The complete nucleotide sequence of the multi-drug resistance-encoding IncL/M plasmid pACM1

• Now completely sequenced, pACM1 is compared to other IncL/M resistance plasmids.
• Its resistance insert is Tn1721-based, with internal copies of Tn1696 and Tn2003.
• The Tn1721–Tn1696 junction is also seen in Acinetobacter baumannii MDR islands.
• Similar IncL/M plasmids with Tn1721 were probably widely distributed in the 1990s.
• The plasmid copy number, two to three per chromosome, affects resistance gene dosage.

The 89,977 bp nucleotide sequence of pACM1, isolated from a 1993 outbreak strain of cephalosporin-resistant Klebsiella oxytoca, has been completed and assigned GenBank accession number KJ541681. The plasmid has a single 31,842 bp mosaic multi-drug resistance-encoding (MDR) region comprising the mer resistance module of Tn1696, two integrons with a total of seven cassettes, one complete copy each of IS1R and IS26, and the bla(SHV-5)-carrying Tn2003 (with defective IS26 termini), all within a Tn1721-like element inserted into the mucB gene of the IncL/M plasmid backbone. The Tn1721–Tn1696 combination resembles sequence found in the chromosomal MDR islands of some Acinetobacter baumannii isolates. Among the completely sequenced IncL/M resistance plasmids, the Tn1721-based MDR region is unique, but data from older studies suggest that this type of plasmid was widespread in the 1990s.Since resistance gene dosage is affected by plasmid copy number (PCN), we used a relatively simple new “efficiency-corrected” qPCR assay to measure the PCN of pACM1. There are approximately three copies per chromosome in an Escherichia coli DH5α host, and two in the original Klebsiella oxytoca isolate. We could not find similar PCN data for other medically important plasmids for comparison. The study of this plasmid property and its effect on resistance levels should be facilitated in the future by the availability of qPCR instruments and complete genome sequences.