In Vivo-Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media

ABSTRACTA critical feature of a potential antimicrobial target is the characteristic of being essential for growth and survival during host infection. For bacteria, genome-wide essentiality screens are usually performed on rich laboratory media. This study addressed whether genes detected in that manner were optimal for the identification of antimicrobial targets since thein vivomilieu is fundamentally different. Mutant derivatives of a clinical isolate ofAcinetobacter baumanniiwere screened for growth on human ascites, anex vivomedium that reflects the infection environment. A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, establishing 18 (53%) of these genes asin vivoessential. The putative gene products all had annotated biological functions, represented unrecognized or underexploited antimicrobial targets, and could be grouped into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. TheseA. baumannii in vivoessential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria catalogued in the Database of Essential Genes (DEG), including those ofAcinetobacter baylyi, a closely related species. However, this finding was not due to the absence of orthologs. None of the 18in vivoessential genes identified in this study, or their putative gene products, were targets of FDA-approved drugs or drugs in the developmental pipeline, indicating that a significant portion of the available target space within pathogenic Gram-negative bacteria is currently neglected.IMPORTANCEThe human pathogenAcinetobacter baumanniiis of increasing clinical importance, and a growing proportion of isolates are multiantimicrobial-resistant, pan-antimicrobial-resistant, or extremely resistant strains. This scenario is reflective of the general problem of a critical lack of antimicrobials effective against antimicrobial-resistant Gram-negative bacteria, such asPseudomonas aeruginosa,Klebsiella pneumoniae,Enterobactersp., andEscherichia coli. This study identified a set ofA. baumanniigenes that are essential for growth and survival during infection and demonstrated the importance of using clinically relevant media andin vivovalidation while screening for essential genes for the purpose of developing new antimicrobials. Furthermore, it established that if a gene is absent from the Database of Essential Genes, it should not be excluded as a potential antimicrobial target. Lastly, a new set of high-value potential antimicrobial targets for pathogenic Gram-negative bacteria has been identified.

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CharacterizingIn VivoPharmacodynamics of Carbapenems against Acinetobacter baumannii in a Murine Thigh Infection Model To Support Breakpoint Determinations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02029-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 599-601 ◽

Cited By ~ 11

Author(s):

Shawn H. MacVane ◽

Jared L. Crandon ◽

David P. Nicolau

Keyword(s):

Acinetobacter Baumannii ◽

Free Drug ◽

Bacterial Density ◽

Infection Model ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Dosing Interval ◽

Drug Concentrations

ABSTRACTPharmacodynamic profiling data of carbapenems forAcinetobacterspp. are sparse. This study aimed to determine the pharmacodynamic targets of carbapenems forAcinetobacter baumanniibased on a range of percentages of the dosing interval in which free drug concentrations remained above the MIC (fT>MIC) in the neutropenic murine thigh infection model.fT>MIC values of 23.7%, 32.8%, and 47.5% resulted in stasis, 1-log reductions, and 2-log reductions in bacterial density after 24 h, respectively. The pharmacodynamic targets of carbapenems forA. baumanniidemonstratedin vivoare similar to those of other Gram-negative bacteria.

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Efficacy of Humanized Cefiderocol Exposures over 72 Hours against a Diverse Group of Gram-Negative Isolates in the Neutropenic Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01040-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01040-18 ◽

Cited By ~ 3

Author(s):

Sean M. Stainton ◽

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Infection Model ◽

Diverse Group ◽

Gram Negative ◽

Content Type ◽

Adaptive Resistance

ABSTRACT Herein, we evaluated sustainability of humanized exposures of cefiderocol in vivo over 72 h against pathogens with cefiderocol MICs of 0.5 to 16 μg/ml in the neutropenic murine thigh model. In Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae displaying MICs of 0.5 to 8 μg/ml (n = 11), sustained kill was observed at 72 h among 9 isolates. Postexposure MICs revealed a single 2-dilution increase in one animal compared with controls (1/54 samples, 1.8%) at 72 h. Adaptive resistance during therapy was not observed.

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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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Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.00243-19 ◽

2019 ◽

Vol 87 (9) ◽

Cited By ~ 5

Author(s):

Chad R. Marion ◽

Jaewook Lee ◽

Lokesh Sharma ◽

Kyong-Su Park ◽

Changjin Lee ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Pulmonary Inflammation ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Toll Like Receptors ◽

Gram Negative ◽

Content Type ◽

Intranasal Introduction

ABSTRACT Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.

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Efficacy of Humanized Exposures of Cefiderocol (S-649266) against a Diverse Population of Gram-Negative Bacteria in a Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01022-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 35

Author(s):

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

David P. Nicolau

Keyword(s):

Antibacterial Activity ◽

Multidrug Resistant ◽

Infection Model ◽

Gram Negative Bacteria ◽

Diverse Population ◽

Gram Negative ◽

Content Type ◽

Log Reduction

ABSTRACT Cefiderocol (S-649266) is a novel siderophore cephalosporin with potent in vitro activity against clinically encountered multidrug-resistant (MDR) Gram-negative isolates; however, its spectrum of antibacterial activity against these difficult-to-treat isolates remains to be fully explored in vivo. Here, we evaluated the efficacy of cefiderocol humanized exposures in a neutropenic murine thigh model to support a suitable MIC breakpoint. Furthermore, we compared cefiderocol's efficacy with humanized exposures of meropenem and cefepime against a subset of these phenotypically diverse isolates. Ninety-five Gram-negative isolates were studied. Efficacy was determined as the change in log10 CFU at 24 h compared with 0-h controls. Bacterial stasis or ≥1 log reduction in 67 isolates with MICs of ≤4 μg/ml was noted in 77, 88, and 85% of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa, respectively. For isolates with MICs of ≥8 μg/ml, bacterial stasis or ≥1 log10 reduction was observed in only 2 of 28 (8 Enterobacteriaceae, 19 A. baumannii, and 1 P. aeruginosa) strains. Against highly resistant meropenem and cefepime organisms, cefiderocol maintained its in vivo efficacy. Overall, humanized exposures of cefiderocol produced similar reductions in bacterial density for organisms with MICs of ≤4 μg/ml, whereas isolates with MICs of ≥8 μg/ml generally displayed bacterial growth in the presence of the compound. Data derived in the current study will assist with the delineation of MIC susceptibility breakpoints for cefiderocol against these important nosocomial Gram-negative pathogens; however, additional clinical data are required to substantiate these observations.

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Caspase-11 Plays a Protective Role in Pulmonary Acinetobacter baumannii Infection

Infection and Immunity ◽

10.1128/iai.00350-17 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 10

Author(s):

Wei Wang ◽

Yue Shao ◽

Shengjun Li ◽

Na Xin ◽

Tingxian Ma ◽

...

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Acinetobacter Baumannii ◽

Pulmonary Infection ◽

Protective Role ◽

Innate Immune ◽

Gram Negative Bacteria ◽

Pathological Changes ◽

Gram Negative ◽

Content Type

ABSTRACT Activation of caspase-11 by some Gram-negative bacteria triggers the caspase-1/interleukin 1β (IL-1β) pathway, independent of canonical inflammasomes. Acinetobacter baumannii is a Gram-negative, conditionally pathogenic bacterium that can cause severe pulmonary infection in hospitalized patients. A. baumannii was revealed to activate canonical and noncanonical inflammasome pathways in bone marrow-derived macrophages (BMDMs). Pulmonary infection of caspase-11−/− mice with A. baumannii showed that caspase-11 deficiency impaired A. baumannii clearance, exacerbated pulmonary pathological changes, and enhanced susceptibility to A. baumannii. These data indicate that the caspase-11-mediated innate immune response plays a crucial role in defending against A. baumannii.

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Structural Characterization of TssL from Acinetobacter baumannii: a Key Component of the Type VI Secretion System

Journal of Bacteriology ◽

10.1128/jb.00210-20 ◽

2020 ◽

Vol 202 (17) ◽

Author(s):

Federico M. Ruiz ◽

Juvenal Lopez ◽

C. Gastón Ferrara ◽

Elena Santillana ◽

Yanis R. Espinosa ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Secretion System ◽

Single Locus ◽

Gram Negative Bacteria ◽

Cytoplasmic Protein ◽

Type Vi Secretion System ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a complex molecular nanomachine used by Gram-negative bacteria to deliver diverse effectors into adjacent cells. A membrane complex (MC) anchors this transport system to the bacterial cell wall. One of the proteins forming the MC is TssL, a cytoplasmic protein bound to the inner membrane through a single transmembrane helix. Here, we report the structure of the cytoplasmic N-terminal region of TssL from Acinetobacter baumannii, a bacterium encoding in a single locus a secretion system that is a special case among other T6SSs. The protein structure, consisting of two antiparallel alpha-helical bundles connected by a short loop, reveals several interesting particularities compared with homologous proteins from other organisms. In addition, we demonstrate the structural significance of residues Asp98 and Glu99, which are strongly conserved among T6SS-encoding Gram-negative bacteria. Mutations in these two residues strongly impact protein dynamics, expression, and functionality. Our results improve our understanding of the T6SS of A. baumannii, which remains largely understudied compared with that of other pathogens. IMPORTANCE Several Acinetobacter species carry one functional type VI secretion system (T6SS). The T6SS is encoded in a single locus containing 16 conserved genes, most of which code for proteins essential to T6SS activity. One of these key components is TssL, a cytoplasmic protein bound to the inner membrane. Despite its importance and its particular characteristics, the structure of T6SS in A. baumannii remains understudied. Here, we present structural, in silico, and in vivo studies of TssL, highlighting the importance of two well-conserved residues and improving our understanding of this secretion system in this bacterium.

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ARGONAUT-I: Activity of Cefiderocol (S-649266), a Siderophore Cephalosporin, against Gram-Negative Bacteria, Including Carbapenem-Resistant Nonfermenters and Enterobacteriaceae with Defined Extended-Spectrum β-Lactamases and Carbapenemases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01801-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 20

Author(s):

Michael R. Jacobs ◽

Ayman M. Abdelhamed ◽

Caryn E. Good ◽

Daniel D. Rhoads ◽

Kristine M. Hujer ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Stenotrophomonas Maltophilia ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Carbapenem Resistant ◽

Extended Spectrum ◽

Mueller Hinton ◽

Mueller Hinton Broth

ABSTRACT The activity of the siderophore cephalosporin cefiderocol is targeted against carbapenem-resistant Gram-negative bacteria. In this study, the activity of cefiderocol against characterized carbapenem-resistant Acinetobacter baumannii complex, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Enterobacteriaceae strains was determined by microdilution in iron-depleted Mueller-Hinton broth. The MIC90s against A. baumannii, S. maltophilia, and P. aeruginosa were 1, 0.25, and 0.5 mg/liter, respectively. Against Enterobacteriaceae, the MIC90 was 1 mg/liter for the group harboring OXA-48-like, 2 mg/liter for the group harboring KPC-3, and 8 mg/liter for the group harboring TEM/SHV ESBL, NDM, and KPC-2.

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Genomic and Resistance Epidemiology of Gram-Negative Bacteria in Africa: a Systematic Review and Phylogenomic Analyses from a One Health Perspective

mSystems ◽

10.1128/msystems.00897-20 ◽

2020 ◽

Vol 5 (6) ◽

Cited By ~ 1

Author(s):

John Osei Sekyere ◽

Melese Abate Reta

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Acinetobacter Baumannii ◽

Vibrio Cholerae ◽

Salmonella Enterica ◽

Animal Health ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type

ABSTRACT Antibiotic resistance (AR) remains a major threat to public and animal health globally. However, AR ramifications in developing countries are worsened by limited molecular diagnostics, expensive therapeutics, inadequate numbers of skilled clinicians and scientists, and unsanitary environments. The epidemiology of Gram-negative bacteria, their AR genes, and geographical distribution in Africa are described here. Data were extracted and analyzed from English-language articles published between 2015 and December 2019. The genomes and AR genes of the various species, obtained from the Pathosystems Resource Integration Center (PATRIC) and NCBI were analyzed phylogenetically using Randomized Axelerated Maximum Likelihood (RAxML) and annotated with Figtree. The geographic location of resistant clones/clades was mapped manually. Thirty species from 31 countries and 24 genera from 41 countries were analyzed from 146 articles and 3,028 genomes, respectively. Genes mediating resistance to β-lactams (including blaTEM-1, blaCTX-M, blaNDM, blaIMP, blaVIM, and blaOXA-48/181), fluoroquinolones (oqxAB, qnrA/B/D/S, gyrA/B, and parCE mutations, etc.), aminoglycosides (including armA and rmtC/F), sulfonamides (sul1/2/3), trimethoprim (dfrA), tetracycline [tet(A/B/C/D/G/O/M/39)], colistin (mcr-1), phenicols (catA/B, cmlA), and fosfomycin (fosA) were mostly found in Enterobacter spp. and Klebsiella pneumoniae, and also in Serratia marcescens, Escherichia coli, Salmonella enterica, Pseudomonas, Acinetobacter baumannii, etc., on mostly IncF-type, IncX3/4, ColRNAI, and IncR plasmids, within IntI1 gene cassettes, insertion sequences, and transposons. Clonal and multiclonal outbreaks and dissemination of resistance genes across species and countries and between humans, animals, plants, and the environment were observed; Escherichia coli ST103, K. pneumoniae ST101, S. enterica ST1/2, and Vibrio cholerae ST69/515 were common strains. Most pathogens were of human origin, and zoonotic transmissions were relatively limited. IMPORTANCE Antibiotic resistance (AR) is one of the major public health threats and challenges to effective containment and treatment of infectious bacterial diseases worldwide. Here, we used different methods to map out the geographical hot spots, sources, and evolutionary epidemiology of AR. Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Neisseria meningitis/gonorrhoeae, Vibrio cholerae, Campylobacter jejuni, etc., were common pathogens shuttling AR genes in Africa. Transmission of the same clones/strains across countries and between animals, humans, plants, and the environment was observed. We recommend Enterobacter spp. or K. pneumoniae as better sentinel species for AR surveillance.

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Four Carbapenem-Resistant Gram-Negative Species Carrying Distinct Carbapenemases in a Single Patient

Journal of Clinical Microbiology ◽

10.1128/jcm.03623-14 ◽

2014 ◽

Vol 53 (3) ◽

pp. 1031-1033 ◽

Cited By ~ 8

Author(s):

Baixing Ding ◽

Fupin Hu ◽

Yang Yang ◽

Qinglan Guo ◽

Jinwei Huang ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Acinetobacter Baumannii ◽

Enterobacter Aerogenes ◽

Single Patient ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

Carbapenem-resistantEscherichia coli,Klebsiella pneumoniae,Enterobacter aerogenes, andAcinetobacter baumanniiwere isolated from a single patient, each producing different carbapenemases (NDM-1, KPC-2, IMP, and OXA-23, respectively). The NDM-1-producingE. colistrain was preceded by a clonally related carbapenem-susceptible strain a month earlier, suggestingin vivoacquisition ofblaNDM-1.

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