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Background Patients with systemic candidiasis often receive prolonged echinocandin therapy in the inpatient or outpatient setting. Rezafungin is a novel echinocandin currently in clinical trials characterized by once-weekly dosing interval. In order to understand the potential benefit of rezafungin to facilitate earlier hospital discharge, the purpose of this project was to better understand barriers to discharge in patients with proven or suspected invasive candidiasis. Methods Electronic health records from two large health systems (20+ hospitals) were reviewed to identify patients given an echinocandin. Patients given an echinocandin until hospital discharge were evaluated for outpatient use as well as barriers that prevented earlier discharge. Identified barriers were developed into a quantitative framework and a qualitative interview guide. Using a constant comparative method, the framework for hospital discharge barriers was constructed using a series of open-ended questions and axial coding to identify discharge barrier themes. Results were integrated to produce a mixed-method model. Results A total of 1,665 echinocandin courses were evaluated. Five hundred and thirty-four patients (32%) received echinocandin therapy until at least the day of hospital discharge of which 328 of 534 (61%) patients were either discharged to home or transferred to another facility. Significant predictors for outpatient echinocandin use were osteomyelitis (OR 4.07, 95% CI: 1.06-15.66; p=0.041) and other deep-seated infection (OR 4.44; 95% CI: 1.65-11.96; p=0.003). Stewardship analysis identified the majority of patients (54%) had the possibility for at least one day earlier discharge (potential earlier discharge:1.65±1.16 days). The quantitative model identified major barriers to be transition of care-, other medical care-, and infectious diseases-related. The qualitative model largely agreed with the quantitative model with additional psychosocial and health care access variables identified. Conclusion Using a mixed method approach, barriers to hospital discharge and potential use of new antifungal therapies were identified. These data could be used to assist transitions of care in patients with invasive candidiasis. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been associated with activation of the E. faecalis (Efs) LiaFSR response and redistribution of APL microdomains (predicted to contain cardiolipin) away from the septum. Efs encodes two putative cardiolipin synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, the exact roles of each enzyme in resistance are unknown. This work aims to establish the contributions for both enzymes in the development of DAP-R. Methods cls1 and cls2 were deleted individually and in tandem from Efs OG117∆liaX (a DAP-R strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS). Results cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant phenotypic changes from the parent strain. Interestingly, qRT-PCR showed that cls2 expression was upregulated upon deletion of cls1 (and vice-versa), suggesting a compensatory role for one enzyme upon deletion of the other (Fig 1). When comparing membrane lipid content between Efs OG117∆liaX∆cls1 and Efs OG117∆liaX∆cls2, there were no significant differences in both the overall amount or species of cardiolipin generated, further supporting a potential redundancy between the cardiolipin synthases (Fig 2). Ultimately, double deletion of both cls genes lowered the DAP MIC relative to the parent strain and restored septal localization of APL microdomains. Conclusion Overall, Cls1 has a predominant role in the development of DAP-R in E. faecalis. However, here, we describe a novel compensatory role for Cls2 under conditions in which there is no functional Cls1 to maintain the DAP-R phenotype. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Weekly oritavancin plus ampicillin continuous infusion combination therapy was used to successfully treat a deep spine vancomycin-resistant Enterococcus faecium infection associated with hardware. Checkerboard and time-kill assays confirmed synergy between these two antibiotics. Further synergies of oritavancin and ampicillin with rifampin or the endogenous human antimicrobial peptide cathelicidin LL-37 were demonstrated.

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

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP-R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR-independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP-R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two-component regulatory system and a putative fatty acid kinase (dak) in both DAP-R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP-R mutant and wild-type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.

Two pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa were isolated from 2 patients after exposure to β-lactams. The genetic basis of ceftolozane/tazobactam resistance was evaluated, and β-lactam-resistant mechanisms were assessed by phenotypic assays. Whole genome sequencing identified mutations in AmpC including the mutation (V213A) and a deletion of 7 amino acids (P210–G216) in the Ω-loop. Phenotypic assays showed that ceftolozane/tazobactam resistance in the strain with AmpCV213A variant was associated with increased β-lactamase hydrolysis activity. On the other hand, the deletion of 7 amino acids in the Ω-loop of AmpC did not display enhanced β-lactamase activity. Resistance to ceftolozane/tazobactam in P. aeruginosa is associated with changes in AmpC; however, the apparent loss of β-lactamase activity in AmpC∆7 suggests that non-AmpC mechanisms could play an important role in resistance to β-lactam/β-lactamase inhibitor combinations.

ObjectivesAmong viridans group streptococcal infective endocarditis (IE), the Streptococcus mitis group is the most common aetiological organism. Treatment of IE caused by the S. mitis group is challenging due to the high frequency of β-lactam resistance, drug allergy and intolerability of mainstay antimicrobial agents such as vancomycin or gentamicin. Daptomycin has been suggested as an alternative therapeutic option in these scenarios based on its excellent susceptibility profile against S. mitis group strains . However, the propensity of many S. mitis group strains to rapidly evolve stable, high-level daptomycin resistance potentially limits this approach.MethodsWe evaluated the activity of 6 mg/kg/day daptomycin alone or in combination with gentamicin, ceftriaxone or ceftaroline against two daptomycin-susceptible S. mitis group strains over 96 h in a pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations.ResultsDaptomycin alone was not bactericidal and high-level daptomycin resistance evolved at 96 h in both organisms. Combinations of daptomycin + ceftriaxone and daptomycin + ceftaroline demonstrated enhanced killing activity compared with each antibiotic alone and prevented emergence of daptomycin resistance at 96 h. Use of gentamicin as an adjunctive agent neither improved the efficacy of daptomycin nor prevented the development of daptomycin resistance.ConclusionsAddition of ceftriaxone or ceftaroline to daptomycin improves the bactericidal activity against S. mitis group strains and prevents daptomycin resistance emergence. Further investigation with combinations of daptomycin and β-lactams in a large number of strains is warranted to fully elucidate the clinical implications of such combinations for treatment of S. mitis group IE.

La daptomicina (DAP) es un lipopéptido cíclico con actividad in vitro contra una variedad de patógenos grampositivos, incluidos los organismos resistentes a múltiples fármacos. Desde su introducción en la práctica clínica en 2003, la DAP se ha convertido en un importante antibiótico clave de primera línea para las infecciones graves o profundas causadas por organismos grampositivos. Desafortunadamente, la resistencia a DAP (DAP-R) se ha documentado ampliamente en organismos clínicamente importantes como Staphylococcus aureus, Enterococcus spp. y Streptococcus spp. Los estudios sobre los mecanismos de DAP-R en Bacillus subtilis y otras bacterias Gram-positivas indican que las vías genéticas de DAP-R son diversas y complejas. Sin embargo, un fenómeno común que surge de estos estudios mecanísticos es que DAP-R está asociado con importantes cambios adaptativos en la homeostasis de la pared celular y la membrana celular con cambios críticos en la fisiología celular. Los hallazgos relacionados con estos cambios adaptativos han brindado nuevos conocimientos sobre la genética y los mecanismos moleculares de la respuesta al estrés de la envoltura celular bacteriana y la forma en que las bacterias grampositivas hacen frente al ataque del péptido antimicrobiano y protegen las estructuras vitales de la envoltura celular, como la membrana celular. membrana. En esta revisión, examinaremos los hallazgos más recientes relacionados con los mecanismos moleculares de resistencia a DAP en patógenos grampositivos relevantes y discutiremos las implicaciones clínicas para la terapia contra estas importantes bacterias. Daptomycin (DAP) is a cyclic lipopeptide with in vitro activity against a variety of Gram‐positive pathogens, including multidrug‐resistant organisms. Since its introduction into clinical practice in 2003, DAP has become an important key frontline antibiotic for severe or deep‐seated infections caused by Gram‐positive organisms. Unfortunately, DAP resistance (DAP‐R) has been extensively documented in clinically important organisms such as Staphylococcus aureus, Enterococcus spp., and Streptococcus spp. Studies on the mechanisms of DAP‐R in Bacillus subtilis and other Gram‐positive bacteria indicate that the genetic pathways of DAP‐R are diverse and complex. However, a common phenomenon emerging from these mechanistic studies is that DAP‐R is associated with important adaptive changes in cell wall and cell membrane homeostasis with critical changes in cell physiology. Findings related to these adaptive changes have provided novel insights into the genetics and molecular mechanisms of bacterial cell envelope stress response and the manner in which Gram‐positive bacteria cope with the antimicrobial peptide attack and protect vital structures of the cell envelope, such as the cell membrane. In this review, we will examine the most recent findings related to the molecular mechanisms of resistance to DAP in relevant Gram‐positive pathogens and discuss the clinical implications for therapy against these important bacteria.