Amplification of Aminoglycoside Resistance Gene aphA1 in Acinetobacter baumannii Results in Tobramycin Therapy Failure

Abstract

Gene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 μg/ml in successive Acinetobacter baumannii isolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1 aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ≤8 μg/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 μg/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ≤8 μg/ml and no fitness cost and were stably maintained in vitro. Exposure in vitro to tobramycin of the initial susceptible isolate and of the A. baumannii AB0057 reference strain led to similar aphA1 amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivo development of antibiotic resistance secondary to gene amplifications resulting in therapy failure.

Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges

“…Furthermore, transposon transfer of resistance can lead to accumulations of large copy numbers of resistance genes or transposons. For example, a patient treated with tobramycin experienced a remarkable rise in resistance concurrent with the isolated strain (172). The MIC increased from 0.5 to 16 g/ml in only 4 days of therapy and was due to amplification of copy numbers of the Tn6020 transposon containing the aphA1 gene mediating resistance to tobramycin.…”

“…Transposon-mediated passage of resistance mechanisms are well described, including those for AmpC cephalosporinases, OXA carbapenemases, KPC serine carbapenemases, and NDM or VIM metallo-carbapenemases, and aminoglycosides (101,135,136,147,148,150,155,158,159,(172)(173)(174)(175)(176)(177)(178). Indeed, transposon-mediated resistance to classically described antibiotic classes, including ␤-lactams, tetracyclines, aminoglycosides, and sulfonamides, had occurred in a global lineage of A. baumannii by the late 1970s; new resistance was subsequently acquired in transposon lineages in the 1980s as new antibiotics became available (136).…”

War Wound Treatment Complications Due to Transfer of an IncN Plasmid Harboring bla OXA-181 from Morganella morganii to CTX-M-27-Producing Sequence Type 131 Escherichia coli

“…Whole-genome sequencing was performed using an Illumina MiSeq desktop sequencer as previously described (27). Newbler version 2.7 (454 Life Sciences, Branford, CT) was used to assemble MiSeq sequencing reads into de novo contigs and scaffolds.…”

Selection and evolution of resistance to antimicrobial drugs

“…In addition, effective antibiotics are essential for treatments requiring the insertion of catheters, a procedure associated with a high risk of bacterial infection. Resistance to antibiotics among human pathogens increases the risks of treatment failure due to the application of an ineffective antibiotic, it also increases the risk of mortality by increasing the time from an initial diagnosis to an effective therapy, and it increases morbidity by increasing the use of more toxic antibiotics as replacements for those rendered ineffective by resistance and imposes an extra healthcare cost and productivity loss that in the EU was estimated as costing at least €1.5 billion in 2007 (Technical Report: The bacterial challenge: time to react (2009) is available on the website of the European Medicines Agency, http://www.ema.europa.eu/docs/en_GB/document_library/Report/2009/11/WC500008770.pdf). The increasing realization that antibiotic resistance imposes a financial burden on society, in addition to exacerbating personal issues of morbidity and mortality, is a strong motivation for governments and other stakeholders to adopt measures to address the problems caused by antibiotic resistance.…”