Insertion sequence IS18 mediates overexpression of blaOXA-257 in a carbapenem-resistant Acinetobacter bereziniae isolate.

Sir, Acinetobacter bereziniae, previously known as Acinetobacter genomic species 10, has been isolated primarily from clinical specimens and the hospital environment and more rarely from various other sources, including vegetables, soil and animals. Antibiotic resistance is rarely reported in this species. Over the last decade, carbapenem resistance in Acinetobacter spp., mainly Acinetobacter baumannii, has emerged as a threat in hospitals around the world. The most widespread mechanism resulting in carbapenem resistance in Acinetobacter spp. is mediated through carbapenemhydrolysing class D b-lactamases, also known as oxacillinases. The overexpression of blaOXA genes is often associated with insertion sequences (IS) located upstream and providing strong promoters. Carbapenem resistance in A. bereziniae has previously been associated with the metallo-b-lactamases IMP, SIM and VIM or overexpression of OXA-229, a variant of the intrinsic OXA-228-like, which was mediated by a mutated promoter. To date, OXA-228-like has not been associated with an IS. In the present study, we investigated a carbapenem-resistant Acinetobacter strain isolated from the bronchial secretions of a patient in Germany in 2012. Isolate KH243 was initially identified as Acinetobacter guillouiae by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. However, rpoB sequencing revealed 100% similarity to the A. bereziniae type strain CIP 70.12 (accession no. DQ207475). By Etest, carbapenem MICs were 12 and .32 mg/L for imipenem and meropenem, respectively. Multiplex PCR for OXA subclasses that are associated with carbapenem resistance in Acinetobacter spp. (OXA-51, OXA-23, OXA-40, OXA-58, OXA-143 and OXA-235) was negative. Based on published A. bereziniae sequences, primers were designed to amplify and sequence the intrinsic blaOXA and its surrounding region from isolate KH243 (Table 1). PCR revealed an unexpected large amplicon of 2.1 kb. Sequencing of the purified PCR product by primer walking identified IS18 40 bp upstream of a novel blaOXA-228 variant, which was numbered blaOXA-257 by the Lahey b-lactamase database (http://www.lahey.org/Studies/) and was submitted to GenBank. OXA-257 possessed six amino acid differences compared with OXA-228. The IS18::blaOXA-257 nucleotide sequence reported in this paper has been submitted to EMBL/GenBank under accession number KC567681. The IS18 insertion element encoded a transposase that harboured eight amino acid changes compared with the IS18 sequence available in the IS database (http://www-is.biotoul.fr/). IS18 was flanked by a 3 bp target site duplication (TTT) and 26 bp imperfect inverted repeats. In Acinetobacter spp., IS are often located upstream of blaOXA genes and, by providing strong promoters, lead to overexpression of the OXA, resulting in carbapenem resistance. For example, the intrinsic blaOXA-51-like and the acquired blaOXA-58-like in A. baumannii are often associated with ISAba1 and ISAba3, respectively. IS18 has also been associated with blaOXA-58-like. 8 Other IS elements include ISAcra1, which was recently identified and overexpressed blaOXA-23 in a carbapenemresistant Acinetobacter radioresistens isolate. Two predicted promoters were found upstream of blaOXA-257 with both 235 boxes located within the right inverted repeat of IS18. One was a hybrid promoter based on those previously described in A. bereziniae isolates Nec (blaOXA-229) and Baz (blaOXA-228). 3 The 235 and 210 boxes were identical to the 235 box in Nec and the 210 box in Baz, respectively, and were