Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes

ABSTRACTA paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR)Acinetobacter baumanniiinfections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection ofA. baumanniistrains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 μg/ml. This concentration significantly reduced bacterial viability, while 40 μg/ml killed all cells of theA. baumanniiATCC 19606Tand ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606Tand ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-μg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival ofGalleria mellonellalarvae infected with ATCC 19606Tor ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrantA. baumanniiinfections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media.

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Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02877-15 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5806-5816 ◽

Cited By ~ 61

Author(s):

James M. Regeimbal ◽

Anna C. Jacobs ◽

Brendan W. Corey ◽

Matthew S. Henry ◽

Mitchell G. Thompson ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Therapeutic Option ◽

Multidrug Resistant ◽

Surrounding Tissue ◽

Content Type ◽

Wound Model ◽

Spread Of Infection ◽

Selection Event

ABSTRACTMultidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages againstAcinetobacter baumanniiand demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growthin vitrovia a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulatedA. baumanniibacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor forA. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in aGalleria mellonellamodel. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

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In VitroandIn VivoAntimicrobial Activities of Gallium Nitrate against Multidrug-Resistant Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01519-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5961-5970 ◽

Cited By ~ 72

Author(s):

Luísa C. S. Antunes ◽

Francesco Imperi ◽

Fabrizia Minandri ◽

Paolo Visca

Keyword(s):

Human Serum ◽

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Therapeutic Potential ◽

Survival Rates ◽

Bloodstream Infections ◽

Multidrug Resistant ◽

Gallium Nitrate ◽

Content Type ◽

Bacterial Physiology

ABSTRACTMultidrug-resistantAcinetobacter baumanniiposes a tremendous challenge to traditional antibiotic therapy. Due to the crucial role of iron in bacterial physiology and pathogenicity, we investigated iron metabolism as a possible target for anti-A. baumanniichemotherapy using gallium as an iron mimetic. Due to chemical similarity, gallium competes with iron for binding to several redox enzymes, thereby interfering with a number of essential biological reactions. We found that Ga(NO3)3, the active component of an FDA-approved drug (Ganite), inhibits the growth of a collection of 58A. baumanniistrains in both chemically defined medium and human serum, at concentrations ranging from 2 to 80 μM and from 4 to 64 μM, respectively. Ga(NO3)3delayed the entry ofA. baumanniiinto the exponential phase and drastically reduced bacterial growth rates. Ga(NO3)3activity was strongly dependent on iron availability in the culture medium, though the mechanism of growth inhibition was independent of dysregulation of gene expression controlled by the ferric uptake regulator Fur. Ga(NO3)3also protectedGalleria mellonellalarvae from lethalA. baumanniiinfection, with survival rates of ≥75%. At therapeutic concentrations for humans (28 μM plasma levels), Ga(NO3)3inhibited the growth in human serum of 76% of the multidrug-resistantA. baumanniiisolates tested by ≥90%, raising expectations on the therapeutic potential of gallium for the treatment ofA. baumanniibloodstream infections. Ga(NO3)3also showed strong synergism with colistin, suggesting that a colistin-gallium combination holds promise as a last-resort therapy for infections caused by pan-resistantA. baumannii.

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Characterization of RelA in Acinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00045-20 ◽

2020 ◽

Vol 202 (12) ◽

Cited By ~ 3

Author(s):

María Pérez-Varela ◽

Aimee R. P. Tierney ◽

Ju-Sim Kim ◽

Andrés Vázquez-Torres ◽

Philip Rather

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

Cell Physiology ◽

Content Type ◽

Content Model ◽

Transposon Insertion ◽

Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

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Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606Twith Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

Infection and Immunity ◽

10.1128/iai.06279-11 ◽

2012 ◽

Vol 80 (3) ◽

pp. 1015-1024 ◽

Cited By ~ 126

Author(s):

Jennifer A. Gaddy ◽

Brock A. Arivett ◽

Michael J. McConnell ◽

Rafael López-Rojas ◽

Jerónimo Pachón ◽

...

Keyword(s):

Epithelial Cells ◽

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Ex Vivo ◽

Iron Acquisition ◽

Alveolar Epithelial Cells ◽

Lung Epithelial Cells ◽

Content Type ◽

Alveolar Epithelial

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606Ttype strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays usingGalleria mellonellalarvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606Tcells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with theseex vivoandin vivoapproaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606Tto establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606Tstrain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin.

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GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.00180-20 ◽

2020 ◽

Vol 89 (1) ◽

pp. e00180-20

Author(s):

Michael J. Gebhardt ◽

Daniel M. Czyz ◽

Shweta Singh ◽

Daniel V. Zurawski ◽

Lev Becker ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

Resistant Isolate ◽

Infection Model ◽

Content Type ◽

Transcription Regulator ◽

Nutritional Immunity ◽

Metabolic Products ◽

Lysr Family

ABSTRACTA critical facet of mammalian innate immunity involves the hosts’ attempts to sequester and/or limit the availability of key metabolic products from pathogens. For example, nutritional immunity encompasses host approaches to limit the availability of key heavy metal ions such as zinc and iron. Previously, we identified several hundred genes in a multidrug-resistant isolate of Acinetobacter baumannii that are required for growth and/or survival in the Galleria mellonella infection model. In the present study, we further characterize one of these genes, a LysR family transcription regulator that we previously named GigC. We show that mutant strains lacking gigC have impaired growth in the absence of the amino acid cysteine and that gigC regulates the expression of several genes involved in the sulfur assimilation and cysteine biosynthetic pathways. We further show that cells harboring a deletion of the gigC gene are attenuated in two murine infection models, suggesting that the GigC protein, likely through its regulation of the cysteine biosynthetic pathway, plays a key role in the virulence of A. baumannii.

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CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii

Infection and Immunity ◽

10.1128/iai.00259-20 ◽

2020 ◽

Vol 88 (12) ◽

Author(s):

John M. Farrow ◽

Greg Wells ◽

Samantha Palethorpe ◽

Mark D. Adams ◽

Everett C. Pesci

Keyword(s):

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Bacterial Pathogen ◽

Multidrug Resistant ◽

Growth Defect ◽

Gene Encoding ◽

Content Type ◽

Indirect Transmission ◽

Mutant Strains ◽

Carbon Storage Regulator

ABSTRACT Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism’s abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

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An Improved Medium for Colistin Susceptibility Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01950-17 ◽

2018 ◽

Vol 56 (5) ◽

Cited By ~ 10

Author(s):

Konrad Gwozdzinski ◽

Saina Azarderakhsh ◽

Can Imirzalioglu ◽

Linda Falgenhauer ◽

Trinad Chakraborty

Keyword(s):

Susceptibility Testing ◽

Acquired Resistance ◽

Multidrug Resistant ◽

Colistin Resistance ◽

Reliable Determination ◽

Content Type ◽

E Coli ◽

Resistant Species ◽

Mueller Hinton ◽

Serovar Typhimurium

ABSTRACTThe plasmid-located colistin resistance genemcr-1confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination ofmcr-1-containingEnterobacteriaceae. Colistin susceptibility testing was performed for 50mcr-1-containingEscherichia coliandKlebsiella pneumoniaeisolates, 7 intrinsically polymyxin-resistant species,K. pneumoniaeandE. coliisolates with acquired resistance to polymyxins due tomgrBandpmrBmutations, respectively, and 32mcr-1-negative, colistin-susceptible isolates ofAcinetobacter baumannii,Citrobacter freundii,Enterobacter cloacae,E. coli,K. pneumoniae, andSalmonella entericaserovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lackingmcr-1. Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for bothmcr-1-containingEnterobacteriaceaeisolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.

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Expression, purification, crystallization and preliminary crystallographic analysis of the phosphoglycerate kinase fromAcinetobacter baumannii

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309112020222 ◽

2012 ◽

Vol 68 (7) ◽

pp. 790-792

Author(s):

Kayla Baretta ◽

Craig Garen ◽

Jiang Yin ◽

Michael N. G. James

Keyword(s):

Synchrotron Radiation ◽

Acinetobacter Baumannii ◽

Light Source ◽

Phosphoglycerate Kinase ◽

Multidrug Resistant ◽

Crystallographic Analysis ◽

Lithium Sulfate ◽

Space Group ◽

Antibiotic Development ◽

Potential Target

Acinetobacter baumanniiis a common multidrug-resistant clinical pathogen that is often found in hospitals. TheA. baumanniiphosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development.AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. TheAbPGK crystals belonged to space groupP2221. They diffracted to a resolution of 2.5 Å using synchrotron radiation at the Canadian Light Source.

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Emergence of a Competence-Reducing Filamentous Phage from the Genome of Acinetobacter baylyi ADP1

Journal of Bacteriology ◽

10.1128/jb.00424-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3209-3219 ◽

Cited By ~ 12

Author(s):

Brian A. Renda ◽

Cindy Chan ◽

Kristin N. Parent ◽

Jeffrey E. Barrick

Keyword(s):

Acinetobacter Baumannii ◽

Vibrio Cholerae ◽

Bacterial Species ◽

Normal Growth ◽

Multidrug Resistant ◽

Filamentous Phage ◽

Acinetobacter Baylyi ◽

Laboratory Evolution ◽

Content Type ◽

Evolution Experiment

ABSTRACTBacterial genomes commonly contain prophage sequences as a result of past infections with lysogenic phages. Many of these integrated viral sequences are believed to be cryptic, but prophage genes are sometimes coopted by the host, and some prophages may be reactivated to form infectious particles when cells are stressed or mutate. We found that a previously uncharacterized filamentous phage emerged from the genome ofAcinetobacter baylyiADP1 during a laboratory evolution experiment. This phage has a genetic organization similar to that of theVibrio choleraeCTXϕ phage. The emergence of the ADP1 phage was associated with the evolution of reduced transformability in our experimental populations, so we named it thecompetence-reducingacinetobacter phage (CRAϕ). Knocking out ADP1 genes required for competence leads to resistance to CRAϕ infection. Although filamentous bacteriophages are known to target type IV pili, this is the first report of a phage that apparently uses a competence pilus as a receptor.A. baylyimay be especially susceptible to this route of infection because every cell is competent during normal growth, whereas competence is induced only under certain environmental conditions or in a subpopulation of cells in other bacterial species. It is possible that CRAϕ-like phages restrict horizontal gene transfer in nature by inhibiting the growth of naturally transformable strains. We also found that prophages with homology to CRAϕ exist in several strains ofAcinetobacter baumannii. These CRAϕ-likeA. baumanniiprophages encode toxins similar to CTXϕ that might contribute to the virulence of this opportunistic multidrug-resistant pathogen.IMPORTANCEWe observed the emergence of a novel filamentous phage (CRAϕ) from the genome ofAcinetobacter baylyiADP1 during a long-term laboratory evolution experiment. CRAϕ is the first bacteriophage reported to require the molecular machinery involved in the uptake of environmental DNA for infection. Reactivation and evolution of CRAϕ reduced the potential for horizontal transfer of genes via natural transformation in our experiment. Risk of infection by similar phages may limit the expression and maintenance of bacterial competence in nature. The closest studied relative of CRAϕ is theVibrio choleraeCTXϕ phage. Variants of CRAϕ are found in the genomes ofAcinetobacter baumanniistrains, and it is possible that phage-encoded toxins contribute to the virulence of this opportunistic multidrug-resistant pathogen.

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In VitroActivity of RX-P873 against Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04840-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2280-2285 ◽

Cited By ~ 5

Author(s):

Robert K. Flamm ◽

Paul R. Rhomberg ◽

Ronald N. Jones ◽

David J. Farrell

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Active Agent ◽

Multidrug Resistant ◽

Surveillance Program ◽

Gram Negative ◽

Content Type ◽

Highly Active ◽

Bacterial Ribosome

ABSTRACTRX-P873 is a novel antibiotic from the pyrrolocytosine series which exhibits high binding affinity for the bacterial ribosome and broad-spectrum antibiotic properties. The pyrrolocytosines have shownin vitroactivity against multidrug-resistant Gram-negative and Gram-positive strains of bacteria known to cause complicated urinary tract, skin, and lung infections, as well as sepsis.Enterobacteriaceae(657),Pseudomonas aeruginosa(200), andAcinetobacter baumannii(202) isolates from North America and Europe collected in 2012 as part of a worldwide surveillance program were testedin vitroby broth microdilution using Clinical and Laboratory Standards Institute (CLSI) methodology. RX-P873 (MIC90, 0.5 μg/ml) was >32-fold more active than ceftazidime and inhibited 97.1% and 99.5% ofEnterobacteriaceaeisolates at MIC values of ≤1 and ≤4 μg/ml, respectively. There were only three isolates with an MIC value of >4 μg/ml (all were indole-positiveProtea). RX-P873 (MIC50/90, 2/4 μg/ml) was highly active againstPseudomonas aeruginosaisolates, including isolates which were nonsusceptible to ceftazidime or meropenem. RX-P873 was 2-fold less active againstP. aeruginosathan tobramycin (MIC90, 2 μg/ml; 91.0% susceptible) and colistin (MIC90, 2 μg/ml; 99.5% susceptible) and 2-fold more potent than amikacin (MIC90, 8 μg/ml; 93.5% susceptible) and meropenem (MIC90, 8 μg/ml; 76.0% susceptible). RX-P873, the most active agent againstAcinetobacter baumannii(MIC90, 1 μg/ml), was 2-fold more active than colistin (MIC90, 2 μg/ml; 97.0% susceptible) and 4-fold more active than tigecycline (MIC90, 4 μg/ml). This novel agent merits further exploration of its potential against multidrug-resistant Gram-negative bacteria.

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