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.

Cefiderocol heteroresistance associated with mutations in TonB-dependent receptor genes in Pseudomonas aeruginosa of clinical origin.

The siderophore-cephalosporin cefiderocol (FDC) presents a promising treatment option for carbapenem-resistant (CR) P. aeruginosa (PA). FDC circumvents traditional porin and efflux-mediated resistance by utilizing TonB-dependent receptors (TBDRs) to access the periplasmic space. Emerging FDC resistance has been associated with loss of function mutations within TBDR genes or the regulatory genes controlling TBDR expression. Further, difficulties with antimicrobial susceptibility testing (AST) and unexpected negative clinical treatment outcomes have prompted concerns for heteroresistance, where a single lineage isolate contains resistant subpopulations not detectable by standard AST. This study aimed to evaluate the prevalence of TBDR mutations among clinical isolates of P. aeruginosa and the phenotypic effect on FDC susceptibility and heteroresistance. We evaluated the sequence of pirR, pirS, pirA, piuA, or piuD from 498 unique isolates collected before the introduction of FDC from four clinical sites in Portland, OR (1), Houston, TX (2), and Santiago, Chile (1). At some clinical sites, TBDR mutations were seen in up to 25% of isolates, and insertion, deletion, or frameshift mutations were predicted to impair protein function were seen in 3% of all isolates (n = 15). Using population analysis profile testing, we found that P. aeruginosa with major TBDR mutations were enriched for a heteroresistant phenotype and undergo a shift in the susceptibility distribution of the population as compared to susceptible strains with wild-type TBDR genes. Our results indicate that mutations in TBDR genes predate the clinical introduction of FDC, and these mutations may predispose to the emergence of FDC resistance.

Multisite Detection of Tn1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida.

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Efficacy, Safety, Pharmacokinetics, and Microbiome Changes of Ibezapolstat in Adults with Clostridioides difficile Infection: A Phase 2a Multicenter Clinical Trial.

BACKGROUND: This study was the first human validation of the gram-positive bacterial DNA polymerase IIIC target in patients with Clostridioides difficile infection. The primary objectives were to assess clinical cure rates and adverse events (AEs). Secondary objectives were to evaluate plasma/fecal pharmacokinetics, microbiologic eradication, microbiome and bile acid effects, and sustained clinical cure (SCC) with ibezapolstat. METHODS: This single-arm, open-label, phase 2a study enrolled adults with C. difficile infection at 4 US centers. Patients received ibezapolstat 450 mg orally every 12 hours for 10 days and followed for an additional 28 days to assess study objectives. RESULTS: Ten patients with a mean (standard deviation [SD]) age of 49 [15] years were enrolled. Seven AEs were reported classified as mild-moderate. Plasma levels of ibezapolstat ranged from 233 to 578 ng/mL while mean (SD) fecal levels were 416 (494) µg/g stool by treatment day 3 and >1000 µg/g stool by days 8-10. A rapid increase in alpha diversity in the fecal microbiome was noted after starting ibezapolstat therapy, which was maintained after completion of therapy. A proportional decrease in Bacteroidetes phylum was observed (mean change [SD], -10.0% [4.8%]; P = .04) with a concomitantly increased proportion of Firmicutes phylum (+14.7% [5.4%]; P = .009). Compared with baseline, total primary bile acids decreased by a mean (SD) of 40.1 (9.6) ng/mg stool during therapy (P < .001) and 40.5 (14.1) ng/mg stool after completion of therapy (P = .007). Rates of both initial clinical cure and SCC at 28 days were 100% (10 of 10 patients). CONCLUSIONS: In this phase 2a study, 10 of 10 patients achieved SCC, demonstrated favorable pharmacokinetics, minimal AEs, and beneficial microbiome and bile acids results. These results support continued clinical development.

Multiomics characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolates with heterogeneous intermediate resistance to vancomycin (hVISA) in Latin America.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. OBJECTIVE: To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. METHODS: 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. RESULTS: Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. CONCLUSIONS: Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Evolution of Cefiderocol Non-Susceptibility in Pseudomonas aeruginosa in a Patient Without Previous Exposure to the Antibiotic.

We report the emergence of non-susceptibility to cefiderocol from a subpopulation of Pseudomonas aeruginosa recovered from a patient without history of cefiderocol exposure. Whole genome sequencing identified mutations in major iron transport pathways previously associated with cefiderocol uptake. Susceptibility testing should be performed before therapy with siderophore cephalosporins.

Candida auris Invasive Infections during a COVID-19 Case Surge.

Clinical cases of C. auris noted during a COVID-19 surge led to an epidemiological, clinical, and genomic investigation. Evaluation identified a close genetic relationship but inconclusive epidemiologic link between all cases. Prolonged hospitalization due to critical illness from COVID-19 and use of antimicrobials may have contributed to clinical infections.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 µg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus ß-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

BACKGROUND: The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown. METHODS: We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and ß-lactam binding affinity in HOU503 versus R497. RESULTS: DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased ß-lactam binding affinity of PBP5 of HOU503 compared with that of R497. CONCLUSIONS: Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus ß-lactam therapy.

Simultaneous Infection with Enterobacteriaceae and Pseudomonas aeruginosa Harboring Multiple Carbapenemases in a Returning Traveler Colonized with Candida auris.

We report our clinical experience treating a critically ill patient with polymicrobial infections due to multidrug-resistant Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in a 56-year-old woman who received health care in India and was also colonized by Candida auris A precision medicine approach using whole-genome sequencing revealed a multiplicity of mobile elements associated with NDM-1, NDM-5, and OXA-181 and, supplemented with susceptibility testing, guided the selection of rational antimicrobial therapy.

Long-Term Compassionate Use of Cefiderocol To Treat Chronic Osteomyelitis Caused by Extensively Drug-Resistant Pseudomonas aeruginosa and Extended-Spectrum-ß-Lactamase-Producing Klebsiella pneumoniae in a Pediatric Patient.

We report a 15 year-old Nigerian adolescent male with chronic osteomyelitis caused by an extensively drug-resistant (XDR) Pseudomonas aeruginosa strain of sequence type 773 (ST773) carrying blaNDM-1 and an extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae strain. The patient developed neurological side effects in the form of circumoral paresthesia with polymyxin B and asymptomatic elevation of transaminases with aztreonam (used in combination with ceftazidime-avibactam). Cefiderocol treatment for 14 weeks plus bone implantation resulted in apparent cure and avoided amputation.

Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

Detection of heterogeneous vancomycin intermediate resistance in MRSA isolates from Latin America.

BACKGROUND: Vancomycin is a common first-line option for MRSA infections. The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype is associated with therapeutic failure. However, hVISA isolates are usually reported as vancomycin susceptible by routine susceptibility testing procedures. OBJECTIVES: To detect and characterize the hVISA phenotype in MRSA isolates causing infections in nine Latin American countries. METHODS: We evaluated a total of 1189 vancomycin-susceptible MRSA isolates recovered during 2006-08 and 2011-14. After an initial screening of hVISA using glycopeptide-supplemented agar strategies, the detection of hVISA was performed by Etest (GRD) and Macro-method (MET). Isolates deemed to be hVISA were subjected to population analysis profile/AUC (PAP/AUC) and WGS for further characterization. Finally, we interrogated alterations in predicted proteins associated with the development of the VISA phenotype in both hVISA and vancomycin-susceptible S. aureus (VSSA) genomes. RESULTS: A total of 39 MRSA isolates (3.3%) were classified as hVISA (1.4% and 5.6% in MRSA recovered from 2006-08 and 2011-14, respectively). Most of the hVISA strains (95%) belonged to clonal complex (CC) 5. Only 6/39 hVISA isolates were categorized as hVISA by PAP/AUC, with 6 other isolates close (0.87-0.89) to the cut-off (0.9). The majority of the 39 hVISA isolates exhibited the Leu-14→Ile (90%) and VraT Glu-156→Gly (90%) amino acid substitutions in WalK. Additionally, we identified 10 substitutions present only in hVISA isolates, involving WalK, VraS, RpoB and RpoC proteins. CONCLUSIONS: The hVISA phenotype exhibits low frequency in Latin America. Amino acid substitutions in proteins involved in cell envelope homeostasis and RNA synthesis were commonly identified. Our results suggest that Etest-based methods are an important alternative for the detection of hVISA clinical isolates.

Species identification of Enterococcus spp: Whole genome sequencing compared to three biochemical test-based systems and two Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) systems.

AIM: Here, we evaluated the performance of two commercial MALDI-TOF MS systems and three biochemical-based systems and compared them to WGS as the gold standard for identifying isolates of vancomycin-resistant enterococci (VRE). METHODS: A total of 87 VRE clinical isolates were included. The mass spectrometers were the Microflex system with Biotyper software 3.1 and the Vitek MS system. The biochemical-based systems included the Vitek 2, Phoenix, and MicroScan WalkAway systems. WGS was performed on an Illumina MiSeq instrument using the MiSeq v3 reagent kit. Vancomycin resistance was determined according to CLSI criteria. RESULTS: Among the 87 VRE, 71 and 16 were identified as Enterococcus faecium and Enterococcus faecalis by WGS. All 71 E faecium were correctly identified by both mass spectrometers, as well as the Vitek 2 and Phoenix instruments. However, only 51 E faecium isolates were correctly identified by the MicroScan system. The most frequent misidentification was Enterococcus casseliflavus (n = 20). For vancomycin-resistant E faecium, the Microflex Biotyper system had the highest sensitivity (85.54%), and all instruments (except for the Microscan) had a 100% specificity and PPV. Up to 87% of E faecalis isolates were misidentified by VITEK MS and VITEK2, 81% by Microscan and Phoenix, and 75% by Bruker biotyper. CONCLUSION: As the coverage of type strain-genome sequence database continues to grow and the cost of DNA sequencing continues to decrease, genome-based identification can be a useful tool for diagnostic laboratories, with its superior accuracy even over MALDI-TOF and database-driven operations.

Disrupting Membrane Adaptation Restores In Vivo Efficacy of Antibiotics Against Multidrug-Resistant Enterococci and Potentiates Killing by Human Neutrophils.

Daptomycin resistance in enterococci is often mediated by the LiaFSR system, which orchestrates the cell membrane stress response. Activation of LiaFSR through the response regulator LiaR generates major changes in cell membrane function and architecture (membrane adaptive response), permitting the organism to survive the antibiotic attack. Here, using a laboratory strain of Enterococcus faecalis, we developed a novel Caenorhabditis elegans model of daptomycin therapy and showed that disrupting LiaR-mediated cell membrane adaptation restores the in vivo activity of daptomycin. The LiaR effect was also seen in a clinical strain of daptomycin-resistant Enterococcus faecium, using a murine model of peritonitis. Furthermore, alteration of the cell membrane response increased the ability of human polymorphonuclear neutrophils to readily clear both E. faecalis and multidrug-resistant E. faecium. Our results provide proof of concept that targeting the cell membrane adaptive response restores the in vivo activity of antibiotics, prevents resistance, and enhances the ability of the innate immune system to kill infecting bacteria.

Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

A data-driven approach to predicting diabetes and cardiovascular disease with machine learning.

BACKGROUND: Diabetes and cardiovascular disease are two of the main causes of death in the United States. Identifying and predicting these diseases in patients is the first step towards stopping their progression. We evaluate the capabilities of machine learning models in detecting at-risk patients using survey data (and laboratory results), and identify key variables within the data contributing to these diseases among the patients. METHODS: Our research explores data-driven approaches which utilize supervised machine learning models to identify patients with such diseases. Using the National Health and Nutrition Examination Survey (NHANES) dataset, we conduct an exhaustive search of all available feature variables within the data to develop models for cardiovascular, prediabetes, and diabetes detection. Using different time-frames and feature sets for the data (based on laboratory data), multiple machine learning models (logistic regression, support vector machines, random forest, and gradient boosting) were evaluated on their classification performance. The models were then combined to develop a weighted ensemble model, capable of leveraging the performance of the disparate models to improve detection accuracy. Information gain of tree-based models was used to identify the key variables within the patient data that contributed to the detection of at-risk patients in each of the diseases classes by the data-learned models. RESULTS: The developed ensemble model for cardiovascular disease (based on 131 variables) achieved an Area Under - Receiver Operating Characteristics (AU-ROC) score of 83.1% using no laboratory results, and 83.9% accuracy with laboratory results. In diabetes classification (based on 123 variables), eXtreme Gradient Boost (XGBoost) model achieved an AU-ROC score of 86.2% (without laboratory data) and 95.7% (with laboratory data). For pre-diabetic patients, the ensemble model had the top AU-ROC score of 73.7% (without laboratory data), and for laboratory based data XGBoost performed the best at 84.4%. Top five predictors in diabetes patients were 1) waist size, 2) age, 3) self-reported weight, 4) leg length, and 5) sodium intake. For cardiovascular diseases the models identified 1) age, 2) systolic blood pressure, 3) self-reported weight, 4) occurrence of chest pain, and 5) diastolic blood pressure as key contributors. CONCLUSION: We conclude machine learned models based on survey questionnaire can provide an automated identification mechanism for patients at risk of diabetes and cardiovascular diseases. We also identify key contributors to the prediction, which can be further explored for their implications on electronic health records.

Influence of Inoculum Effect on the Efficacy of Daptomycin Monotherapy and in Combination with ß-Lactams against Daptomycin-Susceptible Enterococcus faecium Harboring LiaSR Substitutions.

Enterococcus faecium isolates that harbor LiaFSR substitutions but are phenotypically susceptible to daptomycin (DAP) by current breakpoints are problematic, since predisposition to resistance may lead to therapeutic failure. Using a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, we investigated DAP regimens (6, 8, and 10 mg/kg of body weight/day) as monotherapy and in combination with ampicillin (AMP), ceftaroline (CPT), or ertapenem (ERT) against E. faecium HOU503, a DAP-susceptible strain that harbors common LiaS and LiaR substitutions found in clinical isolates (T120S and W73C, respectively). Of interest, the efficacy of DAP monotherapy, at any dose regimen, was dependent on the size of the inoculum. At an inoculum of ∼109 CFU/g, DAP doses of 6 to 8 mg/kg/day were not effective and led to significant regrowth with emergence of resistant derivatives. In contrast, at an inoculum of ∼107 CFU/g, marked reductions in bacterial counts were observed with DAP at 6 mg/kg/day, with no resistance. The inoculum effect was confirmed in a rat model using humanized DAP exposures. Combinations of DAP with AMP, CPT, or ERT demonstrated enhanced eradication and reduced potential for resistance, allowing de-escalation of the DAP dose. Persistence of the LiaRS substitutions was identified in DAP-resistant isolates recovered from the SEV model and in DAP-resistant derivatives of an initially DAP-susceptible clinical isolate of E. faecium (HOU668) harboring LiaSR substitutions that was recovered from a patient with a recurrent bloodstream infection. Our results provide novel data for the use of DAP monotherapy and combinations for recalcitrant E. faecium infections and pave the way for testing these approaches in humans.

Clinical Features and Genomic Epidemiology of Bloodstream Infections due to Enterococcal Species Other Than Enterococcus faecalis or E. faecium in Patients With Cancer.

Background: Non-Enterococcus faecium, non-E. faecalis (NFF) enterococci are a heterogeneous group of clinically pathogenic enterococci that include species with intrinsic low-level vancomycin resistance. Patients with cancer are at increased risk for bacteremia with NFF enterococci, but their clinical and molecular epidemiology have not been extensively described. Methods: We conducted a retrospective review of all patients (n = 70) with NFF bacteremia from 2016 to 2022 at a major cancer center. The main outcomes assessed were 30-day mortality, microbiological failure (positive blood cultures for ≥4 days), and recurrence of bacteremia (positive blood culture <14 days after clearance). Whole-genome sequencing was performed on all available NFF (n = 65). Results: Patients with hematological malignancies made up 56% of the cohort (77% had leukemia). The majority of solid malignancies (87%) were gastrointestinal in origin. The majority of infections (83%) originated from an intra-abdominal source. The most common NFF species were E. gallinarum (50%) and E. casseliflavus (30%). Most (61%) patients received combination therapy. Bacteremia recurred in 4.3% of patients, there was a 30-day mortality of 23%, and 4.3% had microbiological failure. E. gallinarum and E. casseliflavus isolates were genetically diverse with no spatiotemporal clustering to suggest a single strain. Frequencies of ampicillin resistance (4.3%) and daptomycin resistance (1.9%) were low. Patients with hematologic malignancy had infections with NFF enterococci that harbored more resistance genes than patients with solid malignancy (P = .005). Conclusions: NFF bacteremia is caused by a heterogeneous population of isolates and is associated with significant mortality. Hematological malignancy is an important risk factor for infection with NFF resistant to multiple antibiotics.