Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

ABSTRACTAcinetobacter baumanniiis a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources forA. baumanniistrain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that makeA. baumanniia formidable public health threat.IMPORTANCEAcinetobacter baumanniiis one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate ofA. baumanniithat is highly virulent and representative of current outbreak strains.

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The Acinetobacter baumannii Znu System Overcomes Host-Imposed Nutrient Zinc Limitation

Infection and Immunity ◽

10.1128/iai.00746-19 ◽

2019 ◽

Vol 87 (12) ◽

Cited By ~ 4

Author(s):

Laura E. Hesse ◽

Zachery R. Lonergan ◽

William N. Beavers ◽

Eric P. Skaar

Keyword(s):

Acinetobacter Baumannii ◽

Defense Mechanisms ◽

Pathogenic Bacteria ◽

Bacterial Pathogen ◽

Multidrug Resistant ◽

World Health ◽

Content Type ◽

Nutritional Immunity ◽

Essential Nutrient ◽

Health Organization

ABSTRACT Acinetobacter baumannii is an opportunistic bacterial pathogen capable of causing a variety of infections, including pneumonia, sepsis, wound, and burn infections. A. baumannii is an increasing threat to public health due to the prevalence of multidrug-resistant strains, leading the World Health Organization to declare A. baumannii a “Priority 1: Critical” pathogen, for which the development of novel antimicrobials is desperately needed. Zinc (Zn) is an essential nutrient that pathogenic bacteria, including A. baumannii, must acquire from their hosts in order to survive. Consequently, vertebrate hosts have defense mechanisms to sequester Zn from invading bacteria through a process known as nutritional immunity. Here, we describe a Zn uptake (Znu) system that enables A. baumannii to overcome this host-imposed Zn limitation. The Znu system consists of an inner membrane ABC transporter and an outer membrane TonB-dependent receptor. Strains of A. baumannii lacking any individual Znu component are unable to grow in Zn-starved conditions, including in the presence of the host nutritional immunity protein calprotectin. The Znu system contributes to Zn-limited growth by aiding directly in the uptake of Zn into A. baumannii cells and is important for pathogenesis in murine models of A. baumannii infection. These results demonstrate that the Znu system allows A. baumannii to subvert host nutritional immunity and acquire Zn during infection.

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Novel Engineered Peptides of a Phage Lysin as Effective Antimicrobials against Multidrug-Resistant Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02972-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 2671-2679 ◽

Cited By ~ 52

Author(s):

Mya Thandar ◽

Rolf Lood ◽

Benjamin Y. Winer ◽

Douglas R. Deutsch ◽

Chad W. Euler ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Bacterial Pathogen ◽

Antibacterial Activities ◽

Polymyxin B ◽

Multidrug Resistant ◽

Gram Negative ◽

Content Type ◽

Human Red Blood Cells ◽

Phage Lysin

ABSTRACTAcinetobacter baumanniiis a Gram-negative bacterial pathogen responsible for a range of nosocomial infections. The recent rise and spread of multidrug-resistantA. baumanniiclones has fueled a search for alternative therapies, including bacteriophage endolysins with potent antibacterial activities. A common feature of these lysins is the presence of a highly positively charged C-terminal domain with a likely role in promoting outer membrane penetration. In the present study, we show that the C-terminal amino acids 108 to 138 of phage lysin PlyF307, named P307, alone were sufficient to killA. baumannii(>3 logs). Furthermore, P307 could be engineered for improved activity, the most active derivative being P307SQ-8C(>5-log kill). Both P307 and P307SQ-8Cshowed highin vitroactivity againstA. baumanniiin biofilms. Moreover, P307SQ-8Cexhibited MICs comparable to those of levofloxacin and ceftazidime and acted synergistically with polymyxin B. Although the peptides were shown to kill by disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B cells; however, serum was found to be inhibitory to lytic activity. In a murine model ofA. baumanniiskin infection, P307SQ-8Creduced the bacterial burden by ∼2 logs in 2 h. This study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to treat topical infections and remove biofilms caused by Gram-negative pathogens.

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Genomic Analysis of the Multidrug-Resistant Acinetobacter baumannii Strain MDR-ZJ06 Widely Spread in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01134-10 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4506-4512 ◽

Cited By ~ 77

Author(s):

Hua Zhou ◽

Tongwu Zhang ◽

Dongliang Yu ◽

Borui Pi ◽

Qing Yang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Resistance Island

ABSTRACTWe previously reported that the multidrug-resistant (MDR)Acinetobacter baumanniistrain MDR-ZJ06, belonging to European clone II, was widely spread in China. In this study, we report the whole-genome sequence of this clinically important strain. A 38.6-kb AbaR-type genomic resistance island (AbaR22) was identified in MDR-ZJ06. AbaR22 has a structure similar to those of the resistance islands found inA. baumanniistrains AYE and AB0057, but it contained only a few antibiotic resistance genes. The region of resistant gene accumulation as previously described was not found in AbaR22. In the chromosome of the strain MDR-ZJ06, we identified the geneblaoxa-23in a composite transposon (Tn2009). Tn2009shared the backbone with otherA. baumanniitransponsons that harborblaoxa-23, but it was bracketed by two ISAba1elements which were transcribed in the same orientation. MDR-ZJ06 also expressed thearmAgene on its plasmid pZJ06, and this gene has the same genetic environment as thearmAgene of theEnterobacteriaceae. These results suggest variability of resistance acquisition even in closely relatedA. baumanniistrains.

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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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Radiation-Inactivated Acinetobacter baumannii Vaccine Candidates

Vaccines ◽

10.3390/vaccines9020096 ◽

2021 ◽

Vol 9 (2) ◽

pp. 96

Author(s):

Stephen J. Dollery ◽

Daniel V. Zurawski ◽

Elena K. Gaidamakova ◽

Vera Y. Matrosova ◽

John K. Tobin ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Treatment Options ◽

Bacterial Pathogen ◽

Multidrug Resistant ◽

Bacterial Cells ◽

Wound Infections ◽

Protein Profiles ◽

Vaccine Candidates ◽

Rich Media ◽

Life Threatening

Acinetobacter baumannii is a bacterial pathogen that is often multidrug-resistant (MDR) and causes a range of life-threatening illnesses, including pneumonia, septicemia, and wound infections. Some antibiotic treatments can reduce mortality if dosed early enough before an infection progresses, but there are few other treatment options when it comes to MDR-infection. Although several prophylactic strategies have been assessed, no vaccine candidates have advanced to clinical trials or have been approved. Herein, we rapidly produced protective whole-cell immunogens from planktonic and biofilm-like cultures of A. baumannii, strain AB5075 grown using a variety of methods. After selecting a panel of five cultures based on distinct protein profiles, replicative activity was extinguished by exposure to 10 kGy gamma radiation in the presence of a Deinococcus antioxidant complex composed of manganous (Mn2+) ions, a decapeptide, and orthophosphate. Mn2+ antioxidants prevent hydroxylation and carbonylation of irradiated proteins, but do not protect nucleic acids, yielding replication-deficient immunogenic A. baumannii vaccine candidates. Mice were immunized and boosted twice with 1.0 × 107 irradiated bacterial cells and then challenged intranasally with AB5075 using two mouse models. Planktonic cultures grown for 16 h in rich media and biofilm cultures grown in static cultures underneath minimal (M9) media stimulated immunity that led to 80–100% protection.

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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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Genomic Analysis of Multiresistant Staphylococcus capitis Associated with Neonatal Sepsis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00898-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 11

Author(s):

Glen P. Carter ◽

James E. Ussher ◽

Anders Gonçalves Da Silva ◽

Sarah L. Baines ◽

Helen Heffernan ◽

...

Keyword(s):

Neonatal Sepsis ◽

Neonatal Intensive Care ◽

Genomic Analysis ◽

Bloodstream Infections ◽

Multidrug Resistant ◽

Comparative Genomic ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Staphylococcus Capitis ◽

Hospital Infection Control

ABSTRACT Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.

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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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In Vitro Activity of Sulbactam-Durlobactam against Acinetobacter baumannii-calcoaceticus Complex Isolates Collected Globally in 2016 and 2017

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02534-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 4

Author(s):

Sarah M. McLeod ◽

Samir H. Moussa ◽

Meredith A. Hackel ◽

Alita A. Miller

Keyword(s):

Acinetobacter Baumannii ◽

Antibacterial Activities ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Single Amino Acid ◽

Content Type ◽

Level Of Activity ◽

Severe Infections ◽

Sources Of Infection

ABSTRACT Acinetobacter baumannii-calcoaceticus complex (ABC) organisms cause severe infections that are difficult to treat due to preexisting antibiotic resistance. Sulbactam-durlobactam (formerly sulbactam-ETX2514) (SUL-DUR) is a β-lactam–β-lactamase inhibitor combination antibiotic designed to treat serious infections caused by ABC organisms, including multidrug-resistant (MDR) strains. The in vitro antibacterial activities of SUL-DUR and comparator agents were determined by broth microdilution against 1,722 clinical isolates of ABC organisms collected in 2016 and 2017 from 31 countries across Asia/South Pacific, Europe, Latin America, the Middle East, and North America. Over 50% of these isolates were resistant to carbapenems. Against this collection of global isolates, SUL-DUR had a MIC50/MIC90 of 1/2 μg/ml compared to a MIC50/MIC90 of 8/64 μg/ml for sulbactam alone. This level of activity was found to be consistent across organisms, regions, sources of infection, and subsets of resistance phenotypes, including MDR and extensively drug-resistant isolates. The SUL-DUR activity was superior to those of the tested comparators, with only colistin having similar potency. Whole-genome sequencing of the 39 isolates (2.3%) with a SUL-DUR MIC of >4 μg/ml revealed that these strains encoded either the metallo-β-lactamase NDM-1, which durlobactam does not inhibit, or single amino acid substitutions near the active site of penicillin binding protein 3 (PBP3), the primary target of sulbactam. In summary, SUL-DUR demonstrated potent antibacterial activity against recent, geographically diverse clinical isolates of ABC organisms, including MDR isolates.

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