In silico analysis and PCR characterization of non-Tn4401 transposable elements in Pseudomonas aeruginosa

Multiresistance in P. aeruginosa has greatly increased due to the presence of carbapenemases genes such as bla_KPC, discovered in Klebsiella pneumoniae and mobilized by horizontal transfer to other species and genera. Its dissemination has been mainly due to the Tn4401. However, some non-Tn4401 elements (NTE_KPC) associated with bla_KPC have been recently found in K. pneumoniae and other bacteria. The aim of this work was to characterize in silico the transposable elements associated with bla_KPC in P. aeruginosa according to the reports presented in GenBank, to contribute with information that allows elucidating the dissemination mechanisms of this resistance determinant. To identify these elements, a search algorithm was performed using NCBI databases, sequences were filtered, and paired alignments with ACT and EasyFig were performed to characterize the bla_KPC upstream region and to determine the carrier sequences such as Tn4401 or NTE_KPC. When Tn4401 presence was discarded, the genetic environment of bla_KPC was characterized by using specialized databases such as ISFinder, CARD, among others. Using specific regions of the NTE_KPC variants, PCR oligonucleotides were designed targeting these elements and assessed in a cohort of 61 clinical isolates, recovered in 5 Colombian institutions. As a result of the in-silico approach we found 55 bla_KPC positive isolates (after being filtered) of which 73 % were carried in an NTE_KPC. All of them were associated with the bla_KPC-2 variant. These results show NTE_KPC are the most frequently reported environments in this species in the GenBank. On the PCR assay, the results showed that, even though Tn4401 was the most frequent element in Colombia, NTE_KPC-IIf was presented in a significant number of isolates (29.5%) and circulates in different genetic lineages. Interestingly, the NTE_KPC-IIf has not been reported in any other organism. This study contributes to the knowledge of carbapenems resistance dissemination mechanisms in P. aeruginosa.