Persistent Staphylococcus aureus infections treated with prolonged daptomycin (DAP) can select for DAP resistance (DAP-R), reinforcing the need for combination strategies that both improve killing and constrain resistance evolution. Prior work suggests the DAP+ceftaroline (CPT) combination can deliver synergistic killing, prevent emergence of DAP-R, and resensitize DAP-R subpopulations toward a DAP-susceptible (DAP-S) phenotype. Here, using a clinically derived, MSSA isogenic DAP-S (616)/DAP-R (703) strain pair, we evaluated DAP+CPT activity across in vitro assays, ex vivo models, and an in vivo experimental infective endocarditis (IE) model, and integrated ultra-deep targeted sequencing to link phenotypic responses to genomic adaptation. DAP+CPT produced enhanced killing of both strains in vitro and ex vivo, improved target-tissue clearance of the DAP-R strain in vivo, prevented emergence of DAP-R in the DAP-S parental strain in vitro and ex vivo, and resensitized the DAP-R strain toward a DAP-S phenotype ex vivo. Genomically, ultra-deep sequencing of resistance loci (thousands-fold coverage) identified fixed background divergence versus the N315 reference and revealed regimen-dependent selection in membrane-stress pathways, including a high-frequency mixed mprF subpopulation consistent with DAP-driven heterogeneity under monotherapy, contrasted by distinct locus-level changes under combination exposure. In addition, coverage profiling detected a large, combination-associated mobile-element/prophage gene-content event in the 703 background affecting an immune-evasion/β-hemolysin-converting region, highlighting that antibiotic pressure can couple resistance dynamics with pathogenesis-relevant genome remodeling. Together, these data show that DAP+CPT provides potent activity beyond synergistic killing-improving clearance while constraining or reshaping resistance evolution-and they define genomic signatures that help explain divergent evolutionary trajectories under DAP alone versus DAP+CPT.

Purpose:This national survey aimed to describe the work settings, characteristics, employment activities, scope, functions, and challenges of the pharmacist workforce responsible for infectious diseases (ID) and antimicrobial stewardship (AMS)-related tasks in the U.S. Methods:An internet-based Qualtrics XM survey was distributed to 22,749 unique individuals with potential ID or AMS job responsibilities via email listservs for three national pharmacy organizations, and was open from 9/25/2024 to 10/24/2024. A respondent was considered engaged in ID/AMS activities if they reported involvement in at least one of 14 activities directly related to ID/AMS. Results:A total of 796 pharmacists with ID or AMS job responsibilities responded (3.5% response rate), with 607 working clinically or administratively in ID or AMS further categorized in four mutually exclusive groups based on formal and informal ID/AMS responsibilities. Respondents were predominantly female (66%), less than 40 years of age (59%) and white (82%). ID-specific training was completed by 41.8%, and 74% reported having student loan debt at graduation. Work-related activities were diverse and most frequently included: staffing or taking calls on weekends related to ID/AMS, AMS, educating learners or healthcare providers about ID-related topics, precepting learners, and conducting ID-related research and/or quality improvement projects. Respondents frequently indicated they lacked adequate job resources. Conclusions:The results highlight the extensive responsibilities placed on ID/AMS pharmacists to fulfill multiple roles. Pharmacists frequently lack ID-specific training or dedicated time for AMS responsibilities. The workforce is young, suggesting a need for both increased capacity for training programs and strategies for workforce retention.

The differentiation of endovascular sources of Staphylococcus aureus bacteraemia from non-endovascular infection carries significant prognostic information that can be used to therapeutically stratify patients. The endovascular glycocalyx is a protective barrier composed of glycosaminoglycans (GAGs) that can be degraded and excreted in the urine from endothelial injury occurring in endovascular infections, but not in non-endovascular infections. To determine whether patients who had S. aureus endovascular infections, including endocarditis, had increased urinary glycocalyx shedding suggestive of endothelial damage, urine samples from 55 patients with bacteraemia caused by S. aureus were assessed for GAG content. Patients with endovascular source bacteraemia were compared to those from non-vascular source bacteraemia (e.g. prosthetic joint infections, pyomyositis and cellulitis) for GAG content. As expected, patients with S. aureus bacteraemia stemming from endovascular foci of infection showed increased GAG content in the urine (24.11±4.9 g GAG/mol creatinine, n=22) compared to those with non-vascular infections (13.65±1.6 g GAG/mol creatinine, n=33, P=0.024). Further analysis of GAG composition in urine revealed differential presence of GAGs between these two groups. In the first pilot study of its kind, we found that the measurement of GAG in the urine of patients with S. aureus bacteraemia shows promise for clinical risk stratification to identify high-risk endovascular infections in order to guide clinical diagnostic and therapeutic decision-making. Larger studies will be needed to determine relevant quantitative cut-off values.

Staphylococcus aureus bloodstream infections remain a major clinical challenge. A key knowledge gap is how S. aureus adapts to the hostile, nutrient-limited environment of human serum, where immune pressures, such as complement, antimicrobial peptides, and nutritional immunity, restrict bacterial survival. Recent investigations integrating transcriptomic, proteomic, and metabolomic data across five clinically relevant S. aureus lineages revealed coordinated serum-specific metabolic and stress-response adaptations (W. Mujchariyakul, C. J. Walsh, S. Giulieri, C. Cramond, et al., mSystems 11:e01183-25, 2026, https://doi.org/10.1128/msystems.01183-25). Serum triggered increased gluconeogenic and TCA-cycle activity, expanded carbohydrate, amino acid, and lipid utilization, and induction of iron-acquisition systems, nucleotide biosynthesis, and oxidative-stress defenses, while suppressing ribosome biogenesis. Functional validation confirmed key roles for carbon-metabolism genes (gapdhB, sucA), siderophore and iron-uptake systems (sirA, sstD), and the peroxide regulator perR. These findings highlight the metabolic resourcefulness and stress resilience that enable S. aureus survival and persistence despite antibiotic therapy. This work underscores the importance of multiomic approaches across pathogens and physiologic models to reveal new therapeutic targets for bloodstream infections.

ABSTRACT Despite the prevalence and severity of enterococcal bacteremia (EcB), the mechanisms underlying systemic host responses to the disease remain unclear. Here, we present an extensive study that profiles molecular differences in plasma from EcB patients using an unbiased multi-omics approach. We performed shotgun proteomics and metabolomics on 105 plasma samples, including those from EcB patients and healthy volunteers. Comparison between healthy volunteer and EcB-infected patient samples revealed significant disparities in proteins and metabolites involved in the acute phase response, inflammatory processes, and cholestasis. Several features distinguish these two groups with remarkable accuracy. Cross-referencing EcB signatures with those of Staphylococcus aureus bacteremia revealed shared reductions in cholesterol metabolism proteins and differing responses in platelet alpha granule and neutrophil-associated proteins. Characterization of Enterococcus isolates derived from patients facilitated a nuanced comparison between EcB caused by Enterococcus faecalis and Enterococcus faecium, uncovering reduced immunoglobulin abundances in E. faecium cases and features capable of distinguishing the underlying microbe. Leveraging extensive patient metadata, we now have identified features associated with mortality or survival, revealing significant multi-omic differences and pinpointing histidine-rich glycoprotein and fetuin-B as features capable of distinguishing survival status with excellent accuracy. Altogether, this study aims to culminate in the creation of objective risk stratification algorithms—a pivotal step toward enhancing patient management and care. To facilitate the exploration of this rich data source, we provide a user-friendly interface at https://gonzalezlab.shinyapps.io/EcB_multiomics/ . IMPORTANCE Enterococcus infections have emerged as the second most common nosocomial infection, with enterococcal bacteremia (EcB) contributing to thousands of patient deaths annually. To address a lack of detailed understanding regarding the specific systemic response to EcB, we conducted a comprehensive multi-omic evaluation of the systemic host response observed in patient plasma. Our findings reveal significant features in the metabolome and proteome associated with the presence of infection, species differences, and survival outcome. We identified features capable of discriminating EcB infection from healthy states and survival from mortality with excellent accuracy, suggesting potential practical clinical utility. However, our study also established that systemic features to distinguish Enterococcus faecalis from Enterococcus faecium EcB show only a moderate degree of discriminatory accuracy, unlikely to significantly improve upon current diagnostic methods. Comparisons of differences in the plasma proteome relative to healthy samples between bacteremia caused by Enterococcus and Staphylococcus aureus suggest the presence of bacteria-specific responses alongside conserved inflammatory reactions.

This study elucidates potential genetic determinants and mechanisms involved in the synergistic effects of daptomycin (DAP) + fosfomycin (FOF) combination therapy. Among 33 clinically derived DAP-susceptible (S)/DAP-resistant (R) isogenic strain pairs, mutations in the mprF gene occurred in 30/33 DAP-R strains, including polymorphisms of L826F (33%) or T345A/L/I (15%). Strain variants of DAP-S CB1483 serially passaged in vitro for 10 days in DAP +/− FOF identified a key non-synonymous mutation in mprF (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this mprF mutation following 10-day passage. This L826F mprF polymorphism, associated with a “gain-in-function” phenotype, exhibited increased amounts of lysyl-phosphatidylglycerol (L-PG) in the cell membrane (CM). Transcriptomics revealed a relatively modest number (~10) of distinct genes that were significantly up- or downregulated (≥2 log fold) in both the DAP-S and DAP-R strain pairs upon DAP + FOF exposures (vs. DAP or FOF alone). Of note, DAP + FOF decreased expression of lrgAB and sdrE and increased the expression level of fosB. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/− FOF showed significantly reduced lrgB expression in vegetations compared with DAP treatment alone. Overall, these findings indicate that DAP + FOF therapy impacts MRSA through multiple specific mechanisms, enhancing bacterial clearance.

Since its standardization, clinical antimicrobial susceptibility testing (AST) has relied upon a standard medium, Mueller-Hinton Broth/Agar (MHB/A), to determine antibiotic resistance. However, this microbiologic medium bears little resemblance to the host milieu, calling into question the physiological relevance of resistance phenotypes it reveals. Recent studies investigating antimicrobial susceptibility in mammalian cell culture media, a more host-mimicking environment, demonstrate that exposure to host factors significantly alters susceptibility profiles. One such factor is bicarbonate, an abundant ion in the mammalian bloodstream/tissues. Importantly, bicarbonate sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to early-generation β-lactams used for the treatment of methicillin-susceptible S. aureus (MSSA). This “NaHCO3-responsive” phenotype is widespread among US MRSA USA300/CC8 bloodstream and skin and soft tissue infection isolates. Translationally, β-lactam therapy has proven effective against NaHCO3-responsive MRSA in both ex vivo simulated endocarditis vegetation (SEV) and in vivo rabbit infective endocarditis (IE) models. Mechanistically, bicarbonate appears to influence mecA expression and PBP2a production/localization, as well as key elements for PBP2a functionality, including the PBP2a chaperone PrsA, components of functional membrane microdomains (FMMs), and wall teichoic acid (WTA) synthesis. The NaHCO3-responsive phenotype highlights the critical role of host factors in shaping antibiotic susceptibility, emphasizing the need to incorporate more physiological conditions into AST protocols.

Rural and critical access hospitals serve 15% of the United States population and utilize antibiotics at similar rates and spectrum as larger urban hospitals, making them a priority for antimicrobial stewardship. However, barriers such as insufficient personnel, limited electronic health record capabilities, and financial constraints limit stewardship initiatives. Telestewardship partnerships with urban hospitals offer a promising solution; however, a structured process to develop and implement such programs is not established. This perspective focuses on unmet needs in rural hospitals to provide future direction for improved patient care in these settings. In 2024, UW Health engaged leaders of small and rural hospitals to design a telestewardship program that meets regulatory requirements (ie, Joint Commission Standards). Despite these requested services, financial barriers hindered implementation of telestewardship partnerships. This work underscores the opportunities and challenges faced by rural hospitals and the ongoing need for state and national funding to support these communities.

Minocycline activity against Acinetobacter baumannii (AB) in vivo is underestimated by standard methods of susceptibility testing. We examined pharmacologic effects of minocycline on primary immunity that may be contributing to the in vivo vs. in vitro discrepancy of minocycline activity against AB. Minocycline MICs against 10 AB strains were compared in standard bacteriologic media (Mueller-Hinton broth, MHB) and physiologic (RPMI) media. Macrophages were pretreated with minocycline or comparator antibiotics before AB co-culture. Macrophage cytokine production and phagocytosis of AB were measured without and with pre-treatment with minocycline. Two to eight-fold reduction in minocycline MIC against 10 AB strains occurred in RPMI compared to MHB, which was more pronounced than other antibiotic classes. Macrophages pretreated with 1, 5, 10, 30, 50, and 100 μg/mL minocycline before bacterial co-cultures significantly decreased AB inoculum at 6 hours of co-culture in a dose-dependent manner, with no bacterial colonies observed from co-cultures with macrophages pretreated with 30 μg/mL or more of minocycline. Macrophages pretreated with minocycline for 24 hours before zymosan stimulation led to significantly higher levels of phagocytosis. Macrophages treated with minocycline for 24 hours significantly decreased production of IL-6, TNF-α, and MCP-1 in a dose dependent manner. The minocycline in vivo efficacy may be attributed to enhanced activity in nutrient-limited, physiologic medium combined with increased macrophage phagocyte efficiency. Incorporating novel assays that recapitulate the in vivo environment will be important for understanding the host-pathogen-antibiotic relationship toward a goal of improved future drug discovery and overall treatment strategies against AB and other drug-resistant pathogens. ### Competing Interest Statement The authors have declared no competing interest.

Background:Dalbavancin is a long-acting lipoglycopeptide antibiotic that is increasingly utilized for infections that require prolonged treatment durations despite the lack of Food and Drug Administration approval for these indications. There is no consensus regarding optimal dosing of dalbavancin for these infections and no available pharmacokinetic studies to identify optimal dosing for long-term use. Methods:An in silico pharmacokinetic simulation was performed to assess the predicted dalbavancin concentration resulting from commonly utilized dosing regimens, in addition to modified regimens. The primary endpoint evaluated was days of median 24-hour free area under the curve over the minimum inhibitory concentration (AUC/MIC) >27.1, the established PK target. Results:A dosing regimen of 1500 mg on day 0 and day 7 resulted in median AUC/breakpoint value above the target for 57 days (lower 95% confidence interval [CI], 37 days). A modified regimen of 1500 mg on day 0 and day 21 resulted in an additional 11 days of median AUC/breakpoint target attainment. The other standard dosing regimen modeled was 1000 mg on day 0, then 500 mg weekly for 5 doses. This regimen achieved the AUC/breakpoint target for 76 days (lower 95% CI, 59 days). This regimen was modified to 1000 mg on day 0, then 500 mg on days 14 and 28, which shortened the median effective treatment duration by 14 days but required 3 fewer doses. Conclusions:These simulated results, when combined with the favorable observational data, support the use of commonly reported dalbavancin regimens for prolonged therapy durations. In addition, these pharmacokinetic/pharmacodynamic data support extending the dosing interval beyond the frequently reported weekly regimens, which should be investigated further with a clinical trial.

BackgroundPhenome-WideAssociation study (PheWAS) is a powerful tool designed to systematically screen clinical observations derived from medical records (phenotypes) for association with a variable of interest. Despite their usefulness, no systematic screening of phenotypes associated withStaphylococcusaureusinfections(SAIs) has been done leaving potential novel risk factors or complications undiscovered.Method and cohortsWe tailored the PheWAS approach into a two-stage screening procedure to identify novel phenotypes correlating with SAIs. The first stage screened for co-occurrence of SAIs with other phenotypes within medical records. In the second stage, significant findings were examined for the correlations between their age of onset with that of SAIs. The PheWAS was implemented using the medical records of 754,401 patients from the Marshfield Clinic Health System. Any novel associations discovered were subsequently validated using datasets from TriNetX andAll of Us, encompassing 109,884,571 and 118,538 patients respectively.ResultsForty-one phenotypes met the significance criteria of a p-value < 3.64e-5 and odds ratios of > 5. Out of these, we classified 23 associations either as risk factors or as complications of SAIs. Three novel associations were discovered and classified either as a risk (long-term use of aspirin) or complications (iron deficiency anemia and anemia of chronic disease). All novel associations were replicated in the TriNetX cohort. In theAll of Uscohort, anemia of chronic disease was replicated according to our significance criteria.ConclusionsThe PheWAS of SAIs expands our understanding of SAIs interacting phenotypes. Additionally, the novel two-stage PheWAS approach developed in this study can be applied to examine other disease-disease interactions of interest. Due to the possibility of bias inherent in observational data, the findings of this study require further investigation.

Staphylococcus aureus bacteremia continues to be associated with significant morbidity and mortality, despite improvements in diagnostics and management. Persistent infections pose a major challenge to clinicians and have been consistently shown to increase the risk of mortality and other infectious complications. S. aureus, while typically not considered an intracellular pathogen, has been proven to utilize an intracellular niche, through several phenotypes including small colony variants, as a means for survival that has been linked to chronic, persistent, and recurrent infections. This intracellular persistence allows for protection from the host immune system and leads to reduced antibiotic efficacy through a variety of mechanisms. These include antimicrobial resistance, tolerance, and/or persistence in S. aureus that contribute to persistent bacteremia. This review will discuss the challenges associated with treating these complicated infections and the various methods that S. aureus uses to persist within the intracellular space.

Research and development of innovative antimicrobials is paramount to addressing the antimicrobial resistance threat. Although antimicrobial discovery and development has increased, difficulties have emerged in the pharmaceutical industry after market approval. In this minireview, we summarize clinical trial data on recently approved antibiotics, calculate incremental cost-effectiveness ratio (ICER) values, and explore ways to adapt ICER calculations to the limitations of antimicrobial clinical trial design. We provide a systematic review and analysis of randomized, controlled studies of antibiotics approved from 2014 - 2022 and extracted the relevant clinical data. Adapted-ICER (aICER) calculations were conducted using the primary condition -specific outcome that was reported in each study (percent mortality or percent cure rate). The literature search identified 18 studies for the 8 total antibiotics which met inclusion criteria and contained data required for aICER calculation. aICER values ranged from -$17,374 to $4,966 per percent mortality and -$43,931 to $2,529 per percent cure rate. With regards to mortality, ceftolozane/tazobactam and imipenem/cilastatin/relebactam proved cost efficacious, with aICER values of $4,965 per percent mortality and $1,955 per percent mortality respectively. Finding value in novel antibiotic agents is imperative to further justifying their development, and aICER values are the most common method of determining value in healthcare. The current outcomes of clinical trials are difficult to translate to aICER, which most effectively use Quality -Adjusted Life Years (QALY) as the quality standard in other fields such as oncology. Future antimicrobial trials should consider introducing methods of assessing measures of health gain such as QALY to better translate the value of novel antimicrobials in healthcare.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most β-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care β-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by β-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting β-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific β-lactam agents.

The global threat of antimicrobial resistance (AMR) varies regionally. This study explores whether geospatial analysis and data visualization methods detect both clinically and statistically significant variations in antibiotic susceptibility rates at a neighborhood level. This observational multicenter geospatial study collected 10 years of patient-level antibiotic susceptibility data and patient addresses from three regionally distinct Wisconsin health systems (UW Health, Fort HealthCare, Marshfield Clinic Health System [MCHS]). We included the initial Escherichia coli isolate per patient per year per sample source with a patient address in Wisconsin (N = 100,176). Isolates from U.S. Census Block Groups with less than 30 isolates were excluded (n = 13,709), resulting in 86,467 E. coli isolates. The primary study outcomes were the results of Moran’s I spatial autocorrelation analyses to quantify antibiotic susceptibility as spatially dispersed, randomly distributed, or clustered by a range of − 1 to + 1, and the detection of statistically significant local hot (high susceptibility) and cold spots (low susceptibility) for variations in antibiotic susceptibility by U.S. Census Block Group. UW Health isolates collected represented greater isolate geographic density (n = 36,279 E. coli , 389 = blocks, 2009–2018), compared to Fort HealthCare (n = 5110 isolates, 48 = blocks, 2012–2018) and MCHS (45,078 isolates, 480 blocks, 2009–2018). Choropleth maps enabled a spatial AMR data visualization. A positive spatially-clustered pattern was identified from the UW Health data for ciprofloxacin (Moran’s I = 0.096, p = 0.005) and trimethoprim/sulfamethoxazole susceptibility (Moran’s I = 0.180, p < 0.001). Fort HealthCare and MCHS distributions were likely random. At the local level, we identified hot and cold spots at all three health systems (90%, 95%, and 99% CIs). AMR spatial clustering was observed in urban areas but not rural areas. Unique identification of AMR hot spots at the Block Group level provides a foundation for future analyses and hypotheses. Clinically meaningful differences in AMR could inform clinical decision support tools and warrants further investigation for informing therapy options.

Abstract Background The management of multi-drug resistant gram-positive infections such as methicillin-resistant Staphylococcus aureus (MRSA) is a complicated task. This is made more difficult when prolonged treatment durations are required, such as for bacteremia, endocarditis, osteomyelitis, or other deep-seated infections. Dalbavancin (DAL) is a long-acting lipoglycopeptide increasingly being used for these infections and is a favorable option when clinicians desire to avoid central catheter placement. Despite the increasing use of DAL for prolonged therapy, there is a lack of consensus on optimal dosing and pharmacokinetic attainment. Methods An in silico pharmacokinetic/pharmacodynamic simulation was performed to assess the predicted DAL concentration resulting from either two 1500mg doses separated by one week, or a 1000mg loading dose followed by 5 weekly doses of 500mg. Single dose population PK parameters describing concentrations in serum as well as two tissue compartments were used to determine parameter estimates. Both the time above the MIC and 24-hour free DAL AUC/MIC were assessed as the PD target goals. The PK target for DAL was a 24-hour fAUC/MIC of 27.1 µg/mL. Results The two dose DAL dosing regimen (1500mg x2) maintained simulated free serum concentrations above the breakpoint (0.25 ug/mL) for a median of 56 days (lower 95%CI = 33 days) and above the MIC90 (0.06 ug/mL) for > 56 days (lower 95%CI = 43 days). Six dose DAL regimen (1000mg, then 500mg weekly) maintained simulated free serum concentrations above the breakpoint for a median of > 56 days (lower 95%CI = 53 days) and above the MIC90 for > 56 days (lower 95% CI = > 56 days). Both tissue compartments in the 3 compartment PK model demonstrated very similar drug levels to serum. The lower 95th percentile of 24h fAUC/MIC90 was maintained above 27.1 for > 56 days with both the 2-dose and 6-dose regimens. Simulated free serum concentration of dalbavancin for modeled dosing regimens Conclusion Based on these simulations, the majority of patients maintain therapeutic DAL levels for at least 8 weeks using both modeled dosing regimens. These results, when combined with the observational data reporting clinical success, support the use of DAL when long durations of therapy are required, as for osteomyelitis or bacteremia. Disclosures Paul Hutson, PharmD, MS, Revive: Grant/Research Support Warren Rose, PharmD, MPH, Basilea: Honoraria|Ferring: Honoraria|Merck: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Honoraria

Abstract Background Staphylococcus aureus bacteremia (SaB) is a common infection with a high mortality rate. Optimal therapy for SaB depends on susceptibility, and the use of anti-staphylococcal beta-lactams (ASBL) for MSSA bacteremia leads to lower mortality rates compared to vancomycin. For this reason, rapid susceptibility results, such as molecular detection using the Cepheid Xpert MRSA/SA Blood Culture PCR system, are important for patient management. At our institution, providers have varying levels of confidence in this assay, with some preferring to wait for traditional susceptibility results (AST) before narrowing therapy. This study aimed to both assess concordance between PCR results and AST and determine if waiting for AST leads to differences in patient outcomes. Methods All patients diagnosed with SaB at a single US academic medical center during 2019-2022 were included in a retrospective analysis. Data regarding blood culture results, administered antibiotics, and all-cause mortality was collected. For MSSA, optimized therapy was defined as an ASBL (oxacillin or cefazolin). Results A total of 288 patients with SaB were included (22.2% MRSA). Three isolates had discordant PCR and AST, all reported an MSSA isolate as MRSA. For MRSA, the positive-predictive value was 95.5% and negative-predictive value was 100%. The average time between PCR and AST results was 38.3 hours. Amongst 220 patients with MSSA bacteremia, 28 never received optimized therapy due to early mortality or other infections/conditions. Of the remaining 192 patients, 137 started an ASBL based on PCR results. The average time from culture draw to optimized therapy was 28.9 hours. Fifty-five patients did not receive ASBL until after AST was available, which increased the time to optimized therapy to 72.7 hours. This delay in therapy led to a significant increase in 30-day mortality rate (21.8% vs 10.2%, P=0.0337). Conclusion The Cepheid Xpert MRSA/SA Blood Culture had reliable results that led to a faster time to optimized therapy. Lower mortality was observed when providers opted to start an ASBL based on MSSA PCR results instead of waiting for AST. The mortality benefit of rapid PCR testing in SaB appears to stem from earlier initiation of ASBL therapy for MSSA in addition to improving optimization of MRSA therapies. Disclosures Warren Rose, PharmD, MPH, Basilea: Honoraria|Ferring: Honoraria|Merck: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Honoraria

OBJECTIVE:The objective of this systematic review is to summarize in vitro, preclinical, and human data related to omadacycline and Clostridioides difficile infection (CDI). DATA SOURCES:PubMed and Google Scholar were searched for "omadacycline" AND ("Clostridium difficile" OR "C difficile" OR "Clostridioides difficile") for any studies published before February 15, 2022. The US Food and Drug Administration (FDA) Adverse Events Reporting System (AERS) was searched for omadacycline (for reports including "C. difficile" or "CDI" or "gastrointestinal infection"). The publications list publicly available at Paratek Pharmaceuticals, Inc. Web site was reviewed. STUDY SELECTION AND DATA EXTRACTION:Publications presenting primary data on omadacycline and C. difficile published in English were included. DATA SYNTHESIS:Preclinical and clinical evidence was extracted from 14 studies. No case reports in indexed literature and no reports on FDA AERS were found. Omadacycline has potent in vitro activity against many C. difficile clinical strains and diverse ribotypes. In phase 3 studies, there were no reports of CDI in patients who received omadacycline for either community-acquired bacterial pneumonia or acute bacterial skin and skin structure infection. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE:Omadacycline should be considered a low-risk antibiotic regarding its propensity to cause CDI. CONCLUSIONS:Reducing the burden of CDI on patients and the health care system should be a priority. Patients with appropriate indications who are at heightened risk of CDI may be suitable candidates for omadacycline therapy. In these patients, omadacycline may be preferable to antibiotics with a high CDI risk.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing, they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that may explain synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.IMPORTANCEAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for A. baumannii viability. We show that genes involved in aerobic metabolism are central to A. baumannii physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

Antimicrobial susceptibility testing (AST) remains the cornerstone of effective antimicrobial selection and optimization in patients. Despite recent advances in rapid pathogen identification and resistance marker detection with molecular diagnostics (e.g., qPCR, MALDI-TOF MS), phenotypic (i.e., microbial culture-based) AST methods - the gold standard in hospitals/clinics - remain relatively unchanged over the last few decades. Microfluidics-based phenotypic AST has been growing fast in recent years, aiming for rapid (i.e., turnaround time <8 h), high-throughput, and automated species identification, resistance detection, and antibiotics screening. In this pilot study, we describe the application of a multi-liquid-phase open microfluidic system, named under-oil open microfluidic systems (UOMS), to achieve a rapid phenotypic AST. UOMS provides an open microfluidics-based solution for rapid phenotypic AST (UOMS-AST) by implementing and recording a pathogen's antimicrobial activity in micro-volume testing units under an oil overlay. UOMS-AST allows free physical access (e.g., by standard pipetting) to the system and label-free, single-cell resolution optical access. UOMS-AST can accurately and rapidly determine antimicrobial activities [including susceptibility/resistance breakpoint and minimum inhibitory concentration (MIC)] from nominal sample/bacterial cells in a system aligned with clinical laboratory standards where open systems and optical microscopy are predominantly adopted. Further, we combine UOMS-AST with a cloud lab data analytic technique for real-time image analysis and report generation to provide a rapid (<4 h) sample-to-report turnaround time, shedding light on its utility as a versatile (e.g., low-resource setting and manual laboratory operation, or high-throughput automated system) phenotypic AST platform for hospital/clinic use.