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blaCTX-M-62, a C508T variant of blaCTX-M-3b, was transferred from Klebsiella pneumoniae JIE137 on a conjugative plasmid together with a class 1 integron containing the dfrA12-gcuF-aadA2 cassette array, ISCR1, and qnrB2. blaCTX-M-62 lies between intact and rearranged copies of ISEcp1 in a configuration that can be explained by a combination of transposition and homologous recombination and which also illustrates the ability of ISEcp1 to mobilize an adjacent gene as part of transposition units of different sizes.
The insertion sequence ISEcp1 appears to have been responsible for the capture and mobilization of a number of unrelated antibiotic resistance genes, including several of the blaCTX-M groups encoding a family of extended-spectrum β-lactamases that now appear dominant worldwide (18). ISEcp1 is bounded by 14-bp inverted repeats, IRL at the left-hand end with respect to the direction of transcription of the transposase gene and IRR at the right-hand end. However, ISEcp1 appears to use IRL in conjunction with alternative sequences resembling these IR to mobilize adjacent regions, creating 5-bp duplications of flanking sequence on transposition (9, 14, 15, 19). Like ISPme1, which appears to operate in a similar way (2), ISEcp1 can mobilize the same adjacent gene as part of different-size “transposition units,” as illustrated by transposition of “hybrid” units that include adjacent vector sequence from cloned ISEcp1-resistance gene combinations (15, 19).
blaCTX-M genes appear to have been captured from the chromosomes of Kluyvera spp. For example, a chromosomal region of Kluyvera ascorbata (GenBank accession no. AJ632119) is identical to the ISEcp1-associated, plasmid-borne blaCTX-M-3a gene and its flanking regions (17). In this case a transposition unit that includes a 127-bp “spacer” preceding the blaCTX-M-3a gene and 345 bp of the adjacent orf477 appears to have been mobilized by ISEcp1 using an alternative IR (IR1 in Fig. Fig.11 and and2).2). While the blaCTX-M-3a gene appears widespread, CTX-M-3 may also be encoded by the blaCTX-M-3b variant, which has 8 nucleotide differences and to date has been identified only in Japan (1) (GenBank accession no. AB059404, AB168117, AB185834 to AB185841, AB231615, and AB432919) and Taiwan (pK29; accession no. EF382672) (5). Comparison of the only available sequence that includes regions flanking blaCTX-M-3b (pK29) with equivalent regions flanking blaCTX-M-3a revealed additional differences (Fig. (Fig.1),1), including a shorter 45-bp spacer, suggesting that blaCTX-M-3b may have been mobilized from a different strain of Kluyvera. In pK29, the 345 bp of orf477Δ beyond blaCTX-M-3b are followed by a unique 234-bp region that includes a partial open reading frame, designated yhgE. This suggests initial capture of blaCTX-M-3b was followed by mobilization of a longer ISEcp1 transposition unit utilizing a different alternative IR. Beyond the yhgE region, the first 583 bp of ISEcp1 are found in the orientation opposite to that of the intact ISEcp1 that lies upstream of blaCTX-M-3b (Δ1 in Fig. Fig.2A).2A). The ISEcp1-blaCTX-M-3b-Δ1 region is flanked by 5-bp direct repeats (DR) (TACAA) and is apparently inserted in the plasmid backbone of pK29 as an unusual transposition unit bounded by inverted copies of IRL.
A highly ceftazidime-resistant Klebsiella pneumoniae isolate from Sydney, Australia (JIE137), carries a C508T variant (stated incorrectly as G509T in reference 23) of blaCTX-M-3b, encoding a Pro167Ser CTX-M-3 variant (Ambler numbering) designated CTX-M-62 (http://www.lahey.org/studies/). blaCTX-M-62 was transferred to Escherichia coli DH5αRf on a large conjugative plasmid (pJIE137), but PCR-based replicon typing (4) failed to identify any of the Inc groups tested for (23). Here we analyze the genetic environment of blaCTX-M-62 in detail.
ISEcp1 is adjacent to blaCTX-M-62 in pJIE137 (23), and further PCR and sequencing indicated that, like blaCTX-M-3b in pK29, blaCTX-M-62 is flanked by intact and truncated copies of ISEcp1, with the sequences differing only in the C508T substitution. Direct sequencing and PCR mapping of pJIE137 revealed that ISEcp1Δ1 (nucleotides [nt] 1 to 583) downstream of blaCTX-M-62 is followed by the end of ISEcp1 (Δ2; nt 579 to 1656) in the opposite orientation (Fig. (Fig.2A).2A). The 898-bp abutting IRR of ISEcp1Δ2 matches a region found adjacent to an intact ISEcp1 in the IncA/C plasmid pRA1 (FJ705807; Fig. Fig.2A)2A) (7). The identity ends at the boundary of a 2.554-kb ISEcp1 transposition unit in pRA1 that ends with 14 nt resembling the IR of ISEcp1 (IR3 in Fig. Fig.2B)2B) and is flanked by DR (ATTAT). This region includes an open reading frame (ORF) predicted to encode a protein belonging to the β-lactamase superfamily, here designated orfRA1-14.
The association of extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated quinolone resistance determinants is not unusual (e.g., see reference 10), and screening for qnrB genes (qnrB-F/qnrB-R; Table Table1)1) revealed qnrB2 in pJIE137, although both JIE137 and its transconjugant were sensitive to ciprofloxacin (MIC ≤ 0.5 μg/ml). As expected, qnrB2 was located between ISCR1 and a partial copy of the 3′ conserved segment (3′-CS) of class 1 integrons, and the common (13) dfrA12-gcuF-aadA2 cassette array was identified upstream of ISCR1. The chrA-mph(A)-IS26 configuration found in several multiresistance regions (see reference 6) was identified beyond qnrB2. Long PCR linked ISEcp1-blaCTX-M-62-Δ1/Δ2-orfRA1-14 to intI1, and additional PCR mapping enabled the whole resistance region to be sequenced (Fig. (Fig.2A2A).
The region between orfRA1-14 and IRi of the integron contains genes predicted to encode a zinc metalloproteinase (80% identical/89% similar to Mpr of the IncN plasmid R46) and an ArdK-like protein (48% identical/65% similar to ArdK of R46) separated by a gene potentially encoding a single-strand DNA-binding protein (ssb). A gene predicted to encode a protein ~70% identical to plasmid RepA from several different bacterial species follows ardK. This region also appears to have organization similar to that of part of the IncN resistance plasmids (e.g., R46), suggesting that it corresponds to part of a plasmid backbone and that blaCTX-M-62 and qnrB2 are found in separate resistance regions in pJIE137. The sequence adjacent to IRL of intact ISEcp1 upstream of blaCTX-M-62 in pJIE137 includes a gene apparently encoding an ArdB-like protein (49% identical/66% similar to ArdB of R46 ), suggesting it is also part of the plasmid backbone. The 5 bp (ACATA) adjacent to IRL of this ISEcp1 are identical to those immediately after the end of the orfRA1-14 region (as identified by comparison with pRA1), suggesting that a 5.759-kb ISEcp1-blaCTX-M-62-Δ1/Δ2-orfRA1-14 transposition unit was inserted in this plasmid backbone (Fig. (Fig.2A2A).
The scheme in Fig. Fig.2B2B outlines one way in which this complex transposition unit could have arisen. First, ISEcp1 inserts into the Kluyvera chromosome 45 bp from the start of blaCTX-M-3b and uses IRL and IR1 to transfer this gene plus orf477Δ to a new location. Next, a slightly longer region flanked by IRL and IR2 and including the yhgE segment is mobilized by ISEcp1 as part of a 3.2-kb transposition unit. This unit is inserted into a second copy of ISEcp1 upstream of orfRA1-14 (as seen in pRA1), with the ISEcp1 elements in opposite orientations, generating 5-bp DR (TGATA). Homologous recombination between the duplicated, inversely oriented left ends of ISEcp1 would invert the region between them. Transposition of the 5.759-kb region between IRL and IR3, including orfRA1-14, to a new location would give the configuration in pJIE137, and a single point mutation of blaCTX-M-3b to give blaCTX-M-62 is also required. Transposition of the 3.783-kb region bounded by the two inverted copies of IRL to a new location, with duplication of the target sequence (TACAA), yields the related configuration found in pK29.
The configuration of the blaCTX-M-62 region can therefore be explained by several ISEcp1-mediated transposition events utilizing different alternative IR combined with homologous recombination. The relationship of the qnrB2 region to other characterized resistance regions could also be explained by homologous recombination. For example, in the IncN plasmid pLEW517 (GenBank accession no. DQ390454) (21), dfrA12-gcuF-aadA2 is linked to the chrA-mph(A)-IS26 structure, but the ISCR1-qnrB2 region is not present, while pJIBE401 (accession no. AJ609296), an IncL/M plasmid from Sydney (6), has both ISCR1-qnrB2 and chrA-mph(A)-IS26 but the blaIMP-4-qacG-aacA4-catB3 cassette array (Fig. (Fig.2A).2A). Homologous recombination in the duplicated 3′-CS flanking the ISCR1-qnrB2 region can explain its insertion/deletion (12), while double crossover in both the 5′-CS and 3′-CS allows switching of entire cassette arrays between different integrons (11). Thus, the resistance regions of pJIE137 illustrate how combinations of transposition and homologous recombination may be involved in the movement of resistance genes and the evolution of multiresistance regions.
The nucleotide sequence of the blaCTX-M-62 gene available under GenBank accession no. EF219134 has been extended to include sequence obtained here.
This work was supported by grants from NSW Health (to CIDM-Public Health) and the National Health and Medical Research Council of Australia (NHMRC), including partial support of S.R.P. by NHMRC CCRE grant no. 264625. Z.Z. was supported by an Endeavor International Postgraduate Research Scholarship from the Australian Government Department of Education, Science and Training.
Published ahead of print on 26 April 2010.