Tags

Type your tag names separated by a space and hit enter

Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1.
mSphere. 2017 Mar-Apr; 2(2)M

Abstract

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.

Authors+Show Affiliations

Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.Department of Microbiology, Miami University, Oxford, Ohio, USA.Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, USA. Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28497114

Citation

Luna, Brian M., et al. "Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter Baumannii HUMC1." MSphere, vol. 2, no. 2, 2017.
Luna BM, Ulhaq A, Yan J, et al. Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1. mSphere. 2017;2(2).
Luna, B. M., Ulhaq, A., Yan, J., Pantapalangkoor, P., Nielsen, T. B., Davies, B. W., Actis, L. A., & Spellberg, B. (2017). Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1. MSphere, 2(2). https://doi.org/10.1128/mSphere.00140-17
Luna BM, et al. Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter Baumannii HUMC1. mSphere. 2017 Mar-Apr;2(2) PubMed PMID: 28497114.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1. AU - Luna,Brian M, AU - Ulhaq,Amber, AU - Yan,Jun, AU - Pantapalangkoor,Paul, AU - Nielsen,Travis B, AU - Davies,Bryan W, AU - Actis,Luis A, AU - Spellberg,Brad, Y1 - 2017/04/26/ PY - 2017/03/24/received PY - 2017/04/06/accepted PY - 2017/5/13/entrez PY - 2017/5/13/pubmed PY - 2017/5/13/medline KW - Acinetobacter KW - Gram-negative bacteria KW - antibiotic resistance KW - genetics KW - molecular biology JF - mSphere JO - mSphere VL - 2 IS - 2 N2 - 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. SN - 2379-5042 UR - https://www.unboundmedicine.com/medline/citation/28497114/Selectable_Markers_for_Use_in_Genetic_Manipulation_of_Extensively_Drug_Resistant__XDR__Acinetobacter_baumannii_HUMC1_ L2 - https://doi.org/10.1128/mSphere.00140-17 DB - PRIME DP - Unbound Medicine ER -
Try the Free App:
Prime PubMed app for iOS iPhone iPad
Prime PubMed app for Android
Prime PubMed is provided
free to individuals by:
Unbound Medicine.