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

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• The pACM1 plasmid was isolated from a cephalosporin-resistant Klebsiella oxytoca strain in 1993.
• Plasmid sequence data is crucial for understanding antibiotic resistance mechanisms.

Purpose of the Study:

• To complete and analyze the nucleotide sequence of the pACM1 plasmid.
• To characterize the multi-drug resistance (MDR) region of pACM1.
• To measure the plasmid copy number (PCN) of pACM1 using a novel qPCR assay.

Main Methods:

• Whole-genome sequencing of the pACM1 plasmid.
• Bioinformatic analysis to identify resistance genes and genetic elements.
• Quantitative Polymerase Chain Reaction (qPCR) assay to determine PCN.

Main Results:

• The 89,977 bp sequence of pACM1 (GenBank accession KJ541681) was determined, featuring a unique 31,842 bp MDR region with multiple resistance cassettes and transposons.
• The MDR region contains mer, integrons, IS1R, IS26, and Tn2003 (blaSHV-5) within a Tn1721-like element inserted into the mucB gene of an IncL/M plasmid.
• The PCN of pACM1 was found to be approximately three copies per chromosome in E. coli DH5α and two copies in the original K. oxytoca isolate.

Conclusions:

• The pACM1 plasmid possesses a unique MDR region, distinct from other sequenced IncL/M plasmids.
• The developed qPCR assay provides a reliable method for determining PCN, which influences resistance gene dosage.
• Further studies on PCN and its impact on resistance levels are facilitated by complete genome sequences and qPCR technology.