Mechanisms of IS-Mediated Amplification of the Gene Leading to Tobramycin Resistance in an Acinetobacter baumannii Isolate.

Enhanced levels of resistance to antibiotics arising from amplification of an antibiotic resistance gene that impact therapeutic options are increasingly observed. Amplification can also disclose novel phenotypes leading to treatment failure. However, the mechanism is poorly understood. Here, the route to amplification of the kanamycin and neomycin resistance gene during tobramycin treatment of an Acinetobacter baumannii clinical isolate, leading to tobramycin resistance and treatment failure, was investigated. In the tobramycin-susceptible parent isolate, MRSN56, a single copy of is present in the pseudocompound transposon PTn, bounded by directly oriented copies of IS. For two clinical resistant isolates, new long-read sequence data were combined with available short-read data to complete the genomes. Comparison to the completed genome of MRSN56 revealed that, in both cases, IS had generated a circular translocatable unit (TU) containing PTn and additional adjacent DNA. In one case, this TU was reincorporated into the second product generated by the deletion that formed the TU via the targeted conservative route and amplified about 7 times. In the second case, the TU was incorporated at a new location via the copy-in route and amplified about 65 times. Experimental amplification by subjecting MRSN56 to tobramycin selection pressure yielded different TUs, which were incorporated at either the original location or a new location and amplified many times. The outcomes suggest that when IS is involved, amplification occurs via rolling circle replication of a newly formed TU coupled to the IS-mediated TU formation or reincorporation step. Heteroresistance, a significant issue that is known to impact antibiotic treatment outcomes, is caused by the presence of spontaneously arising cells with elevated levels of resistance to therapeutically important antibiotics in a population of susceptible cells. Gene amplification is one well-documented cause of heteroresistance, but precisely how extensive amplification occurs is not understood. Here, we establish the case for the direct involvement of IS activity in the amplification of the gene to disclose resistance to tobramycin. The gene is usually found associated with IS in Gram-negative pathogens and is commonly found in extensively resistant Acinetobacter baumannii strains. IS and related IS cause adjacent deletions, forming a nonreplicating circular molecule known as a translocatable unit (TU), and amplification via a rolling circle mechanism appears to be coupled to either IS-mediated TU formation or reincorporation. Related IS found in Gram-positive pathogens may play a similar role.