two daptomycin-susceptible clinical VRE strains (E. faecium S447 and E. faecalis S613). Resistant strains derived from these models were then subjected to a variety of phenotypic and genotypic analyses to evaluate characteristics associated with development of daptomycin resistance. MATERIALS AND METHODSBacterial strains. Two daptomycin-susceptible and vancomycin-resistant enterococcal strains, E. faecalis S613 and E. faecium S447 recovered from the bloodstream and urine, respectively, of patients before daptomycin therapy, were evaluated (11, 12). Both isolates developed daptomycin-resistant derivatives in vivo during daptomycin therapy (11, 12) and