In-patient evolution of a high-persister Escherichia coli strain with reduced in vivo antibiotic susceptibility.

Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Despite appropriate antibiotic treatment, relapse of GNB-BSI with the same bacterial strain is common and associated with poor clinical outcomes and high healthcare costs. The role of persister cells, which are sub-populations of bacteria that survive for prolonged periods in the presence of bactericidal antibiotics, in relapse of GNB-BSI is unclear. Using a cohort of patients with relapsed GNB-BSI, we aimed to determine how the pathogen evolves within the patient between the initial and subsequent episodes of GNB-BSI and how these changes impact persistence. Using Escherichia coli clinical bloodstream isolate pairs (initial and relapse isolates) from patients with relapsed GNB-BSI, we found that 4/11 (36%) of the relapse isolates displayed a significant increase in persisters cells relative to the initial bloodstream infection isolate. In the relapsed E. coli strain with the greatest increase in persisters (100-fold relative to initial isolate), we determined that the increase was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. The ptsI mutant was equally virulent in a murine bacteremia infection model but exhibited 10-fold increased survival to antibiotic treatment. This work addresses the controversy regarding the clinical relevance of persister formation by providing compelling data that not only do high-persister mutations arise during bloodstream infection in humans but also that these mutants display increased survival to antibiotic challenge in vivo.

Clinical and Molecular Analyses of Recurrent Gram-Negative Bloodstream Infections.

BACKGROUND: The causes and clinical characteristics of recurrent gram-negative bacterial bloodstream infections (GNB-BSI) are poorly understood. METHODS: We used a cohort of patients with GNB-BSI to identify clinical characteristics, microbiology, and risk factors associated with recurrent GNB-BSI. Bacterial genotyping (pulsed-field gel electrophoresis [PFGE] and whole-genome sequencing [WGS]) was used to determine whether episodes were due to relapse or reinfection. Multivariable logistic regression was used to identify risk factors for recurrence. RESULTS: Of the 1423 patients with GNB-BSI in this study, 60 (4%) had recurrent GNB-BSI. Non-White race (odds ratio [OR], 2.35; 95% confidence interval [CI], 1.38-4.01; P = .002), admission to a surgical service (OR, 2.18; 95% CI, 1.26-3.75; P = .005), and indwelling cardiac device (OR, 2.73; 95% CI, 1.21-5.58; P = .009) were associated with increased risk for recurrent GNB-BSI. Among the 48 patients with recurrent GNB-BSI whose paired bloodstream isolates underwent genotyping, 63% were due to relapse (30 of 48) and 38% were due to reinfection (18 of 48) based on WGS. Compared with WGS, PFGE correctly differentiated relapse and reinfection in 98% (47 of 48) of cases. Median time to relapse and reinfection was similar (113 days; interquartile range [IQR], 35-222 vs 174 days; IQR, 69-599; P = .13). Presence of a cardiac device was associated with relapse (relapse: 7 of 27, 26%; nonrelapse: 65 of 988, 7%; P = .002). CONCLUSIONS: In this study, recurrent GNB-BSI was most commonly due to relapse. PFGE accurately differentiated relapse from reinfection when compared with WGS. Cardiac device was a risk factor for relapse.

Vancomycin Heteroresistance and Clinical Outcomes in Bloodstream Infections Caused by Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are a common etiology of serious and recurrent infections in immunocompromised patients. Although most isolates appear susceptible to vancomycin, a single strain might have a subpopulation of resistant bacteria. This phenomenon is termed heteroresistance and may adversely affect the response to treatment. A retrospective cohort study was performed of pediatric patients with leukemia treated at St. Jude Children's Research Hospital who developed CoNS central line-associated bloodstream infection (CLABSI). Available isolates were sequenced and tested for vancomycin heteroresistance by population analysis profiling. Risk factors for heteroresistance and the association of heteroresistance with treatment failure (death or relapse of infection) or poor clinical response to vancomycin therapy (treatment failure or persistent bacteremia after vancomycin initiation) were evaluated. For 65 participants with CoNS CLABSI, 62 initial isolates were evaluable, of which 24 (39%) were vancomycin heteroresistant. All heteroresistant isolates were of Staphylococcus epidermidis and comprised multiple sequence types. Participants with heteroresistant bacteria had more exposure to vancomycin prophylaxis (P = 0.026) during the 60 days prior to infection. Of the 40 participants evaluable for clinical outcomes, heteroresistance increased the risk of treatment failure (P = 0.012) and poor clinical response (P = 0.001). This effect persisted after controlling for identified confounders. These data indicate that vancomycin heteroresistance is common in CoNS isolates from CLABSIs in pediatric patients with leukemia and is associated with poor clinical outcomes. Validation of these findings in an independent cohort and evaluation of alternative antibiotic therapy in patients with heteroresistant infections have the potential to improve care for serious CoNS infections.

Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement.

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae.

PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.

Incorporation of extracellular fatty acids by a fatty acid kinase-dependent pathway in Staphylococcus aureus.

Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4 ) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

FabH mutations confer resistance to FabF-directed antibiotics in Staphylococcus aureus.

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus.

Discovery of novel bacterial elongation condensing enzyme inhibitors by virtual screening.

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.

Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors.

The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target for antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular fatty acids. The regulation of FASII by extracellular fatty acids was examined in Staphylococcus aureus and Streptococcus pneumoniae, representing two important groups of pathogens. Both bacteria use the same enzymatic tool kit for the conversion of extracellular fatty acids to acyl-acyl carrier protein, elongation, and incorporation into phospholipids. Exogenous fatty acids completely replace the endogenous fatty acids in S. pneumoniae but support only 50% of phospholipid synthesis in S. aureus. Fatty acids overcame FASII inhibition in S. pneumoniae but not in S. aureus. Extracellular fatty acids strongly suppress malonyl-CoA levels in S. pneumoniae but not in S. aureus, showing a feedback regulatory system in S. pneumoniae that is absent in S. aureus. Fatty acids overcame either a biochemical or a genetic block at acetyl-CoA carboxylase (ACC) in S. aureus, confirming that regulation at the ACC step is the key difference between these two species. Bacteria that possess a stringent biochemical feedback inhibition of ACC and malonyl-CoA formation triggered by environmental fatty acids are able to circumvent FASII inhibition. However, if exogenous fatty acids do not suppress malonyl-CoA formation, FASII inhibitors remain effective in the presence of fatty acid supplements.

Female Sex and Mortality in Patients with Staphylococcus aureus Bacteremia: A Systematic Review and Meta-analysis.

Importance: Staphylococcus aureus is the leading cause of death due to bacterial bloodstream infection. Female sex has been identified as a risk factor for mortality in S aureus bacteremia (SAB) in some studies, but not in others. Objective: To determine whether female sex is associated with increased mortality risk in SAB. Data Sources: MEDLINE, Embase, and Web of Science were searched from inception to April 26, 2023. Study Selection: Included studies met the following criteria: (1) randomized or observational studies evaluating adults with SAB, (2) included 200 or more patients, (3) reported mortality at or before 90 days following SAB, and (4) reported mortality stratified by sex. Studies on specific subpopulations (eg, dialysis, intensive care units, cancer patients) and studies that included patients with bacteremia by various microorganisms that did not report SAB-specific data were excluded. Data Extraction and Synthesis: Data extraction and quality assessment were performed by 1 reviewer and verified by a second reviewer. Risk of bias and quality were assessed with the Newcastle-Ottawa Quality Assessment Scale. Mortality data were combined as odds ratios (ORs). Main Outcome and Measures: Mortality at or before 90-day following SAB, stratified by sex. Results: From 5339 studies retrieved, 89 were included (132 582 patients; 50 258 female [37.9%], 82 324 male [62.1%]). Unadjusted mortality data were available from 81 studies (109 828 patients) and showed increased mortality in female patients compared with male patients (pooled OR, 1.12; 95% CI, 1.06-1.18). Adjusted mortality data accounting for additional patient characteristics and treatment variables were available from 32 studies (95 469 patients) and revealed a similarly increased mortality risk in female relative to male patients (pooled adjusted OR, 1.18; 95% CI, 1.11-1.27). No evidence of publication bias was encountered. Conclusions and Relevance: In this systematic review and meta-analysis, female patients with SAB had higher mortality risk than males in both unadjusted and adjusted analyses. Further research is needed to study the potential underlying mechanisms.

Staphylococcus aureus fatty acid auxotrophs do not proliferate in mice.

Inactivation of acetyl-coenzyme A (acetyl-CoA) carboxylase confers resistance to fatty acid synthesis inhibitors in Staphylococcus aureus on media supplemented with fatty acids. The addition of anteiso-fatty acids (1 mM) plus lipoic acid supports normal growth of ΔaccD strains, but supplementation with mammalian fatty acids was less efficient. Mice infected with strain RN6930 developed bacteremia, but bacteria were not detected in mice infected with its ΔaccD derivative. S. aureus bacteria lacking acetyl-CoA carboxylase can be propagated in vitro but were unable to proliferate in mice, suggesting that the acquisition of inactivating mutations in this enzyme is not a mechanism for the evasion of fatty acid synthesis inhibitors.

Perturbation of Staphylococcus aureus gene expression by the enoyl-acyl carrier protein reductase inhibitor AFN-1252.

This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC(0-48)]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro. The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro. These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria.

Phosphatidylglycerol homeostasis in glycerol-phosphate auxotrophs of Staphylococcus aureus.

BACKGROUND: The balanced synthesis of membrane phospholipids, fatty acids and cell wall constituents is a vital facet of bacterial physiology, but there is little known about the biochemical control points that coordinate these activities in Gram-positive bacteria. In Escherichia coli, the glycerol-phosphate acyltransferase (PlsB) plays a key role in coordinating fatty acid and phospholipid synthesis, but pathogens like Staphylococcus aureus have a different acyltransferase (PlsY), and the headgroup of the major membrane phospholipid, phosphatidylglycerol (PtdGro), is used as a precursor for lipoteichoic acid synthesis. RESULTS: The PlsY acyltransferase in S. aureus was switched off by depriving strain PDJ28 (ΔgpsA) of the required glycerol supplement. Removal of glycerol from the growth medium led to the rapid cessation of phospholipid synthesis. However, the continued utilization of the headgroup caused a reduction in PtdGro coupled with the accumulation of CDP-diacylglycerol and phosphatidic acid. PtdGro was further decreased by its stimulated conversion to cardiolipin. Although acyl-acyl carrier protein (ACP) and malonyl-CoA accumulated, fatty acid synthesis continued at a reduced level leading to the intracellular accumulation of unusually long-chain free fatty acids. CONCLUSIONS: The cessation of new phospholipid synthesis led to an imbalance in membrane compositional homeostasis. PtdGro biosynthesis was not coupled to headgroup turnover leading to the accumulation of pathway intermediates. The synthesis of cardiolipin significantly increased revealing a stress response to liberate glycerol-phosphate for PtdGro synthesis. Acyl-ACP accumulation correlated with a decrease in fatty acid synthesis; however, the coupling was not tight leading to the accumulation of intracellular fatty acids.

Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia: Host, Pathogen, and Treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) is a devastating pathogen responsible for a variety of life-threatening infections. A distinctive characteristic of this pathogen is its ability to persist in the bloodstream for several days despite seemingly appropriate antibiotics. Persistent MRSA bacteremia is common and is associated with poor clinical outcomes. The etiology of persistent MRSA bacteremia is a result of the complex interplay between the host, the pathogen, and the antibiotic used to treat the infection. In this review, we explore the factors related to each component of the host-pathogen interaction and discuss the clinical relevance of each element. Next, we discuss the treatment options and diagnostic approaches for the management of persistent MRSA bacteremia.

Inflammasome-mediated glucose limitation induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen that frequently causes relapsing infections. The failure of antibiotics to eradicate infection contributes to infection relapse. Host-pathogen interactions have a substantial impact on antibiotic susceptibility and the formation of antibiotic tolerant cells. In this study, we interrogate how a major S. aureus virulence factor, α-toxin, interacts with macrophages to alter the microenvironment of the pathogen, thereby influencing its susceptibility to antibiotics. We find α-toxin-mediated activation of the NLRP3 inflammasome induces antibiotic tolerance. Induction of tolerance is driven by increased glycolysis in the host cells, resulting in glucose limitation and ATP depletion in S. aureus. Additionally, inhibition of NLRP3 activation improves antibiotic efficacy in vitro and in vivo, suggesting that this strategy has potential as a host-directed therapeutic to improve outcomes. Our findings identify interactions between S. aureus and the host that result in metabolic crosstalk that can determine the outcome of antimicrobial therapy.

Antibiotic-induced accumulation of lipid II synergizes with antimicrobial fatty acids to eradicate bacterial populations.

Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection.

Membrane disruption by antimicrobial fatty acids releases low-molecular-weight proteins from Staphylococcus aureus.

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.

Association of Follow-up Blood Cultures With Mortality in Patients With Gram-Negative Bloodstream Infections: A Systematic Review and Meta-analysis.

Importance: Obtaining follow-up blood cultures (FUBCs) in patients with Staphylococcus aureus bloodstream infection (BSI) is standard practice, although its utility in patients with gram-negative bacterial BSI (GN-BSI) is unclear. Objective: To examine whether obtaining FUBCs is associated with decreased mortality (key question [KQ] 1) and whether positive vs negative FUBCs are associated with increased mortality (KQ2). Data Sources: MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, Web of Science, and gray literature were searched from inception to March 11, 2022. Study Selection: Two investigators used predefined eligibility criteria to independently screen titles, abstracts, and relevant full texts. Randomized clinical trials or observational studies that matched or statistically adjusted for differences in, at minimum, level of acute illness between patients in the intervention (eg, FUBCs obtained) and control (eg, FUBCs not obtained) groups were included in primary analyses. Articles published in languages other than English were excluded. Data Extraction and Synthesis: Data abstraction and quality assessments were performed by one investigator and verified by a second investigator. Risk of bias was assessed with the Newcastle-Ottawa Scale. Effect sizes were pooled using random-effects models. The study followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting guideline. Main Outcomes and Measures: Mortality before hospital discharge or up to 30 days from the index blood culture. Results: From 3495 studies, 15 were included (all nonrandomized). In the 5 studies (n = 4378 patients) that met criteria for the KQ1 primary analysis, obtaining FUBCs was associated with decreased mortality (hazard ratio, 0.56; 95% CI, 0.45-0.71). For KQ2, 2 studies met criteria for the primary analysis (ie, matched or statistically adjusted for differences in patients with positive vs negative FUBCs), so an exploratory meta-analysis of all 9 studies that investigated KQ2 (n = 3243 patients) was performed. Positive FUBCs were associated with increased mortality relative to negative blood cultures (odds ratio, 2.27; 95% CI, 1.54-3.34). Limitations of the literature included a lack of randomized studies and few patient subgroup analyses. Conclusions and Relevance: In this systematic review and meta-analysis, obtaining FUBCs in patients with GN-BSI was associated with decreased mortality. The benefit of FUBCs may stem from identification of patients with positive FUBCs, which was a poor prognostic marker.