Characterization of Two Multidrug-Resistant IncA/C Plasmids from the 1960s by Using the MinION Sequencer Device

Abstract

Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C 2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARI R16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes bla TEM-1D and 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 the kfrA gene. ARI IP40a carrying bla TEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARI R16a . 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. The 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-number plasmids and their rearrangement products.

pIP40a, a type 1 IncC plasmid from 1969 carries the integrative element GI sul2 and a novel class II mercury resistance transposon

“…The remaining two base pair differences result in the leucine usually found at position 113 of Int being replaced with an arginine, and the lysine at position 293 of Dcm3 being replaced with an arginine. The same study also reported the sequence of R16a (Szabo et al, 2016), recovered three years earlier in 1966, from the St-Antoine Hospital in Paris (Chabbert et al, 1972). This type 1 IncC plasmid also includes a complete copy of GIsul2 and does not contain an ARI-A island.…”

“…Subsequent to the release of our pIP40a sequence (Harmer and Hall, 2016), another pIP40a sequence was reported (Szabo et al, 2016) (GenBank accession number KX156772). Interestingly, whilst the sequences are very similar, there are some differences that are likely to have accumulated over time, during storage and passage of the plasmid in different laboratories.…”

Social behaviour involving drug resistance: the role of initial density, initial frequency and population structure in shaping the effect of antibiotic resistance as a public good

“…RP4 was isolated from Pseudomonas aeruginosa (Datta et al, 1971), R1 from Salmonella enterica serovar paratyphi (Anderson and Datta, 1965) and R16a from Providencia stuartii (Chabbert et al, 1972). The three plasmids are low copy number (Grinsted et al, 1972;Uhlin and Nordstrom, 1975;Szabo et al, 2016) and encode TEM βlactamases (Matthew and Hedges, 1976;Szabo et al, 2016), so the three enzymes are very similar. It has been reported that the level of transcription of RP4 β-lactamase is higher than that of R1 plasmid (Crowlesmith and Howe, 1980), which explains the higher levels of protection displayed by RP4 in our results and the predominance of RP4 in our stepwise analysis (Table 2).…”

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates

“…The Oxford Nanopore Technologies (ONT) MinION sequencer is a compact device that produces sequencing reads that are thousands to hundreds of thousands of nucleotides in length with real-time, read-by-read data availability (6). This rapid sequencing technology has many promising applications for the clinical microbiology laboratory and has been used successfully to sequence genomes of viruses, bacteria, and fungi (7)(8)(9)(10)(11)(12)(13)(14), to identify viruses and bacteria in primary clinical specimens (15)(16)(17)(18)(19)(20)(21), and to characterize AMR genes in bacterial isolates and primary specimens (7,9,17,(20)(21)(22)(23)(24). Though MinION single-read error rates are higher than those for Illumina short reads, higher accuracy can be achieved by generating consensus sequences from multiple reads or by complementation with high-accuracy short reads (5,6,8,9,25,26).…”