Characterization of two multidrug-resistant IncA/C plasmids from the 1960s by using Oxford Nanopore MinION sequencer device

摘要

Two IncA/C family plasmids from the 1960s have been sequenced and classified into A/C2 Type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 island not previously found in the family. In the 173.1 kb R16a the 29.9 kb ARI is located in a unique backbone position not utilized by ARIs. ARIR16a consists of Tn1, Tn6020, Tn6333 harboring the resistance genes blaTEM-1D, aphA1b and a mer module, respectively, a truncated Tn5393 copy and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6 kb ARI inserted into kfrA gene. ARIIP40a carrying blaTEM-1D and aphA1b genes is composed of Tn1 with Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186 and Tn1000 insertions scattered in the backbone, an IS150 copy in GIsul2 and a complete Tn6333 encoding a mer module at the position of ARIR16a. Loss of resistance markers in R16a, IP40a and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy plasmids and their rearrangement products.