Megaplasmids on the Rise: Combining Sequencing Approaches to Fully Resolve a Carbapenemase-Encoding Plasmid in a Proposed NovelPseudomonasSpecies

Abstract

27Horizontal transfer of plasmids plays a pivotal role in the dissemination of antibiotic 28 resistance genes and emergence of multidrug-resistant bacteria. Sequencing of plasmids 29 is thus paramount for the success of accurate epidemiological tracking strategies in the 30 hospital setting and routine surveillance. Here, we combine Nanopore and Illumina 31 sequencing to fully assemble a carbapenemase-encoding megaplasmid carried by a 32 clinical isolate belonging to a putative novel Pseudomonas species. FFUP_PS_41 has a 33 multidrug resistance phenotype and was initially identified as Pseudomonas putida, but 34 an average nucleotide identity below the cut-off for species delineation suggests a new 35 species related to the P. putida phylogenetic group. FFUP_PS_41 harbors a 498,516-bp 36 untypable megaplasmid (pJBCL41) with low similarity compared with publicly 37 available plasmids. pJBCL41 contains a full set of genes for self-transmission and genes 38 predicted to be responsible for plasmid replication, partitioning, maintenance and heavy 39 metal resistance. pJBCL41 carries a class 1 integron with the |aacA7|bla VIM-2 |aacA4| 40 cassette array (In103) located within a defective Tn402-like transposon that forms part 41 of a 50,273-bp mosaic region bound by 38-bp inverted repeats typical of the Tn3 family 42 and flanked by 5-bp direct repeats. This region is composed of different elements, 43 including additional transposon fragments, five insertion sequences and a Tn3-Derived 44Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach resulted in 45 contiguous assemblies and allowed us to fully resolve a carbapenemase-encoding 46 megaplasmid from Pseudomonas spp. The identification of novel megaplasmids will 47 shed a new light on the evolutionary effects of gene transfer and the selective forces 48 driving AR. 49