Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1

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 t...

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Autores principales:	Brian M. Luna, Amber Ulhaq, Jun Yan, Paul Pantapalangkoor, Travis B. Nielsen, Bryan W. Davies, Luis A. Actis, Brad Spellberg
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Acinetobacter antibiotic resistance genetics Gram-negative bacteria molecular biology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/83a4f617d2854795966090823a1b017a
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spelling	oai:doaj.org-article:83a4f617d2854795966090823a1b017a2021-11-15T15:21:45ZSelectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC110.1128/mSphere.00140-172379-5042https://doaj.org/article/83a4f617d2854795966090823a1b017a2017-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00140-17https://doaj.org/toc/2379-5042ABSTRACT 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.Brian M. LunaAmber UlhaqJun YanPaul PantapalangkoorTravis B. NielsenBryan W. DaviesLuis A. ActisBrad SpellbergAmerican Society for MicrobiologyarticleAcinetobacterantibiotic resistancegeneticsGram-negative bacteriamolecular biologyMicrobiologyQR1-502ENmSphere, Vol 2, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Acinetobacter antibiotic resistance genetics Gram-negative bacteria molecular biology Microbiology QR1-502
spellingShingle	Acinetobacter antibiotic resistance genetics Gram-negative bacteria molecular biology Microbiology QR1-502 Brian M. Luna Amber Ulhaq Jun Yan Paul Pantapalangkoor Travis B. Nielsen Bryan W. Davies Luis A. Actis Brad Spellberg Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
description	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.
format	article
author	Brian M. Luna Amber Ulhaq Jun Yan Paul Pantapalangkoor Travis B. Nielsen Bryan W. Davies Luis A. Actis Brad Spellberg
author_facet	Brian M. Luna Amber Ulhaq Jun Yan Paul Pantapalangkoor Travis B. Nielsen Bryan W. Davies Luis A. Actis Brad Spellberg
author_sort	Brian M. Luna
title	Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
title_short	Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
title_full	Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
title_fullStr	Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
title_full_unstemmed	Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1
title_sort	selectable markers for use in genetic manipulation of extensively drug-resistant (xdr) <named-content content-type="genus-species">acinetobacter baumannii</named-content> humc1
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/83a4f617d2854795966090823a1b017a
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Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) <named-content content-type="genus-species">Acinetobacter baumannii</named-content> HUMC1

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