Attention Seeker: Production, Modification, and Release of Inflammatory Peptidoglycan Fragments in Neisseria Species

ABSTRACT Maintenance of the structural macromolecule peptidoglycan (PG), which involves regulated cycles of PG synthesis and PG degradation, is pivotal for cellular integrity and survival. PG fragments generated from the degradation process are usually efficiently recycled by Gram-negative bacteria. However, Neisseria gonorrhoeae and a limited number of Gram-negative bacteria release PG fragments in amounts sufficient to induce host tissue inflammation and damage during an infection. Due to limited redundancy in PG-modifying machineries and genetic tractability, N. gonorrhoeae serves as a great model organism for the study of biological processes related to PG. This review summarizes the generation, modification, and release of inflammatory PG molecules by N. gonorrhoeae and related species and discusses these findings in the context of understanding bacterial physiology and pathogenesis.

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Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough

Journal of Bacteriology ◽

10.1128/jb.00286-17 ◽

2017 ◽

Vol 199 (23) ◽

Author(s):

Lucy Zeng ◽

Etsuko Wooton ◽

David A. Stahl ◽

Peter J. Walian

Keyword(s):

Electron Microscopy ◽

Sequence Analysis ◽

Model Organism ◽

Desulfovibrio Vulgaris ◽

Gram Negative Bacteria ◽

Diverse Range ◽

Gram Negative ◽

Content Type ◽

Sulfate Reducing

ABSTRACT Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes. IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.

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Improved Antimicrobial Activities of Synthetic-Hybrid Bacteriocins Designed from Enterocin E50-52 and Pediocin PA-1

Applied and Environmental Microbiology ◽

10.1128/aem.03477-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1661-1667 ◽

Cited By ~ 20

Author(s):

Santosh Kumar Tiwari ◽

Katia Sutyak Noll ◽

Veronica L. Cavera ◽

Michael L. Chikindas

Keyword(s):

Micrococcus Luteus ◽

Electrical Potential ◽

Gram Negative Bacteria ◽

Additional Advantage ◽

Gram Negative ◽

Content Type ◽

Intracellular Atp ◽

Hybrid Peptides ◽

C Terminus ◽

N Terminus

ABSTRACTTwo hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such asMicrococcus luteus,Salmonella entericaserovar Enteritidis 20E1090, andEscherichia coliO157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach

eLife ◽

10.7554/elife.44341 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 29

Author(s):

Mark Austin Hanson ◽

Anna Dostálová ◽

Camilla Ceroni ◽

Mickael Poidevin ◽

Shu Kondo ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Fungal Pathogens ◽

Gene Editing ◽

Cationic Peptides ◽

Gram Negative Bacteria ◽

Biological Processes ◽

Gram Negative ◽

Bacteria And Fungi

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete most known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking these ten AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.

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Microbiological pattern of prosthetic hip and knee infections: a high-volume, single-centre experience in China

Journal of Medical Microbiology ◽

10.1099/jmm.0.001305 ◽

2021 ◽

Author(s):

Yali Yu ◽

Yiyi Kong ◽

Jing Ye ◽

Aiguo Wang ◽

Wenteng Si

Keyword(s):

Antibiotic Treatment ◽

Prolonged Treatment ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Musculoskeletal Infection ◽

Gram Negative ◽

Content Type ◽

Empirical Antibiotic Treatment ◽

Link Type

Introduction. Prosthetic joint infection (PJI) is a serious complication after arthroplasty, which results in high morbidity, prolonged treatment and considerable healthcare expenses in the absence of accurate diagnosis. In China, microbiological data on PJIs are still scarce. Hypothesis/Gap Statement. The incidence of PJI is increasing year by year, and the proportion of drug-resistant bacteria infection is nicreasing, which brings severe challenges to the treatment of infection. Aim. This study aimed to identify the pathogens in PJIs, multi-drug resistance, and evaluate the effect of the treatment regimen in patients with PJI. Methodology. A total of 366 consecutive cases of PJI in the hip or knee joint were admitted at the Orthopedic Surgery Center in Zhengzhou, China from January 2012 to December 2018. Infections were confirmed in accordance with the Infectious Diseases Society of America and the Musculoskeletal Infection Society (MSIS) criteria. Concurrently, patient demographic data, incidence and antibiotic resistance were investigated. Statistical differences were analysed using Fisher’s exact test or chi-square test. Results. Altogether, 318 PJI cases satisfying the inclusion criteria were enrolled in this study, including 148 with hip PJIs and 170 with knee PJIs. The average age of patients with hip PJIs was lesser than that of patients with knee PJIs (56.4 vs. 68.6 years). Meanwhile, coagulase-negative staphylococcus (CNS, n=81, 25.5 %) was the predominant causative pathogen, followed by Staphylococcus aureus (n=67, 21.1 %). Methicillin-resistant Staphylococcus (MRS) was identified in 28.9 % of PJI patients. In addition, fungus accounted for 4.8 % (n=15), non-tuberculosis mycobacterium accounted for 1.6 % (n=5), polymicrobial pathogens accounted for 21.7 % (n=69), and Gram-negative bacteria accounted for 7.9 % (n=25) of the total infections. The results of antibiotic susceptibility testing showed that gentamicin and clindamycin β-lactam antibiotics were poorly susceptible to Gram-positive isolates, but they were sensitive to rifampicin, linezolid and vancomycin. While antibiotics such as amikacin and imipenem were effective against Gram-negative bacteria, there was a high resistance rate of other pathogens to gentamicin, clindamycin and some quinolone antibacterial drugs. Empirical antibiotic treatment should combine vancomycin and cephalosporin, levofloxacin or clindamycin. When the pathogen is confirmed, the treatment should be individualized. Conclusions. The prevalence of culture-negative PJIs is still very high. Gram-positive bacteria are still the main type of pathogens that cause PJIs. Attention should be paid to the high incidence of MRS, such as MRSA and MR-CNS, among PJI patients. Empirical antibiotic treatment should cover Gram-positive isolates, especially Staphylococcus .

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YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽

10.1128/mbio.00598-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 10

Author(s):

Randi L. Guest ◽

Daniel Samé Guerra ◽

Maria Wissler ◽

Jacqueline Grimm ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

Essential Protein ◽

Ftsh Protease ◽

Acyl Chains ◽

Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

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Robust Suppression of Lipopolysaccharide Deficiency in Acinetobacter baumannii by Growth in Minimal Medium

Journal of Bacteriology ◽

10.1128/jb.00420-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 1

Author(s):

Emma Nagy ◽

Richard Losick ◽

Daniel Kahne

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Minimal Medium ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Growth Defects ◽

Outer Leaflet ◽

Morphological Defects ◽

Outer Membrane Biogenesis

ABSTRACT Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii. Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium. IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

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Inactivation of Polymyxin by Hydrolytic Mechanism

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02378-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 1

Author(s):

Jianhua Yin ◽

Gang Wang ◽

Dan Cheng ◽

Jianv Fu ◽

Juanping Qiu ◽

...

Keyword(s):

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Peptide Antibiotics ◽

Peptide Bonds ◽

Gram Negative ◽

Content Type ◽

Gram Positive Bacteria ◽

Polymyxin E ◽

Cyclic Heptapeptide ◽

Hydrolytic Mechanism

ABSTRACTPolymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxin-inactivating enzyme fromBacillus licheniformisstrain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other betweenl-Thr andl-α-γ-diaminobutyric acid (l-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, includingBacillusandPaenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis.

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β-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate

Applied and Environmental Microbiology ◽

10.1128/aem.00318-19 ◽

2019 ◽

Vol 85 (15) ◽

Author(s):

Marietta John-White ◽

James Gardiner ◽

Priscilla Johanesen ◽

Dena Lyras ◽

Geoffrey Dumsday

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gram Negative Bacteria ◽

Diverse Range ◽

Α Subunit ◽

Gram Negative ◽

Content Type ◽

Proteolytic Resistance ◽

Insight Into ◽

Unique Capability

ABSTRACT β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus. Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions. IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

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Comparable Efficacy and Better Safety of Double β-Lactam Combination Therapy versus β‑Lactam plus Aminoglycoside in Gram-Negative Bacteria in Randomized, Controlled Trials

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00425-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 4

Author(s):

Yuanyuan Jiao ◽

Bartolome Moya ◽

Mong-Jen Chen ◽

Alexandre P. Zavascki ◽

Hsinyin Tsai ◽

...

Keyword(s):

Clinical Trials ◽

Clinical Response ◽

Risk Ratio ◽

Sensitivity Analyses ◽

Secondary Outcome ◽

Controlled Trials ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Randomized Controlled

ABSTRACTThere is a great need for efficacious therapies against Gram-negative bacteria. Double β-lactam combination(s) (DBL) are relatively safe, and preclinical data are promising; however, their clinical role has not been well defined. We conducted a metaanalysis of the clinical and microbiological efficacy of DBL compared to β-lactam plus aminoglycoside combinations (BLAG). PubMed, Embase, ISI Web of Knowledge, and Cochrane Controlled Trials Register database were searched through July 2018. We included randomized controlled clinical trials that compared DBL with BLAG combinations. Clinical response was used as the primary outcome and microbiological response in Gram-negative bacteria as the secondary outcome; sensitivity analyses were performed forPseudomonas aeruginosa,Klebsiellaspp., andEscherichia coli. Heterogeneity and risk of bias were assessed. Safety results were classified by systems and organs. Thirteen studies evaluated 2,771 cases for clinical response and 665 cases for microbiological response in various Gram-negative species. DBL achieved slightly, but not significantly, better clinical response (risk ratio, 1.05; 95% confidence interval [CI], 0.99 to 1.11) and microbiological response in Gram-negatives (risk ratio, 1.11; 95% CI, 0.99 to 1.25) compared with BLAG. Sensitivity analyses by pathogen showed the same trend. No significant heterogeneity across studies was found. DBL was significantly safer than BLAG regarding renal toxicity (6.6% versus 8.8%,P = 0.0338) and ototoxicity (0.7 versus 3.1%,P = 0.0137). Other adverse events were largely comparable. Overall, empirically designed DBL showed comparable clinical and microbiological responses across different Gram-negative species, and were significantly safer than BLAG. Therefore, DBL should be rationally optimized via the latest translational approaches, leveraging mechanistic insights and newer β-lactams for future evaluation in clinical trials.

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