Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by efflux

Abstract

Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.

FIG. 1.

Graphical map of pIP1206. The color code for the various gene functions is indicated below the map. The origin of transfer (oriT) is marked by a black arrow. The G+C plot is indicated on the inner circle (mean, 51%). G+C contents that were >2 standard deviations above and below the mean are indicated in dark orange and in yellow, respectively.

FIG. 2.

(A) Schematic representation of the 19-kb fragment of pIP1206 containing the RepFII replicons and the genes for the arginine deiminase pathway. The RepFII genes are shown in blue, those of the arginine pathway in brown, IS elements in red, transposase in pink, and ORFs for hypothetical proteins in black. The OriV-2 replication origin is shown in dark blue. (B) Comparison of the RepFII regions of pRSB107, pIP106, and pAPEC-01-ColBM. The genes in pIP1206 exhibiting the highest level of identity with pRSB107 are indicated in blue, and those with identify with pAPEC-01-ColBM are in green. Insertion sequences are in red. Asterisks indicate a 4-bp deletion in insB′ of IS1 sequences IS1-2 and IS1-3. Arrows represent coding sequences and indicate the direction of transcription. The Δ symbol denotes a deletion.

FIG. 3.

Sequence comparison of the oriT regions of plasmids R100 and pIP1206. The TraI recognition site (sbi) and the TraY (sbyA), TraM (sbmA, -B, -C, and -D), and integration host factor (ihfA and -B) binding sites are indicated and are as defined previously (1). The vertical arrow indicates the oriT origin of DNA transfer.

FIG. 4.

Schematic representation of antibiotic resistance regions of pIP1206 to tetracycline and chloramphenicol (A), to sulfonamides, quaternary ammonium, streptomycin/spectinomycin, and trimethoprim (B), and to β-lactams, aminoglycosides, and fluoroquinolones (C). (D) Nucleotide sequences flanking the mobile elements. DR1 and DR2 flank at one end IS26-1 and IS26-5. Genes for resistance are indicated in yellow, those for transposases/resolvases are in pink, integrases are in green, IS are in red, IRs and DRs are in blue, and ORFs for hypothetical proteins are in black. Arrows represent coding sequences and indicate the direction of transcription. The percent G+C content is indicated under the arrows. Truncated genes are indicated by a Δ symbol.

FIG. 5.

Schematic representation of the 7-kb fragment containing the Hsd restriction-modification system (A) and HsdS of pIP1206 (B). The percent G+C content is indicated within the arrows. The highest amino acid identity is indicated in bold. A dash indicates absence of the gene.