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. IMPORTANCE A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.

FIG 1

Dendrogram (a) and  optical genome map (b) of clinical isolates of A. baumannii and reference strains. Strain relatedness was calculated using the unweighted-pair group method using average linkages (UPGMA) (5). Optical maps were compared with in silico optical maps of A. baumannii reference strains AB0057, AYE, and ACICU generated from GenBank sequences. Blue areas represent regions of alignment compared to isolate MRSN 3361, and white areas represent unaligned regions. Vertical black bars indicate NcoI restriction sites. The areas corresponding to the amplified regions are enclosed in red rectangles.

FIG 2

Schematic representation of AbaR28 and amplified units (a) and the genetic environment of Tn6020 (top) and the amplified unit (bottom) in A. baumannii isolate MRSN 58 (b). (a) Resistance island AbaR28 is indicated by a thick black line at the top of the panel. Amplified units are indicated by thin black lines. The sizes of the resistance island or amplified units are in kilobases. The open arrows represent coding sequences (yellow arrows, IS26; red arrows, aminoglycoside resistance genes; blue arrows, genes associated with DNA mobility) and indicate the direction of transcription. The notched arrows represent the truncated comM and sup genes. The chromosomal genes flanking AbaR28 are indicated in black. AbaR28 is a truncated version of AbaR3 generated by a 5′-ward deletion mediated by the IS26 copy located at the left end of Tn6020 which is now the extremity of the island. This element is known to create deletions in adjacent regions (20, 37). The deletion removed the 5′ end of Tn6019 and the 5′ portion of the comM target gene. The right end of AbaR28 corresponds to the 26-bp terminal right inverted repeat (IRR) of Tn6019, known to be associated with AbaRs (arrow) (11), and flanked by a 5-bp (ACCGC) duplication of target DNA (5 bp), the characteristic footprint of AbaR3 insertions (20). (b) The vertical black arrow indicates the insertion site of Tn6020 in isolate MRSN 58. The duplicated target sequence is in blue lettering and the sequence carried over 8 bp is in red. Tn6020 is indicated by a double-head arrow, and its size is shown in kilobases. Gene nomenclature was assigned based on the closest BLAST match from GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed February 2014) using reference strain AB0057.

FIG 3

Relative gene copy numbers. Number of copies of aphA1 in isolates MRSN 57 (blue line) and MRSN 58 (red line) as determined by qPCR during growth with (solid line) and without (dashed line) tobramycin (4 μg/ml). Data are the averages from three independent experiments; error bars represent 1 standard deviation.

FIG 4

In vitro induction of gene amplification. (a and b) Number of copies of aphA1 (blue), aacC1 (red), uspA (green), and comM (purple) genes as determined by qPCR in isolate MRSN 56 (a) and strain AB0057 (b) following growth on increasing concentrations of tobramycin. Data are the averages of three independent experiments; error bars represent 1 standard deviation. No growth was observed for MRSN 56 on 16 μg/ml of tobramycin.

FIG 5

Biological cost and resistance associated with gene amplification. Tobramycin MICs (filled circles) and relative growth rates (open circles) of individual colonies of isolate MRSN 58 containing various copy numbers of the aphA1 gene. Eighteen individual colonies were grown at 37°C up to an OD₆₀₀ of 0.9 in BHI broth, and the growth rates and tobramycin MICs by microdilution were determined. The same bacterial suspension was used to purify DNA for aphA1 gene copy number determination by qPCR. Experiments were performed at least three times independently. The logarithmic tendency curves have R² values of 0.9 (solid line) and 0.8 (dashed line).