IS26-Associated In4-Type Integrons Forming Multiresistance Loci in Enterobacterial Plasmids

Abstract

Three distinct multiresistant loci from enterobacterial plasmids each comprised an integron and an IS26-associated sequence. Sequence comparison suggested a common ancestral structure that derived from an IS26 insertion into the 5′ conserved segment of an In4-type integron and evolved through acquisition of gene cassettes and IS26-mediated recruitment of additional resistance genes of diverse origin.

Resistance to multiple antibiotics in enterobacteria is largely attributed to acquisition of multiresistance plasmids (MRPs) (20). Sequencing data from MRPs have shown that resistance genes often occur in clusters carried by class 1 integrons. The latter, though not mobile themselves, are commonly associated with various transposons, such as Tn21, Tn1696, and Tn1412 of the Tn3 family (1, 13, 17).

We have previously reported on three MRPs: the SHV-5-encoding pSEM from Salmonella enterica serovar Typhimurium that belonged to IncL/M and carried In-t3 (18), the IBC-1-encoding pAK33 from Klebsiella pneumoniae, assigned to the IncL/M incompatibility group (data not shown), carrying the In-111 integron (19), and the VIM-1-encoding p541, an IncN plasmid from Escherichia coli including In-e541 (9, 10). Notwithstanding the different variable regions (Fig. 1), in each integron the first 113 bp of the common 5′ conserved segment (5′CS) (89 bp from the stop codon of intI) had been deleted due to an IS26 insertion (IS26/Δ5′CS). Also, the sequences adjacent to this IS26 (IS26-1) included additional resistance genes (aphA in pAK33, bla_CMY-13 in p541, and bla_SHV-5 in pSEM). These similarities prompted us to further investigate these structures by sequencing and comparison of the integron flanking regions.

FIG. 1.

Graphic presentation of the multiresistant loci found in the enterobacterial plasmids pSEM, pAK33, and p541. Inverted terminal repeats (IRt) at the 3′CS boundaries of the In4-like integrons and target site duplications (TSD) are indicated. The shaded parts represent the common IS26/Δ5′CS sequences.

Sequences flanking the 3′ end of integrons.

To determine DNA sequences flanking the 3′ end of In-t3, In-111, and In-e541, the respective plasmids were partially digested with various endonucleases and the fragments were ligated into the chloramphenicol-resistant phagemid pBC-SK(+) (Stratagene, La Jolla, Calif.). E. coli DH5α was used as a host of recombinant plasmids. Recombinant clones were screened by PCR assays using various combinations of oligonucleotide primers specific for class 1 integrons (7), IS26 (11), and gene cassettes carried by In-t3, In-111, and In-e541 (9, 18, 19). Sequencing was performed at both strands of selected clones and PCR products using an ABI PRISM 377 sequencer (Applied Biosystems, Foster City, Calif.).

Intact 3′CS sequences, including orf5, orf6, and the terminal inverted repeat (IRt) typical of many class 1 integrons, were identified in In-t3 and In-111. Both integrons also carried a complete copy of IS6100 adjacent to the 3′CS (Fig. 1). The 3′CS of In-e541 included a truncated orf5 (orf5Δ1) and an IS26 in opposite orientation with respect to IS26-1. This element was juxtaposed to codon 40 of orf5, and its inverted terminal repeat on the left (ITR_L) was deleted due to insertion of an IS1 that included insA and a truncated insB gene. A 6.0-kb sequence comprising three genes encoding an EcoRII cytocine methylase (GenBank X05050), an EcoRII endonuclease (GenBank M26404), and a Uvp1 invertase-resolvase (GenBank X16119) as well as an additional IS26 copy and the remaining 3′CS of In-e541 (3′ end of orf5, orf6, the internal copy of IRt, and an IS6100) was found downstream IS1. Identical 8-bp sequences, apparently representing the target site duplication generated by IS26-mediated transposition, were identified at the boundaries of orf5Δ1 and orf5Δ2 (Fig. 1). These experiments showed that the three integrons were associated with IS6100 and, therefore, belonged to a lineage of class 1 integrons related to In4 from Tn1696 (14).

Sequences flanking the 5′ end of integrons.

To determine the sequences flanking the IS26/Δ5′CS region of the three integrons, PCR assays and cloning procedures as above were applied. In In-111 (pAK33), an aphA variant (816 bp) encoding an aminoglycoside phosphotransferase was adjacent to IS26/Δ5′CS. The 3′ end of aphA was bounded by a complete copy of IS26 in direct orientation with IS26-1 (Fig. 1). A similar structure, resembling the kanamycin-resistant transposon Tn4352 (21), was also identified at the left-hand end of the Citrobacter freundii chromosomal fragment containing bla_CMY-13, located in the p541 plasmid adjacent to the VIM-1-encoding In-e541 (Fig. 1). In pSEM, a second IS26, occurring as a direct repeat copy of IS26-1, was identified downstream of bla_SHV-5. The IS26-bounded sequence (7,996 bp) comprised eight open reading frames: truncated putative endonuclease (ΔygbM), fuculose-1-phosphate aldolase (fucA), putative tRNA synthase (ygbK), putative oxidoreductase (ygbJ), putative transcriptional regulator (ygbl), bla_SHV-5, recF, and a truncated lactose transport gene (ΔlacY) (Fig. 1). It exhibited >90% homology with a chromosomal segment of K. pneumoniae including the intrinsic bla_SHVgene (www.genome.wustl.edu/projects/bacterial/kpneumoniae/).Sequences upstream the extreme left IS26 (at least 60 bp in each locus), compared with the relevant flanks of the other IS26 elements, did not include duplications characteristic of IS26-mediated transposition.

A GenBank search revealed two more MRPs resembling pSEM, the IncFII p1658/97 plasmid from E. coli (AF550679; unpublished data) and the IncL/M pACM1 from Klebsiella oxytoca (U90945, AY081221, AY309067, AY309066) (15, 16). They both carried In4-type integrons similar to In-t3 in which the 5′CS was truncated by an IS26 at the same position as in the integrons discussed here. Additionally, these integrons were associated with SHV-5-encoding sequences flanked by IS26 that, however, were differently oriented than those in pSEM.

Formation and spread of IS26/Δ5′CS-containing multiresistant loci.

It is likely that the mobile elements of the IS6 family, including IS26, do not exhibit any marked target site specificity (8). Therefore, the hypothesis of independent IS26 insertions into the same “hot-spot” of different class 1 integrons that, additionally, all belonged to the In4 family was discarded. The key features of the loci described here were the following: (i) the occurrence of the IS26-In4 structure in distinct replicons and (ii) the presence of multiple copies of IS26 in the sequences adjoining the 5′CS that were, most probably, inserted independently as indicated from the absence of target site duplications. Based also on the properties of IS26 (2, 4, 5, 8), it can be proposed that the IS26/Δ5′CS-containing multiresistant loci derived from a common structure. The initial step would be the insertion in the 5′CS of a plasmid-borne In4-type integron of an IS26-1 derived either by intramolecular transposition or by transposition of an element located on a different replicon. IS26-1 probably facilitated recruitment of diverse IS26-flanked sequences, such as aphA and the β-lactamase-encoding chromosomal fragments observed here, by homologous recombination as suggested by the lack of target site duplications. Also, IS26-mediated cointegration of different replicons, subsequently resolved by RecA-dependent homologous recombination, can explain mobilization of IS26-In4 among distinct MRPs.

IS26 is widely spread among plasmids (2, 6, 12, 21) and implicated in the dissemination of resistance genes in several ways. Compound IS26 transposons carrying from one to nine resistance genes have been described previously (5, 12). Also, IS26 elements seem to facilitate mobilization of chromosomal sequences containing resistance genes (3, 6). The findings of this study suggest that association of IS26 with a class 1 integron of the In4 lineage was probably a critical step in the evolution of diverse multiresistance plasmids found in clinical enterobacteria.

Nucleotide sequence accession numbers.

The GenBank accession numbers of the sequences presented here are AJ245670 and AJ009829 (pSEM), AY260546 (pAK33), and AY339625 and AY340637 (p541).