blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids

Abstract

Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

FIG. 1.

Major accessory gene elements found in IncA/C plasmids peH4H, pAM04528, pAR060302, and pSN254. Shown are the floR resistance region (found in all four plasmids), the aadA resistance region (found in peH4H, pAR060302, and pSN254), the kan resistance region (found in peH4H only), and the merA resistance region (found in all four plasmids). H, hypothetical open reading frames.

FIG. 2.

Schematic diagram of full-length, IncA/C, and bla_CMY-2 plasmids. (A) The aadA region is absent from pAM04528. (B) The traD, traL, traE, and traK genes are absent from peH4H. (C) A 22-kbp region downstream from the bla_CMY-2 region is absent from peH4H. (D) The kan region is present in peH4H between insB3 and insB4. (E) IS1294 is found only in pAR060302. (F) Only a single copy of bla_CMY-2 is present in pAR060302. The portions of the sequence that are not highlighted are identical in these plasmids, except for minor differences noted in Table 2. Bars, 10,000 bp.

FIG. 3.

Schematic diagram showing different bla_CMY-2 regions. The primary element consists of eight genes downstream of traA, as shown for pAR060302. A second copy of this segment (inverted) is inserted immediately upstream of the original segment in pSN254 and pAM04528. Note that the proposed reversal in directionality of tnpA is suspect due to the sequence symmetry in this region (see Discussion). A duplicated bla_CMY-2 region is also present in peH4H as a tandem repeat downstream of the original element. Gene names with a (t) suffix are truncated relative to the full length shown for pAR060302. H1 and H2 refer to hypothetical open reading frames.

FIG. 4.

Schematic diagram highlighting several conserved segments and variable regions relative to antimicrobial resistance for pAR060302 and other IncA/C plasmids from Yersinia ruckeri (pYR1) (33), Photobacterium damselae (pP99-018) (17), and Y. pestis (pIP1202) (33). Relative to pYR1, plasmid pP99-018 (also see pP91278 [17]) has the addition of insB1 and insB2 sequences associated with tet(C) and tet(D). The tet(C) and tet(D) genes are relocated in pIP1202 (between insB3and insB4), while strA and strB are found between insB1 and insB2. In contrast, the bla_CMY-2 plasmids (from pSN254, peH4H, pAM04528, and pAR060302) harbor no inserts between insB1 and insB2 (Fig. 2); if present, the region between insB3 and insB4 is occupied by the kan region. In addition, these plasmids include a floR region, a merA region, and, with the exception of pAM04528, an aadA region (associated with insD1), as shown for pAR060302. Locus pSN254(115) is shown here as a reference point, and this sequence is identical for all plasmids considered in this study except for a 3-prime truncation in pYR-1 and a 5-prime truncation in peH4H.

FIG. 5.

(A) Bootstrap consensus tree for a neighbor-joining algorithm showing relative genetic relatedness between eight IncA/C plasmids. The tree was based on sequences from 91 conserved genes (BLAST score threshold = 0.3) (see Table S1 in the supplemental material; see Materials and Methods for details), with pIP1202 arbitrarily chosen as the reference plasmid. (B) Subtree for bla_CMY-2 plasmids based on 25 conserved genes (BLAST score threshold = 0.3) (see Table S2 in the supplemental material; see Materials and Methods for details), with pSN254 used as the reference sequence. (C) Subtree for three additional plasmids based on 49 conserved genes (BLAST score threshold = 0.3; see Table S3 in the supplemental material; see Materials and Methods for details), with pIP1202 used as the reference sequence. Neighbor-joining consensus trees were calculated from 500 bootstrap iterations, with numbers at nodes representing percent correspondence. The scale bars represent the number of base substitutions per site (32).