The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication

Abstract

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis. IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

“…1, red arrowheads). We demonstrated previously that these increases are associated with a DNA gyrase block in a fluoroquinolone-sensitive background, and the LEV data here mimic the position-specific fitness data observed previously with ciprofloxacin (CIP) 20 .…”

“…DNA was isolated from samples taken at the start and end of this outgrowth, and transposon insertion sites were polymerase chain reaction (PCR)-amplified and enumerated by massively parallel sequencing. Read counts mapping to the chromosome and plasmid pAB3 were used to calculate a normalized value of the fitness of each individual transposon mutant relative to the entire pool using established methods 20 .…”

“…Among the ten genes are known determinants of multidrug defense including each component of the adeIJK multidrug efflux system 26 , and the bfmR envelope regulator that we have previously shown modulates survival after antibiotic exposure 27 . Additional candidate broad susceptibility determinants controlling envelope-level processes were genes encoding the putative periplasmic protease CtpA 20,27 , lipooligosaccharide (LOS) synthesis enzymes LpsB and LpxL 28,29 , and BlhA, a protein of unknown function involved in cell division 20,30 . An uncharacterized gene present in plasmid pAB3 (ACX60_RS18565) was also detected as modulating defense against several antibiotics.…”

Spermidine and spermine are the natural substrates of theAcinetobacter baumanniiAmvA multidrug efflux pump

“…spermine ~6 mM, putrescine ~20 mM amvA expression is regulated in response to these molecules, and 4) regulation of amvA involves binding of these putative substrates to its cognate regulator, AmvR. Note that amvA expression is not induced by its other substrates including ciprofloxacin (30), deoxycholate (31), chlorhexidine (32) or benzalkonium chloride (33).…”

Essential gene analysis inAcinetobacter baumanniiby high-density transposon mutagenesis and CRISPR interference

“…To determine gene essentiality across the A. baumannii genome, we performed saturating random transposon mutagenesis in strain ATCC 17978 and identified mutations that allow colonies to form on rich medium. To this end, we used two independent transposition systems which enable random insertional mutagenesis at high efficiency at different sites in the genome: a Himar1 mariner transposon system (5), which generates insertions at TA dinucleotides (13,14), and a Tn10-altered target specificity (Tn10-ATS) system, which generates insertions effectively at random due to relaxed site specificity (13,15). We generated two separate, highly saturated mutant libraries from ~550,000 and ~300,000 mutant colonies with each system, respectively (Materials and Methods).…”