In34, a complex In5 family class 1 integron containing orf513 and dfrA10

Abstract

A complex class 1 integron, In34, found in a conjugative plasmid from a multidrug-resistant Klebsiella pneumoniae strain isolated in 1997 at a hospital in Sydney, Australia, was shown to have a backbone related to that of In2, which belongs to the In5 family. In In34, the aadB gene cassette replaces the aadA1a cassette in In2, and two additional resistance genes, dfrA10 and aphA1, that are not part of a gene cassette are present. The aphA1 gene is in a Tn4352-like transposon that is located in the tniA gene. The dfrA10 gene lies adjacent to a 2,154-bp DNA segment, known as the common region, that contains an open reading frame predicting a product of 513 amino acids (Orf513). Orf513 is 66 and 55% identical to the products of two further open reading frames that, like the common region, are found adjacent to antibiotic resistance genes. A 27-bp conserved sequence was found at one end of each type of common region. The loss of dfrA10 due to homologous recombination between flanking direct repeats and incorporation of the excised circle by homologous recombination were demonstrated. Part of In34 is identical to the sequenced portion of In7, which is from a multidrug-resistant Escherichia coli strain that had been isolated 19 years earlier in the same hospital. In34 and In7 are in plasmids that contain the same six resistance genes conferring resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, neomycin, tobramycin, trimethoprim, and sulfonamides, but the plasmid backbones appear to be unrelated, suggesting that translocation of a multiple-drug-resistance-determining region as well as horizontal transfer may have occurred.

FIG. 1.

Structures of class 1 integrons. (A) In4 and In5, representing the main backbone types; (B) class 1 integrons containing the common region. IRi and IRt are shown as vertical bars labeled i and t, respectively, and the attI1 site is shown as a narrow open box. The CR is represented as a shaded box, and insertion sequences are represented as numbered open boxes, with Δ indicating a partial copy of IS6100. The extents of the 5′-CS (medium-thickness lines), the 3′-CS (thin lines), and the tni module (thick line) present in published sequences are indicated, with the numbers referring to positions in standard versions of these sequences. Unique regions are shown as hatched boxes, and genes are indicated by horizontal arrows. The shaded boxes above the end of the complete CR in In6 represent duplications of the final 393 and 28 bp of the CR. The cassettes present at the positions marked by vertical arrows are as follows: In5, aacA(IIa); In4, aacC1-orfE-aadA2-cmlA1; In6, aacA4-aadA2; In7, aadB. The sequences used are from GenBank accession nos. M29695 and U38230 and standard sequences for In5; U12338 for In4; sequences compiled from U38230L06822, U04277, U04278, and X68227, together with standard and unpublished sequences, for In6; and L06418 for In7.

FIG. 2.

Right-hand boundary of the CR. (A) Sequences downstream of orf513 in In6, In7, and pSAL-1 are aligned to show the boundary between CR and unique sequences or CR duplications. (B) Alignment of sequences downstream of orfA and orf2 showing the equivalent boundaries. Uppercase letters represent CR sequences (CR1, orf513; CR2, orfA; CR3, orf2), and lowercase letters represent unique sequences, with vertical arrows indicating the positions of the boundaries. Colons indicate identical bases, and bases in boldface type are conserved in all three CRs. The sequences used in panel A are listed in Fig. 1 plus pSAL-1 integron (GenBank accession no. AJ237702), and the sequences in panel B were obtained from the GenBank accession numbers indicated on the right-hand side. (C) Comparison of the boundaries of the three CRs. The sequences at the right-hand boundaries of CR1, CR2, and CR3 are aligned, with identical nucleotides boxed.

FIG. 3.

Structure of In34 and relationship to In2. (A) Structures of In34 and In2. The features are as described in the legend to Fig. 1, with gene cassettes represented as open boxes and an adjacent filled box (the 59-be). The aphA1 region is shown as a narrow open box. tnp₂₁, tnp₁₆₉₆, and mer₂₁ represent the tnp regions of Tn21 and Tn1696 and the mer region of Tn21, respectively. (B) Movement of CR1-dfrA10 by homologous recombination. The line diagrams show the inserts in the named plasmids, and the gel shows NcoI digests of these plasmids. The 4.0-kb NcoI fragment that forms the circular molecule and that is acquired and lost by homologous recombination is indicated in the line diagram (bar) and on the gel (horizontal arrow). The sizes of markers are given in kilobases, and restriction enzyme site abbreviations are as follows: B, BamHI; N, NcoI; P, PstI; and X, XhoI.

FIG. 4.

Antibiotic resistance genes adjacent to CRs. The ends of the CRs associated with orf513 (CR1), orfA (CR2), and orf2 (CR3) are shaded boxes; and the unique regions are open boxes, with antibiotic resistance genes indicated by horizontal arrows. For unique regions adjacent to CR1, a closed end indicates the end of the unique region; and the adjacent number is the position of the first base of 3′-CS2 in the standard 3′-CS, or the −45 indicates 45 bp of a 59-be preceding bp 1 of the standard 3′-CS. In one case IRi marks this boundary. The sequences used here are from the GenBank accession numbers listed in Fig. 1 plus the following: ampC/R, GenBank accession no. AJ237702; bla_CMY-9, AB061794; bla_CTX-M-2, AJ311891; bla_CTX-M-9, AF174129; dfrA19, AJ310778; qnr, AY070235; sul2, D37825; dfrA9, X57730; flo, AF231986; tetR/A (31), AJ250203; dfrA18, AY034138; ereB, X03988; tetR/A(G), AF261825.