Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii

ABSTRACT The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii. In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii. In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application. IMPORTANCE Acinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

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Complete Genome Sequences of Four Extensively Drug-Resistant Acinetobacter baumannii Isolates from Thailand

Microbiology Resource Announcements ◽

10.1128/mra.00949-20 ◽

2020 ◽

Vol 9 (40) ◽

Author(s):

Peechanika Chopjitt ◽

Thidathip Wongsurawat ◽

Piroon Jenjaroenpun ◽

Parichart Boueroy ◽

Rujirat Hatrongjit ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Complete Genome ◽

Sequence Type ◽

Clinical Isolates ◽

Drug Resistant ◽

Genome Sequences ◽

Content Type ◽

Type Isolate ◽

Resistant Genes ◽

Extensively Drug Resistant

ABSTRACT Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.

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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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Complete Genome Sequence of Acinetobacter radioresistens Strain LH6, a Multidrug-Resistant Bacteriophage-Propagating Strain

Microbiology Resource Announcements ◽

10.1128/mra.00929-18 ◽

2018 ◽

Vol 7 (5) ◽

Cited By ~ 2

Author(s):

Clay S. Crippen ◽

Steven Huynh ◽

William G. Miller ◽

Craig T. Parker ◽

Christine M. Szymanski

Keyword(s):

Antimicrobial Resistance ◽

Acinetobacter Baumannii ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Related Species ◽

Complete Genome ◽

Multidrug Resistant ◽

Drug Resistant ◽

Content Type ◽

Resistance Determinants

Antimicrobial resistance is a major problem worldwide. Understanding the interplay between drug-resistant pathogens, such as Acinetobacter baumannii and related species, potentially acting as environmental reservoirs is critical for preventing the spread of resistance determinants.

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Draft Genome Sequences of Clinical Isolates of Multidrug-Resistant Acinetobacter baumannii

Genome Announcements ◽

10.1128/genomea.01547-16 ◽

2017 ◽

Vol 5 (5) ◽

Author(s):

Keesha E. Erickson ◽

Nancy E. Madinger ◽

Anushree Chatterjee

Keyword(s):

Acinetobacter Baumannii ◽

Resistance Genes ◽

Draft Genome ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Genome Sequences ◽

Content Type ◽

University Of Colorado ◽

The University

ABSTRACT We report here the draft genome sequences of two clinically isolated Acinetobacter baumannii strains. These samples were obtained from patients at the University of Colorado Hospital in 2007 and 2013 and encode an estimated 20 and 13 resistance genes, respectively.

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Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

mSphere ◽

10.1128/msphere.00140-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 6

Author(s):

Brian M. Luna ◽

Amber Ulhaq ◽

Jun Yan ◽

Paul Pantapalangkoor ◽

Travis B. Nielsen ◽

...

Keyword(s):

Molecular Biology ◽

Acinetobacter Baumannii ◽

Genetic Manipulation ◽

Multidrug Resistant ◽

Molecular Techniques ◽

Drug Resistant ◽

Selectable Markers ◽

Content Type ◽

Extensively Drug Resistant ◽

Selection Of

ABSTRACT Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

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Real-Time Sequencing To Decipher the Molecular Mechanism of Resistance of a Clinical Pan-Drug-Resistant Acinetobacter baumannii Isolate from Marseille, France

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01314-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 592-596 ◽

Cited By ~ 48

Author(s):

Jean-Marc Rolain ◽

Seydina M. Diene ◽

Marie Kempf ◽

Gregory Gimenez ◽

Catherine Robert ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Molecular Mechanisms ◽

Multidrug Resistant ◽

Drug Resistant ◽

Two Component System ◽

Genome Sequences ◽

Component System ◽

Content Type ◽

Two Component ◽

Baumannii Isolate

ABSTRACTWe compare the whole-genome sequences of two multidrug-resistant clinicalAcinetobacter baumanniiisolates recovered in the same patient before (ABIsac_ColiS susceptible to colistin and rifampin only) and after (ABIsac_ColiR resistant to colistin and rifampin) treatment with colistin and rifampin. We decipher all the molecular mechanisms of antibiotic resistance, and we found mutations in therpoBgene and in the PmrAB two-component system explaining resistance to rifampin and colistin in ABIsac_ColiR, respectively.

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Globally Expanding Carbapenemase Finally Appears in Spain: Nosocomial Outbreak of Acinetobacter baumannii Producing Plasmid-Encoded OXA-23 in Barcelona, Spain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01486-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 5155-5157 ◽

Cited By ~ 25

Author(s):

Noraida Mosqueda ◽

Paula Espinal ◽

Clara Cosgaya ◽

Sergio Viota ◽

Virginia Plasensia ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Molecular Characterization ◽

Multidrug Resistant ◽

Sequence Type ◽

Clinical Isolates ◽

Nosocomial Outbreak ◽

Content Type ◽

Hospital Outbreak

ABSTRACTResistance ofAcinetobacter baumanniiclinical isolates to carbapenems is on the rise worldwide mainly in association with the production of OXA-23. Until recently, however, OXA-23 was absent in Spain. In this work, we report the molecular characterization of a hospital outbreak of OXA-23-producingA. baumanniiin Barcelona caused by a multidrug-resistant (MDR) clone belonging to international clone IC-II/sequence type ST85 between October 2010 and May 2011.blaOXA-23was carried in a plasmid of 90 kb and located within the composite transposon Tn2006.

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In VitroSusceptibility of Mycobacterium tuberculosis Isolates to an Oral Carbapenem Alone or in Combination with β-Lactamase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03539-14 ◽

2014 ◽

Vol 58 (11) ◽

pp. 7010-7014 ◽

Cited By ~ 21

Author(s):

Yasuhiro Horita ◽

Shinji Maeda ◽

Yuko Kazumi ◽

Norio Doi

Keyword(s):

Mycobacterium Tuberculosis ◽

Human Blood ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

ABSTRACTWe evaluated the antituberculosis (anti-TB) activity of five β-lactams alone or in combination with β-lactamase inhibitors against 41 clinical isolates ofMycobacterium tuberculosis, including multidrug-resistant and extensively drug-resistant strains. Of those, tebipenem, an oral carbapenem, showed the most potent anti-TB activity against clinical isolates, with a MIC range of 0.125 to 8 μg/ml, which is achievable in the human blood. More importantly, in the presence of clavulanate, MIC values of tebipenem declined to 2 μg/ml or less.

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Synergistic Effects and Antibiofilm Properties of Chimeric Peptides against Multidrug-Resistant Acinetobacter baumannii Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02473-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1622-1629 ◽

Cited By ~ 41

Author(s):

Ramamourthy Gopal ◽

Young Gwon Kim ◽

Jun Ho Lee ◽

Seog Ki Lee ◽

Jeong Don Chae ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Synergistic Effects ◽

Antibacterial Activities ◽

Multidrug Resistant ◽

Drug Resistant ◽

Antibacterial Effects ◽

Content Type ◽

Chimeric Peptides ◽

Novel Antibiotics

ABSTRACTThe increasing prevalence of drug-resistant pathogens highlights the need to identify novel antibiotics. Here we investigated the efficacies of four new antimicrobial peptides (AMPs) for potential drug development. The antibacterial activities, synergistic effects, and antibiofilm properties of the four chimeric AMPs were tested againstAcinetobacter baumannii, an emerging Gram-negative, nosocomial, drug-resistant pathogen. NineteenA. baumanniistrains resistant to ampicillin, cefotaxime, ciprofloxacin, tobramycin, and erythromycin were isolated at a hospital from patients with cholelithiasis. All four peptides exhibited significant antibacterial effects (MIC = 3.12 to 12.5 μM) against all 19 strains, whereas five commercial antibiotics showed little or no activity against the same pathogens. An exception was polymyxin, which was effective against all of the strains tested. Each of the peptides showed synergy against one or more strains when administered in combination with cefotaxime, ciprofloxacin, or erythromycin. The peptides also exhibited an ability to prevent biofilm formation, which was not seen with cefotaxime, ciprofloxacin, or erythromycin, though polymyxin also inhibited biofilm formation. Indeed, when administered in combination with ciprofloxacin, the AMP HPMA exerted a potent synergistic effect againstA. baumanniibiofilm formation. Collectively, our findings indicate that the AMPs tested have no cytotoxicity but possess potent antibacterial and antibiofilm activities and may act synergistically with commercial antibiotics.

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Distribution of Intrinsic Plasmid Replicase Genes and Their Association with Carbapenem-Hydrolyzing Class D β-Lactamase Genes in European Clinical Isolates of Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01661-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2154-2159 ◽

Cited By ~ 32

Author(s):

Kevin J. Towner ◽

Benjamin Evans ◽

Laura Villa ◽

Katrina Levi ◽

Ahmed Hamouda ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Horizontal Transmission ◽

Antibiotic Resistance Genes ◽

General System ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Content Type ◽

Class D ◽

Genes Encoding ◽

Plasmid Mobilization

ABSTRACTNinety-six genetically diverse multidrug-resistant clinical isolates ofAcinetobacter baumanniifrom 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D β-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups ofrepgenes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (includingrepAci1 [67 isolates] andrepAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations inrepgene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of therepAci1,repAci3,repAci4, andrepAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated withrepAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated withrepAci1. Most intrinsicAcinetobacterplasmids are non-self-transferable, but the almost ubiquitousrepAci6 gene was strongly associated with a potentialtralocus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains ofA. baumannii.

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