Genetic Determinants of Acinetobacter baumannii Serum-Associated Adaptive Efflux-Mediated Antibiotic Resistance

Abstract

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism’s adaptive efflux-mediated resistance phenotype.

Novel roles for pirin proteins and a 2-ketoglutarate: ferredoxin oxidoreductase ortholog inBacteroides fragiliscentral metabolism and comparison of metabolic mutants susceptibility to metronidazole and amixicile

“…13. These findings agree with reports demonstrating a role for pirin in the control of TCA cycle switching to fermentation metabolism or to aerobic respiration in aerobic and facultatively anaerobic bacteria (Soo et al, 2007; Hansen et al, 2012; Tala et al, 2018; Young et al, 2023). It remains to be defined if B. fragilis pirins undergo Fe(II) to Fe(III) redox changes during oxygen exposure or iron-limiting conditions to modulate protein-protein interactions and conformational dynamics (Ahsan et al, 2023); Barman & Hamelberg, 2016; Liu et al, 2013).…”

“…The involvement of pirin in modulating central metabolism of prokaryotes was also demonstrated in Streptomyces ambofaciens (Talà et al, 2018) and Aliivibrio salmonicida (Hansen et al, 2012). In Acinetobacter baumanii , pirin regulates expression of adaptative efflux mediated antibiotic resistance (Young et al, 2023). In Bacteroides thetaiotaomicron, recombinant pirin exerts immunomodulating actions preventing the induction of pro-inflammatory interleukins in animal model of Crohn’s disease (Delday et al, 2019).…”