Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2022

Sequencing of Acinetobacter baumannii isolate MRSN56 reveals a novel route to antibiotic resistance via IS26-mediated gene amplification (82301)

Christopher J Harmer 1 , Francois Lebreton 2 , Jason Stam 2 , Patrick T McGann 2 , Ruth M Hall 1
  1. University Of Sydney, Darlington, NSW, Australia
  2. Multidrug Resistant Organism Repository and Surveillance Network, Walter Reed Army Institute of Research, Silver Spring, MD, USA

IS26 plays a major role in the mobilisation and accumulation of antibiotic resistance determinants in Gram negative bacteria. Much of the focus has been on the ability of IS26 to create complex mosaic resistance regions containing several different antibiotic resistance determinants by employing an arsenal of different movement mechanisms. However, the role that IS26 plays in amplifying individual resistance genes had not been explored.

 

MRSN56 is an extensively resistant GC1 Acinetobacter baumannii isolate recovered in 2010 from the wounds of a 20-year-old soldier injured in Afghanistan and returned to the USA for treatment. Treatment with tobramycin, one of the few remaining options, led to the emergence of tobramycin resistance, though no known tobramycin resistance genes were identified. Subsequent examination showed that substantial amplification of Tn6020, which carries the aphA1 resistance gene (kanamycin and neomycin resistance), had led to aphA1 giving an unexpected tobramycin resistance phenotype. However, only short-read data were available for MRSN56 which limited the ability to examine the mechanics of amplification.

 

The original tobramycin-sensitive MRSN56, two tobramycin resistant clinical derivatives, and a resistant experimental derivative were sequenced on an Oxford Nanopore MinION platform, and the long-read data was combined in a hybrid assembly with available Illumina Miseq data to generate complete genomes. In each resistant isolate, amplification of aphA1 involved the action of IS26. In one instance, homologous recombination between the two copies of IS26 in Tn6020 to generate a circular translocatable unit (TU) followed by IS26-mediated copy-in cointegration had generated up to 77 copies of Tn6020 in a tandem array. In the other two instances, two different IS26-mediated adjacent deletions which created a circular TU were followed by IS26-mediated targeted conservative cointegration to generate 7-65 copies of Tn6020.

 

Tandem amplification of resistance genes is an important and under-appreciated route to the emergence of novel resistance phenotypes. The role that IS26 plays is likely to be substantial and requires further examination.