RESUMEN
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.IMPORTANCEAcinetobacter 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.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Acinetobacter baumannii/aislamiento & purificación , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Bleomicina/farmacología , Clonación Molecular , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Vectores Genéticos , Humanos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Regiones Promotoras Genéticas , Alineación de Secuencia , Transformación BacterianaRESUMEN
Acinetobacter baumannii is considered a problematic Gram-negative pathogen due to its widespread resistance to antibiotics. Understanding of resistance mechanisms in A. baumannii is critical for designing new and effective therapeutic options. However, this is hampered by the lack of tools to carry out genetic manipulations in A. baumannii. Here, we describe methods to use a chromosomal mini-Tn7-based single-copy gene expression system in A. baumannii. This system can be effectively used for performing genetic complementation studies, for tagging with fluorescent proteins, or for reporter fusion assays.
