Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a Pseudomonas shirazica...

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Horizontal plasmid transfer is a key driver of antibiotic resistance gene dissemination.
• Multidrug-resistant bacteria pose a significant threat to public health, necessitating robust surveillance.
• Accurate plasmid sequencing is crucial for epidemiological tracking and understanding resistance mechanisms.

Purpose of the Study:

• To fully assemble and characterize a carbapenemase-encoding megaplasmid from a clinical isolate.
• To identify the species and phylogenetic group of the multidrug-resistant bacterium.
• To investigate the genetic elements and evolutionary context of the novel megaplasmid.

Main Methods:

• Combined Nanopore and Illumina sequencing for complete plasmid assembly.
• Average nucleotide identity analysis for bacterial species identification.
• Bioinformatic analysis to identify genes, mobile genetic elements, and resistance cassettes within the megaplasmid.

Main Results:

• Successfully assembled a 498,516-bp megaplasmid (pJBCL41) from Pseudomonas shirazica FFUP_PS_41.
• Identified a carbapenem and aminoglycoside resistance gene cassette (|aacA7|blaVIM-2|aacA4|) within a class 1 integron.
• Characterized a complex 50,273-bp region containing transposons, insertion sequences, and a miniature inverted-repeat transposable element.

Conclusions:

• The hybrid sequencing approach enabled complete resolution of a novel, large carbapenemase-encoding megaplasmid.
• The identification of pJBCL41 provides insights into the evolution of antibiotic resistance and gene transfer mechanisms.
• This study highlights the importance of studying novel megaplasmids in understanding bacterial adaptation and resistance spread.