Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978

PLOS ONE, Nov 2019

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4’,6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.

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Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978

May Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978 Felise G. Adams 0 1 2 3 Uwe H. Stroeher 0 1 2 3 Karl A. Hassan 0 1 3 Shashikanth Marri 0 1 3 Melissa H. Brown 0 1 2 3 0 Current address: School of Environmental and Life Science, University of Newcastle , Callaghan, NSW , Australia 1 Australian National Health and Medical Research Council (Project Grant 1047509) and a Flinders Medical Research Foundation Grant to MHB 2 College of Science and Engineering, Flinders University , Adelaide, SA , Australia , 2 Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney, NSW , Australia , 3 College of Medicine and Public Health, Flinders University , Adelaide, SA , Australia 3 Editor: Eric Cascales, Centre National de la Recherche Scientifique , Aix-Marseille Universite , FRANCE In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4',6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were 2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions. - UHS. FGA was supported by AJ and IM Naylon and Playford Trust Ph.D. Scholarships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Acinetobacter baumannii causes a range of disease states including hospital-acquired pneumonia, blood stream, urinary, wound and bone infections, and is responsible for epidemic outbreaks of infection worldwide [ 1 ]. Such infections are often very difficult to treat due to the multidrug resistant (MDR) character of isolates displayed by this organism [ 2, 3 ]. In addition to the impressive propensity of the organism to acquire genetic elements carrying resistance determinants [ 2, 4, 5 ], up-regulation resulting in overproduction of resistance nodulation celldivision (RND) drug efflux systems through integration of insertion sequence elements or mutations in regulatory genes, has also been deemed a major contributor to the MDR phenotype [6±9]. The best studied RND efflux systems in A. baumannii include AdeABC [10], AdeFGH [ 11 ] and AdeIJK [ 12 ]. Of particular interest is the AdeABC system which affords resistance to diverse antibiotics, biocides and dyes [10, 13±15], and has gained attention due to its high incidence of over-expression across many MDR A. baumannii clinical isolates, primarily from incorporation of point mutations in the genes encoding its positive regulator, AdeRS [ 6, 8, 13, 16 ]. Typically RND pumps consist of three proteins that form a complex; the absence of any of these components renders the entire complex non-functional [17]. Interestingly, deletion of adeC in the A. baumannii strain BM4454 did not affect resistance towards two substrates of the pump suggesting that AdeAB can utilize an alternative outer membrane protein (OMP) to efflux antimicrobial compounds [ 13 ]. The genetic arrangement of the AdeABC system places adeABC in an operon that is divergently transcribed to the regulatory adeRS two component signal transduction system (TCSTS). Expression of adeABC occurs by binding of AdeR to a ten base-pair direct repeat motif found within the intercistronic region separating these operons [ 18, 19 ]. Many clinical A. baumannii isolates harbor different genetic arrangeme (...truncated)


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Felise G. Adams, Uwe H. Stroeher, Karl A. Hassan, Shashikanth Marri, Melissa H. Brown. Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978, PLOS ONE, 2018, Volume 13, Issue 5, DOI: 10.1371/journal.pone.0197412