Analysis of potential mechanisms of non-carbapenemase mediated carbapenem resistance in Acinetobacter baumannii

ObjectivesAcinetobacter baumannii is a Gram-negative nosocomial pathogen that plays an important role in the context of bacterial multidrug-resistance. Increasing resistance to carbapenems in particular is of high therapeutic relevance, as in this case only a few antibiotics remain for treatment. The mechanisms of carbapenem resistance in A. baumannii are mainly based upon carbapenemases and the mechanisms such as porin loss, efflux pumps and altered PBPs have been poorly studied to date. MethodsThe A. baumannii reference strain ATCC 17978 was artificially mutated by selection pressure with increasing meropenem concentrations until carbapenem resistance was achieved. Growth analyses were carried out with the mutants and MICs for relevant antibiotics were determined. In addition, the mutants were whole genome sequenced, and the sequences were compared with the wild type. As various mutagenesis attempts for targeted construction of these respective mutants were unfortunately not successful, the strain collection of the NRC was screened for isolates that showed carbapenem resistance without a detectable carbapenemase. These isolates were sequenced and analysed for abnormalities in PBPs and porins in comparison to the sequence of the reference strain ATCC 17978 and were compared to other strains that possessed a carbapenemase. ResultsIn three of the resulting ATCC 17978 mutants, a mutation of PBP2 was observed (W366L). Theses mutants were carbapenem resistant and were not affected by avibactam in contrast to the wild type ATCC 17978. Growth experiments indicated a fitness loss compared to the wild type. As W366L was not found in the clinical isolates, we looked for other abnormalities in various genes associated with carbapenem resistance. Mutations were primarily found in the PBPs, with a mutation in PBP3 (A515V) occurring particularly often. ConclusionThe results of this work support the prevailing thesis that PBP mutations in A. baumannii can lead to carbapenem resistance. Since there are hardly any studies on this hypothesis and for the most part only using outdated methods, these results are of particular relevance and further studies on this topic are recommended.