pEl1573 Carrying bla_IMP-4, from Sydney, Australia, Is Closely Related to Other IncL/M Plasmids

Abstract

Complete sequencing of pEl1573, a representative IncL/M plasmid carrying bla_IMP-4 from Sydney, Australia, revealed an ∼60-kb backbone almost identical to those of IncL/M plasmids pCTX-M3, from Poland, and pCTX-M360, from China, and less closely related to pNDM-HK, pOXA-48a, and pEL60, suggesting different lineages. The ∼28-kb Tn2-derived multiresistance region in pEl1573 is inserted in the same location as those in pCTX-M3 and pNDM-HK and shares some of the same components but has undergone rearrangements.

TEXT

Genes encoding metallo-β-lactamases (MBL) potentially conferring resistance to carbapenems have been identified in Enterobacteriaceae worldwide, but with different geographical distributions. bla_VIM genes are common in southern Europe, bla_NDM-1 is being detected globally but mostly with links to India or the Balkan region, and bla_IMP genes, originally associated with Japan, are now more widespread (15). bla_IMP and bla_VIM genes are both found as gene cassettes in class 1, or occasionally class 3, integrons. bla_IMP-4, first identified in China (3, 10) and initially apparently restricted to Asia and the Pacific but recently also found in the United States (12), is the most commonly reported MBL gene in Enterobacteriaceae in Australia.

bla_IMP-4 was detected in Enterobacteriaceae at similar times in both Sydney (4) and Melbourne (22, 24) as part of the same cassette array (bla_IMP-4-qacG-aacA4-catB3), but the structure and context of the class 1 integron and the plasmid vehicles were different (5). In Sydney, bla_IMP-4 was (5) and remains (26) associated with IncL/M plasmids, which have a broad host range. Here, a representative Sydney bla_IMP-4 IncL/M plasmid was completely sequenced, analyzed, and compared with other fully sequenced IncL/M plasmids available in GenBank (Table 1).

Table 1.

Characterized IncL/M plasmids

Plasmid	GenBank	Size (kb)	No. of primase gene repeatsa	Species	Location	Year	Reference
pEl1573	JX101693	87.731	6	Enterobacter cloacae	Sydney, Australia	2004	This paper
pCTX-M3	AF550415	89.468	5	Citrobacter freundii	Poland	1996	9
pCTX-M360	EU938349	68.018	5b	Klebsiella pneumoniae	China	2001	27
pNDM-HK	HQ451074	88.803	12	Escherichia coli	Hong Kong	2009	11
pOXA-48a	JN626286	61.881	7c	Klebsiella pneumoniae	Turkey	2001	23
pEL60	AY422214	60.145	9	Erwinia amylovora	Lebanon	1998/9	6
pACM1d	AF139719		7	Klebsiella oxytoca	USA	1995	25

^aNumber of repeats of the consensus sequence AAryyGyGymGGTGkymArCrCyCCyGmwGCTGmyGAAsyGGwGG in the primase gene. The final repeat in each plasmid is missing the last two bases. Variable positions are in lowercase: r = A or G; y = C or T; m = A or C; k = G or T; s = C or G; w = A or T.

^bThis difference in the primase genes of pCTX-M-360 and pEL60 was noted as a 180-bp deletion (four 45-bp repeats) in reference 27.

^cThe third repeat has an internal deletion of bases 11 to 21 in the above sequence, creating an in-frame stop codon that splits the primase protein in two, but this may be a sequencing artifact.

^dNot fully sequenced. The GenBank accession number is for the sequence of the primase region only, and seven copies of the repeat were noted in reference 25.

We had previously mapped and partially sequenced the multiresistance region (MRR) of pJIBE401 from the index isolate Klebsiella pneumoniae Kp1239 (5), but gel electrophoresis suggested that Escherichia coli UB5201Rf transconjugants of Kp1239 (4) harbored multiple plasmids. A transconjugant carrying bla_IMP-4 from Enterobacter cloacae El1573, isolated in 2004 from the same Sydney outbreak as Kp1239 (5), appeared to contain only a single plasmid equivalent to pJIBE401. S1 nuclease digestion and pulsed-field gel electrophoresis (2, 21) of this transconjugant revealed a single ∼85-kb plasmid (data not shown), which was designated pEl1573 and sequenced. DNA was extracted, amplified (GenomiPhi v2 DNA kit; GE Healthcare, NJ), quantified, and sequenced (GS-FLX; Roche 454 life sciences, Mannheim, Germany) as described previously (17) at the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Sydney, Australia). Contigs of >1 kb generated by Newbler version 2.3 (Roche) were annotated using RAST (1).

Fifteen contigs with 17- to 81.5-fold coverage (0.25 to 27.5 kb) appeared to be plasmid sequence, while those with <10-fold coverage corresponded to the host E. coli chromosome. Three additional contigs making up the insertion sequence IS5075 had 360- to 430-fold coverage, and circular copies (18) appeared to be present in addition to a linear copy in pEl1573, possibly as a result of the GenomiPhi amplification. PCR (see Table S1 in the supplemental material) guided by the map of the pJIBE401 MRR (5) and available IncL/M resistance plasmid sequences (Table 1), with additional Sanger sequencing to confirm boundaries between contigs, allowed assembly of an 87.731-kb plasmid. Annotation by RAST and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and comparison with other IncL/M plasmids revealed a 60.2-kb backbone carrying a 27.6-kb MRR (Fig. 1).

Fig 1.

(A) Tn2 and Tn2-derived MRR in IncL/M plasmids. IS are labeled with their number or name, with the pointed end indicating IR_R or oriIS for ISCR1. Tall bars represent the 38-bp IR of Tn2 (designated IR_TEM and IR_tnp) and the 38-bp IR of the transposon carrying the chrA gene (IR_chrA) (16), as labeled. Shorter pink bars represent the 25-bp IR of class 1 integrons (IRi, IR at intI1 end; IRt, at tni end). The extents and directions of antibiotic resistance (thick arrows) and other selected genes are indicated. Cassette arrays in class 1 integrons are indicated by letters in boxes: A, dfrA12-gcuF-aadA2; B, bla_IMP-4-qacG-aacA4-catB3; BΔ, bla_IMP-4Δ-intron-ΔcatB3. Filled circles on stalks represent DRs (black, TTATT; blue, GTGAC). The extent of Tn1548 in pCTX-M3 is indicated. pNDM-HK is missing 563 bp of the Tn2 tnpA gene next to IS26, as indicated, which may be the result of an IS26-mediated deletion (16). The solid line below the pEl1573 diagram indicates the region mapped in reference 5, in which the IS interrupting the 38-bp IR_chr was erroneously reported as IS4321 (97% identical to IS5075); both pEl1573 and pJIBE401 have IS5075. (B) Rearrangements in the pEl1573 MRR could give a configuration more closely related to pCTX-M3. The two copies of the 135-bp segment of the IR_TEM end of Tn2 present in inverse orientation at both ends of the MRR are indicated by red horizontal lines. Dotted lines indicate the extent of segments inverted by homologous recombination (HR) between inverse repeats. (C) Related MRR in pJIE137. (D) Related partial structure carrying bla_IMP-4 in pIMP-4. Diagrams were drawn from sequences available under the GenBank accession numbers listed in Table 1 plus EF219134 for pJIE137 and FJ384365 for pIMP-4.

In addition to the bla_IMP-4-qacG-aacA4-catB3 cassette array, sul1 (two copies), qnrB2, and the mph(A) region identified previously, the pEL1573 MRR contains the aac(3)-IId (16) and bla_TEM-1b genes and is related to the insertions in three other IncL/M plasmids. In pCTX-M360 (24), a complete Tn2, consisting of transposase (tnpA) and resolvase (tnpR) genes, a resolution (res) site, and bla_TEM-1b bounded by identical 38-bp inverted repeats (IR_tnp and IR_TEM here) (Fig. 1A), is flanked by 5-bp direct repeats (DRs) characteristic of transposition. The MRR of pCTX-M3 (9) and pNDM-HK (11) are bounded by the outer ends of Tn2 and are inserted in the same position as Tn2 in pCTXM-360 and are flanked by the same DRs but contain additional resistance modules, most of which are common to both plasmids (Fig. 1A). The pEl1573 MRR is also inserted in this same position, is flanked by the same 5-bp DRs, and has modules in common with pCTX-M3 and pNDM-HK but is bounded by two copies of the IR_TEM end of Tn2 rather than the IR_TEM and IR_tnp ends (the absence of Tn2 tnpA was confirmed by PCR with suitable positive controls; see Table S1 in the supplemental material). Some modules are also in the opposite orientation relative to the plasmid backbone, but this could be explained by homologous recombination between the two copies of the 135-bp IR_TEM end of Tn2 that are present in inverse orientation at each end of this MRR and between the two IS26 elements, which would give a configuration matching part of the pCTX-M3 MRR except for the different cassette arrays (Fig. 1B).

It is difficult to predict exactly how these different Tn2-derived MRR may have arisen, but possible sources of some pre-existing combinations of modules are known. For example, parts of the composite transposon Tn1548 (Fig. 1A) have been found on plasmids belonging to different Inc groups (8). Replacement of the 5′-conserved segment and cassette array in pCTX-M3 by the ISAba125Δ-bla_NDM-1-bla_DHA-1Δ region in pNDM-HK could be due to a double crossover in IS26 and one of the shared downstream components. Part of the pEl1573 MRR matches much of the MRR of pJIE137, an IncN-related plasmid also isolated in Sydney (19) (Fig. 1C), and homologous recombination could have linked this segment to the bla_TEM-1b/aac(3)-IId region and can also explain the different cassette arrays (16). Like pJIE137, one bla_IMP-4 positive Sydney isolate (Ca3927) lacks IS5075 (5), suggesting that this might be a later addition. The partial sequence of pIMP-4 from K. pneumoniae isolated in China (FJ384365) (13) is also related to these structures, although the Inc group was not reported. In the available sequence, a truncated bla_IMP-4 cassette and partial catB3 attC site are separated by a group II intron, suggesting derivation from bla_IMP-4-qacG-aacA4-catB3 by intron-mediated deletion (20). The remainder of the sequenced region is also related to the pCTX-M3 and pEl1573 MRR (Fig. 1D), and the isolate also carries bla_TEM-1, consistent with Tn2 being present.

The pEl1573 backbone (Fig. 2) has only 10 single-nucleotide changes from pCTX-M3 (an additional three bases in pCTX-M3 may be sequencing artifacts, as they are not found in any other IncL/M plasmid sequences). The functions of this backbone have already been analyzed in detail (9), and the replication (rep), partition/stability (parAB nuc), and postsegregational killing (pemIK) systems and host range have been examined (14). The pCTX-M-360 backbone (27) has a few additional nucleotide differences and a short deletion compared with pCTX-M3. The backbones of other sequenced IncL/M plasmids are less similar to pCTX-M3, suggesting the existence of more than one lineage. The backbone of pOXA-48a, which carries bla_OXA-48 in the composite transposon Tn1999 (23), has several short deletions compared with pCTX-M3 and some regions of much lower nucleotide identity. pEL60, from the plant pathogen Erwinia amylovora (6), does not carry any resistance genes, and is less closely related to the other plasmids. The pNDM-HK backbone appears to be a hybrid structure, as the MRR and surrounding regions are closely related to pCTX-M3, while other backbone regions are more divergent (Fig. 2). Apart from pCTX-M3 and pCTX-M360, these plasmids also all have different numbers of copies of a 45-bp repeat within the primase (traC) gene (Table 1). Primases generate short RNA primers for initiating synthesis of the complementary DNA strand during conjugation, but any phylogenetic and/or functional significance of the repeat region, which codes for 15 small hydrophobic amino acids, is unclear.

Fig 2.

Comparison of the pNDM-HK, pOXA-48a, and pEL60 backbones with the pEl1573 backbone. The pCTX-M3 backbone is almost identical to the pEl1573 backbone, and the pCTX-360 backbone is also closely related to these two, except that a short region downstream of the ssb gene (shown as a small black box) is missing. The extents and directions of selected genes in the pEl1573 sequence are shown by labeled arrows, with tra genes indicated by the appropriate letters and open reading frames of unknown function indicated by numbers. Percentages on the right indicate the range of identity. Vertical arrows indicate the insertion points of mobile elements and/or MRR that were removed, along with one copy of any DR, before alignment. The triangle above the primase gene in the pEl1573 diagram indicates the repeat region. Sequences were obtained from the accession numbers listed in Table 1. The figure was drawn using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) (7) using a calculation window of 100 bp.

pCTX-M360 carries ISEcp1-bla_CTX-M-3 in addition to the intact Tn2 and is presumably a predecessor of pCTX-M3 (27), which has ISEcp1-bla_CTX-M-3 inserted in the same position plus a Tn2-derived MRR (Fig. 2). pNDM-HK lacks ISEcp1-bla_CTX-M-3, and the Tn2-derived MRR has presumably become associated with this plasmid by homologous recombination in the backbone. pEl1573 has a pCTX-M3-like MRR and appears to be more closely related to pCTX-M3 than to pCTX-M360 but lacks ISEcp1-bla_CTX-M-3. Homologous recombination in the backbone could also explain the loss of this insertion (and seems more likely than precise deletion of ISEcp1-bla_CTX-M-3) but may not be apparent due to the generally high levels of identity.

This analysis illustrates the importance of homologous recombination in the creation of different MRR, in moving these regions between plasmids, and in creating variation in plasmid backbones. Sequencing and/or mapping of additional IncL/M plasmids may reveal additional “intermediate” structures that help to explain the evolution of the different Tn2-derived MRR and will provide information about backbone diversity.

Nucleotide sequence accession number.

The complete sequence of pEl1573 has been submitted to GenBank under accession no. JX101693. The partial sequence of pJIBE401 in GenBank accession no. AJ609296.3 has been corrected to give version AJ609296.3.