pJIE137 Carrying bla_CTX-M-62 Is Closely Related to p271A Carrying bla_NDM-1

Abstract

Complete sequencing of pJIE137 revealed a backbone closely related to p271A, encoding a novel RepA protein but with a similar organization and up to ∼70% nucleotide identity to IncN plasmids. A region in pJIE137 resembling the IncN CUP regulon is mostly missing from p271A, presumably due to recombination. The class 1 In/Tn and ISEcp1-bla_CTX-M-62 transposition unit in pJIE137 and a putative transposon carrying bla_NDM-1 in p271A are inserted in different locations in the plasmid backbone.

TEXT

Resistance to clinically important extended-spectrum β-lactams and carbapenems in the Enterobacteriaceae is often encoded by genes carried on plasmids (3), some of which cannot be typed using available PCR-based replicon typing methods (PBRT) (5, 8). We identified bla_CTX-M-62, encoding a Pro167Ser variant (Ambler numbering) of CTX-M-3, in Klebsiella pneumoniae JIE137. Transconjugants of JIE137 with Escherichia coli DH5αRf were obtained by filter mating with selection on ceftazidime (2 μg/ml) and rifampin (80 μ/ml) at a frequency of 1.33 × 10⁻⁶/recipient and carried bla_CTX-M-62 but gave no amplicons by PBRT (19). Mapping and sequencing revealed a rearranged ISEcp1-bla_CTX-M-62 transposition unit separated from a class 1 integron by a region related to IncN plasmids (18). S1 nuclease digestion and pulsed-field gel electrophoresis (2, 12) of a selected transconjugant carrying bla_CTX-M-62 (Tx37) gave a single band of ∼60 kb (data not shown). Here we present the complete sequence of this plasmid, pJIE137.

(Part of this work was presented at the 21st European Congress of Clinical Microbiology and Infectious Disease/27th International Congress of Chemotherapy, Milan, Italy, 7 to 10 May 2011, poster 1811.)

DNA was extracted from Tx37 and amplified, quantified, and sequenced (GS-FLX; Roche 454 life sciences, Mannheim, Germany) as described previously (11). Newbler (version 2.3; Roche) assembled seven contigs (0.5 to 37 kb, 56× to 128× coverage), and RAST annotation (1) identified the largest as plasmid backbone. Comparison with the existing partial sequence identified three contigs as repeated regions and the other three as segments separated by these repeats, allowing assembly of a 58,107-kb plasmid.

BLASTn searches (http://blast.ncbi.nlm.nih.gov/) with the pJIE137 backbone revealed >90% identity to p271A, which carries bla_NDM-1, from E. coli 271 (Fig. 1) (13). Although both plasmids were isolated in Sydney, Australia, isolate JIE137 was obtained in May 2006 while isolate 271 was obtained in December 2009 (P. Taylor, personal communication) from a patient at a different hospital who had been transferred from Bangladesh (14), suggesting that similar plasmids may have acquired different antibiotic resistance genes in different geographic locations.

Fig 1.

(A) Comparison of the backbones of plasmids R46, pJIE137, and p271A. Broken shading indicates ∼70% identity between R46 (GenBank accession no. AY046276) and pJIE137 (not shown for CUP region). Gray shading shows 90 to 97% identity between pJIE137 and p271A (JF785549). Extents and orientations of various genes are shown by arrows labeled with the gene name (letter only for tra genes). CUP repeats, as defined in reference 6 and annotated under AY046276, are represented by black boxes. Gray boxes indicate 582-bp repeats in pJIE137 that encompass CUP-related regions. Vertical arrows with the following labels indicate insertions: “In+,” class 1 In/Tn plus tet(C) region and ars operon; “In,” class 1 In/Tn; “insertions,” approximate positions of different insertions in the fipA region of IncN plasmids pMAK2 (AB366441), pKP96 (EU195449), pS12 (FJ223605), pNL194 (GU585907), pKOX105 (HM126016), pKC394 and pKC396 (HM138652 and HM138653, respectively) (accession numbers are given in parentheses). (B) Expanded diagram of CUP regions. CUP and related repeats are numbered, with “5” in pJIE137 indicating a proposed ArdK binding site. Unlabeled arrows indicate genes with no equivalent in IncN plasmids (see Table S1 in the supplemental material). Recombination could explain the deletions in pLEW517 (DQ390454) and p271A (dotted lines). The insertion points of Tn2 in pLEW517 and the ISEcp1-bla_CTX-M-62 transposition unit (TU) in pJIE137 are indicated. (C) Details of resistance regions inserted in pJIE137 and p271A. IS are shown as pointed boxes labeled with their name/number (125, ISAba125). Ecp1Δ and Δ represent the right (1,078 bp) and left (583 bp) ends of ISEcp1, respectively. Small open and filled boxes represent gene cassettes and associated attC sites, respectively. Tall bars represent IRi and IRt of class 1 In/Tn and 38-bp terminal IR of Tn3-like transposons, as indicated, with their relative directions shown by arrowheads. The sequences of the 38-bp IR in p271A are shown on the right, with the nucleotides that differ from those in IR_A and IR_R of Tn5403 indicated by lowercase type. DR, or sequences of expected DR length, are shown. Duplications of the 8-bp sequence CAGGGGTC in p271A noted in reference 13 overlap IR_A and IR₂ (underlined bases) and are unlikely to be true DR indicative of insertion.

pJIE137 and p271A backbones.

The pJIE137 and p271A (13) backbones are organized similarly to the backbones of IncN plasmids, typified by plasmid R46 (Fig. 1A). Many of the proteins that they encode are also most closely related to those from IncN plasmids (see Table S1 in the supplemental material), but nucleotide identity is limited (Fig. 1) and the replicon regions and RepA proteins of pJIE137 and p271A are unrelated to those of IncN plasmids.

The p271A backbone lacks a 5.2-kb region found in pJIE137 that corresponds to the CUP (conserved upstream repeat)-controlled regulon of the IncN plasmid R46 (6). The CUP repeats contain a strong promoter and in R46 are associated with the ardA and ardB genes, encoding antirestriction proteins, several other CUP-controlled genes (ccg), and ardK and ardR, which encode regulatory proteins that bind to the CUP repeats (6). Some IncN plasmids have fewer CUP repeats and only subsets of these genes (4) (Fig. 1B), which could be explained by recombination between repeats (6). In pJIE137, two copies of a 582-bp region (14 nucleotide differences) encompassing a shorter segment related to IncN CUP repeats are present, plus two partial copies of this shorter segment (Fig. 1B). These repeats are interspersed with some genes equivalent to those in R46 and other hypothetical genes (see Table S1 in the supplemental material) and contain a promoter and other features found in CUP repeats (see Fig. S1 in the supplemental material). p271A includes only one partial repeat; the rest of the region present in pJIE137 may have been lost by recombination (Fig. 1B).

ArdA and ArdB provide protection from the restriction enzymes of the recipient during conjugation (17). pJIE137 lacks ardA, and ardB is separated from the promoter in the adjacent repeat by insertion of ISEcp1-bla_CTX-M-62, while p271A has neither ardA nor ardB, which may affect conjugation efficiency. pJIE137 and p271A also lack homologs of the IncN mucAB genes, which confer increased resistance to UV radiation and increased susceptibility to mutagenesis (16). However, both pJIE137 and p271A include a gene (ssb) with no equivalent in characterized IncN plasmids which is predicted to encode a single-stranded DNA-binding protein that may have a protective function in conjugation (6).

Insertions in pJIE137 and p271A.

pJIE137 was known to carry a class 1 integron, but the available sequence ended within IS26 (18). Here the remainder of a class 1 In/Tn structure (10) was identified, bounded by the 25-bp inverted repeats IRi (at the intI1 end) and IRt (at the tni end) and flanked by 5-bp direct repeats (DR) (Fig. 1C). These DR indicate insertion by transposition, and it is possible that the whole structure was inserted en bloc, but a simpler class 1 In/Tn structure may have been inserted first, with subsequent acquisition/exchange of internal components by homologous recombination (10). As in several IncN plasmids, the class 1 In/Tn in pJIE137 is inserted upstream of the resP resolvase gene, despite these resP genes being quite different (see Table S1 in the supplemental material) and in opposite orientations with respect to other backbone genes (Fig. 1A). This is explained by Tn402-like transposons and their derivatives, i.e., class 1 In/Tn structures, targeting resolvase genes, including those on plasmids (9). The complex ISEcp1-bla_CTX-M-62 transposition unit (18) is inserted in the repeat upstream of ardB, and one IncN plasmid, pLEW517 (DQ390454), has Tn2 inserted between CUP repeats (Fig. 1B).

Previous analysis of p271A identified a truncated ISAba125 (interrupted by ISEc33) on one side of bla_NDM-1 and ISSen4 and the Tn3-like transposon Tn5403, bounded by a 38-bp IR, on the other (13). Comparison with pJIE137 confirmed that the outermost IR of Tn5403 (IR_A in Fig. 1C) defined one end of the insert in p271A but revealed a third Tn3-like 38-bp IR (IR₁) defining the other end. A fourth 38-bp IR found 181 bp away (IR₂) truncates ISAba125, and a fifth is present near ISSen4 (IR₃). This structure could have resulted from progressive insertions of different transposons with concomitant deletions or from insertion of a hybrid transposon flanked by IR₁ and IR_A created elsewhere. A 755-bp region, including the 3′-end of the fipA gene, is missing from p271A compared with pJIE137, presumably due to transposon-mediated deletion. Insertions in the fipA region are also common in IncN plasmids (Fig. 1A), and since FipA (fertility inhibition protein) inhibits conjugation of certain other plasmids (15), such insertions may have a beneficial effect (13). This analysis shows that important antibiotic resistance genes, carried by different mobile elements and inserted in different locations, have been acquired by a plasmid backbone type that current surveys may be missing.

Nomenclature of pJIE137, p271A, and other plasmids.

Given their organizational similarity to IncN plasmids, it has been suggested that p271A and pJIE137 constitute an “IncN2” subgroup (13). While plasmid Inc groups have been subdivided, with Roman and Arabic numerals and Greek letters (or combinations of these) used to indicate various subgroups (which may or may not be compatible with one another), the meanings of the different symbols seem to lack consistency, highlighting the difficulties inherent in adapting a system that was originally based on phenotypic incompatibility to the classification of plasmids based on sequence comparisons. Classification using relaxase proteins rather than replicons has been suggested (7), and pJIE137 and p271A (with TraI 68% identical to R46 [see Table S1 in the supplemental material]) would presumably be placed in the MOB_F11 group, along with IncN, IncW, and IncP-9 plasmids. Although screening based on relaxases rather than replicons may allow detection of a greater variety of plasmids, there are still problems with how to indicate “mosaic” or “hybrid” plasmids, such as p271A and pJIE137. As increasing numbers of plasmids, including those that are currently “untypeable,” are sequenced, better ways of indicating relationships between plasmids will need to be developed.

Nucleotide sequence accession number.

GenBank accession no. EF219134 has been extended to include the complete nucleotide sequence of pJIE137.