Comparative Genomics of IncL/M-Type Plasmids: Evolution by Acquisition of Resistance Genes and Insertion Sequences

LETTER

IncL/M-type plasmids R446b and R471a were originally isolated from Morganella morganii (formerly Proteus morganii) as the first members of the IncL/M group of multidrug resistance (MDR) plasmids (1, 2). IncL/M plasmids are now commonly identified among environmental and clinical isolates (3–5). This group of plasmids can be considered an emerging threat since it has been increasingly identified as a source of broad-spectrum β-lactam resistance, encoding either the metallo-β-lactamase NDM-1 or the class D carbapenemase OXA-48 and also being responsible for the dissemination of extended-spectrum β-lactamase genes such as bla_CTX-M-3 (5–8). In this study, we analyzed the sequence of plasmid pNDM-OM, harboring the bla_NDM-1 gene, that was recovered from a clinical Klebsiella pneumoniae isolate from the Sultanate of Oman, providing new insights into the evolution of MDR IncL/M-type plasmids.

The complete sequencing workflow of the Illumina Genome Analyzer IIx system (Illumina Inc., San Diego, CA) was performed by the DNAVision company (Gosselles, Belgium). Plasmid pNDM-OM was 87,185 bp in size with an average GC content of 52% and contained 98 open reading frames. It possessed a complete array of genes involved in replication, conjugation, and partition (see Table S1 in the supplemental material). The plasmid architecture observed in pNDM-OM was similar to that of other IncL/M plasmids previously sequenced (Fig. 1). It exhibited a very high gene synteny with plasmid pNDM-HK, which also encodes NDM-1 with only very few differences, consisting of one to four nucleotide changes/deletions at three different locations together with the lack of two insertion sequences identified in pNDM-HK, namely, IS26 and IS186 (Fig. 1). In addition, part of the tnpA gene of Tn2 (563 bp) in pCTX-M-3 and pNDM-OM was missing in pNDM-HK.

Fig 1.

Major structural features of plasmid pNDM-OM compared to those of IncL/M-type plasmids pCTX-M3 (GenBank accession number AF550415), pEL60 (GenBank accession number AY422214), pCTX-M360 (GenBank accession number EU938349), pOXA-48a (GenBank accession number JN626286), and pNDM-HK (GenBank accession number HQ451074). White boxes indicate plasmid scaffold regions that are common among the plasmids. The tra locus is indicated within the box. Resistance genes are indicated by orange boxes, except the β-lactamase genes, which are indicated by blue boxes. Transposon-related genes (tnpA, tnpR, tnpM) and insertion sequences are indicated by red boxes. Replicase genes are indicated by purple boxes.

Overall, in silico analysis of the GenBank databases revealed that IncL/M plasmids have evolved through the acquisition of resistance genes and insertion sequences. The 60,145-bp plasmid pEL60, isolated from Erwinia amylovora, can be considered the typical IncL/M backbone since it does not possess any resistance gene or insertion sequence (see Table S1 in the supplemental material) (Fig. 1 and 2) (4). Plasmid pOXA-48a differed from pEL60 by the following two main additional elements: (i) the integration of composite transposon Tn1999 carrying the bla_OXA-48 carbapenemase gene in the tir gene that encodes a transfer inhibitory protein and (ii) a recombination event resulting in the exchange of three genes within the tra operon (namely, traX, traY, and excA) (8). Plasmid pCTX-M3 differs from pEL60 (9) by the acquisition of the ISEcp1-bla_CTX-M-3-carrying transposon and by the acquisition of a large resistance region constituted by transposon Tn2 in which a Tn1548-like transposon, carrying the 16S rRNA methylase armA gene, was inserted. The structure of pCTX-M360 can be considered an intermediate between plasmids pEL60 and pCTX-M3 since it possesses the typical IncL/M backbone with the insertion of ISEcp1-bla_CTX-M-3 and the insertion of a native Tn2 (Fig. 2) (9). On the other hand, plasmids pNDM-HK and pNDM-OM possess a synteny similar to that of pCTX-M3, including the insertion of Tn2 and the insertion of a Tn1548-like transposon within the Tn2 resolvase gene, but a likely recombination event resulted in the insertion of the NDM module within the Tn1548-borne class 1 integron, leading to the replacement of the integrase gene and the gene cassette array by that module.

Fig 2.

Schematic representation of insertions in the IncL/M backbone. A proposed backbone, namely, pEL60, is represented as in Fig. 1. pOXA48a-specific insertion and recombination are shown below the backbone, while specific insertion and recombination of pCTX-M360, pCTX-M3, pNDM-HK, and pNDM-OM are shown above the backbone. The type of modification, either transposition or recombination, is indicated in the figure.

The integration hot spot we identified among the IncL/M-type plasmids was located between the replication locus and the trbC gene that encodes part of the Trb transfer operon (Fig. 2). First, transposon Tn2 carrying the bla_TEM-1 β-lactamase gene was inserted within this region, and then IS26-mediated insertions may have occurred, leading to a deletion of the resolvase gene and the 5′ end of the Tn2 transposase gene. These events gave rise to a complex structure carrying many resistance determinants, including those compromising the activity of carbapenems, broad-spectrum cephalosporins, aminoglycosides, quinolones, trimethoprim, and sulfonamides (6–8, 10–12). The acquisition of the bla_NDM-1 gene in pNDM-OM resulted from a recombination event leading to an exchange of a class 1 integron within the Tn1548 transposon by the NDM module. This acquisition was probably recent, since the resistance loci of pNDM-OM, pNDM-HK, and pCTX-M-3 derive from a common ancestor (Fig. 1). A second integration hot spot has been identified near the pemI/pemK locus, in which the ISEcp1-bla_CTX-M-3 transposon was inserted. This transposon, conferring resistance to broad-spectrum cephalosporins, was widely distributed among clinical isolates (5). Surprisingly, pNDM-OM and pNDM-HK did not harbor the ISEcp1-bla_CTX-M transposon despite the fact that they are very likely derivatives of pCTX-M3 based on the synteny and phylogenetic analysis of the transfer operon.

This further strengthens the evolutive potential of IncL/M-type plasmids that constitute currently efficient vectors for emerging and clinically relevant resistance genes.

Nucleotide sequence accession number.

The nucleotide and protein sequences corresponding to plasmid pNDM-OM have been deposited in GenBank under accession number JX988621.