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In silico analysis and PCR characterization of non-Tn4401 transposable elements in Pseudomonas aeruginosa
* , * , , , , , ,
1  Bacterial Molecular Genetics Laboratory- El Bosque University
2  Ithree institute, Sidney, Australia
Academic Editor: Marc Maresca


Multiresistance in P. aeruginosa has greatly increased due to the presence of carbapenemases genes such as blaKPC, 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 (NTEKPC) associated with blaKPC 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 blaKPC 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 blaKPC upstream region and to determine the carrier sequences such as Tn4401 or NTEKPC. When Tn4401 presence was discarded, the genetic environment of blaKPC was characterized by using specialized databases such as ISFinder, CARD, among others. Using specific regions of the NTEKPC 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 blaKPC positive isolates (after being filtered) of which 73 % were carried in an NTEKPC. All of them were associated with the blaKPC-2 variant. These results show NTEKPC 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, NTEKPC-IIf was presented in a significant number of isolates (29.5%) and circulates in different genetic lineages. Interestingly, the NTEKPC-IIf has not been reported in any other organism. This study contributes to the knowledge of carbapenems resistance dissemination mechanisms in P. aeruginosa.

Keywords: Multiresistance; Pseudomonas aeruginosa; blaKPC; NTEKPC; Carbapenemases; Tn4401