Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance

Abstract

Introduction: blaOXA-48, blaNDM-1 and blaCTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described blaOXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the blaSHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania).

Methods: Sequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank.

Results: Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the blaOXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible.

Conclusion: This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.

Fig 1. EcoRI, EcoRV and BamHI restriction profiles of purified IncL/M plasmid DNAs, analyzed on 0.8% agarose gel.

DNA on the gel: DNA ladder XV (Roche Diagnostic, GmbH, Mannheim, Germany); E. coli pKPN-El-Nr.7 plasmid; E. coli R69 (IncM reference plasmid); plasmid purified from the R69 x pKPN-El-Nr.7 transconjugant.

Fig 2. Major structural features of IncM and IncL plasmids.

p202c, R69, R471 and pKPN-El-Nr.7 plasmids sequenced in this study are compared with pNDM-OM from K. pneumoniae 601 [7] and pEL60 [26]. In the in scale schematic representation, the open reading frames identified in the sequence are represented with arrows of different colors: the tra and trb transfer loci are in green; the traX, traY, excA entry exclusion genes of the IncM type are in purple, whereas those of the IncL type are in pale blue; resistance genes are in red; transposon-related genes [tnpA, tnpR, tnpM], insertion sequences, integrase and resolvase genes are in yellow; the replicase gene is in blue; partitioning genes, toxin-antitoxin and other stabilization systems are in brown; additional genes of unknown function are in white. The line above each plasmid represents the core genome used in comparative analysis.

Fig 3. Homology trees of the IncL and IncM core genomes.

Core genomes contain the entire scaffold, excluding antimicrobial resistance determinants and genes that were not present in all the plasmids under study. The percentage of nucleotide identity among the compared plasmid core genomes is shown on each branch of the trees. Panels highlight the IncM and IncL branch separation on the tree. The plasmids positive for the bla _OXA-48 gene are highlighted by a pale blue panel.

Fig 4. Homology trees of the ExcA, TraX, TraY protein sequences.

The deduced protein sequences of the proteins from each respective plasmid were downloaded from the GenBank or deduced from the DNA sequences of plasmids performed in this study. The percentage of amino acid identity among the compared protein sequences is shown on each branch of the trees. Plasmids positive for the bla _OXA-48 gene are highlighted by pale blue panels.

Fig 5. Phylogenetic tree and nucleotide sequence alignment of the major stem-and-loop incRNA of IncL and IncM plasmid.

The incRNA sequences were downloaded from the GenBank or identified in plasmids sequenced in this study. The unrooted phylogenetic tree inferred the evolutionary relationships among the various incRNAs based upon similarities and differences in their nucleotide sequences. The part of the phylogeny tree corresponding to plasmids positive for the bla _OXA-48 gene is highlighted by pale blue panels. In the panel showing the nucleotide alignment, the residues showing 100% identity have been shaded. The white parts of the alignment show the mismatches identified among the IncL and IncM incRNA sequences.