Distribution of Class 1 Integrons with IS26-Mediated Deletions in Their 3′-Conserved Segments in Escherichia coli of Human and Animal Origin

Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes

Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an hemolytic Escherichia coli O108:H-human clinical isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and y origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes

Features and characterization of typical class 1 integrons.

<p>(<b>A</b>) Structure of a typical class 1 integron showing the 5′- and 3′-CS. The location and the direction of transcription of genes are indicated. The class 1 integrase gene <i>intI1</i> (black arrow) and <i>attI1</i> integron-integration site (vertical arrow) are located in the integron 5′-CS. The <i>qacEΔ1</i> gene and the <i>sul1</i> gene (grey arrows), and the open reading frame <i>orf5</i> (not shown) are located in the 3′-CS. Inserted gene cassettes are represented by unfilled arrows and their associated 59-be are indicated by vertical arrows. L1, R1 and L2, L3 primer annealing sites are indicated by small horizontal arrows. (<b>B</b>) A diagram showing the genetic structure of the gene cassette arrays amplified using standard PCR primers that target the 5′- and 3′-CS of typical class 1 integrons (cassette arrays detected: <i>dfrA5</i>, <i>dfrA7</i>, <i>aadA1</i>, <i>aadA2</i>, <i>dfrA1/aadA1</i>, <i>dfrA17/aadA5</i> and <i>dfrA12/orfF/aadA2</i>). The following features are indicated: <i>attI1</i> recombination sites (black filled boxes), gene cassette arrays (unfilled arrows) and 59-bes (grey filled boxes). All diagrams are drawn to scale.</p

<i>E. coli</i> strains examined for class 1 integron carriage.

<p>Abbreviations: UTI, Urinary tract infection; SIDS, sudden infant death syndrome; HUS, hemolytic uremic syndrome.</p

Features of <i>E. coli</i> strains harbouring integron-IS<i>26</i> elements.

A<p>The genetic structure of the integron-<i>dfrA5</i>-IS<i>26</i> element and flanking regions of <i>E. coli</i> strain D22 is illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012754#pone-0012754-g002" target="_blank">Figure 2</a>.</p>B<p>Moderate level of resistance. Resistance to antibiotics is indicated by (+) and susceptibility is indicated by (−). Abbreviations of antibiotics: A, ampicillin (32 µg/ml); S, streptomycin (25 µg/ml); T, tetracycline (20 µg/ml); C, chloramphenicol (10 µg/ml); Su, sulfathiazole (550 µg/ml); Tm, trimethoprim (50 µg/ml); K, kanamycin (10 µg/ml); Na, nalidixic acid (50 µg/ml); Sp, spectinomycin (50 µg/ml); G, gentamicin (2.5 µg/ml); and Cp, ciprofloxacin (2 µg/ml). The source of <i>E. coli</i> strains is indicated by B, Bovine or H, human. The location of cattle properties is indicated by P1, Kameruka; P2, Cowra; P3, Eden; P4, Dungog; P5, Finley; P6, Gerringong; P7, Bega; P8, Canowindra and P9, Richmond. Human-derived <i>E. coli</i> strains were sourced from the Melbourne Diagnostic Unit (MDU) from patients with bloody diarrhoea (BD) or urinary tract infections (UTI).</p

An atypical class 1 integron with an IS<i>26</i>-mediated deletion in the integron 3′-CS.

<p>Genetic structure of an atypical class 1 integron located on a plasmid isolated from the bovine-derived <i>E. coli</i> strain, D22. The genetic map represents 8756 bp of nucleotide sequence. All genes are indicated by arrows and shaded in grey, except the class 1 integron <i>intI1</i> gene and <i>dfrA5</i> cassette gene (black) and the Tn<i>21</i> transposition genes <i>tnpM</i>, <i>tnpR</i> and <i>tnpA</i> (unfilled). Other genes identified include the entry exclusion proteins <i>exc1</i> and <i>exc2</i> (truncated) and the kanamycin resistance gene, <i>aphA1</i> (partial sequence). Features involved in recombination include the 59-be (black filled box), IS<i>26</i> transposase gene, <i>tnpA</i>, and IS<i>1</i> genes <i>insA</i> and <i>insB</i>. The position of PCR primers used to screen <i>E. coli</i> strains for this atypical class 1 integron is shown.</p

Illustration of plasmid pECTm80 isolated from <i>E. coli</i> strain 80 (GenBank no. FJ914220).

<p>Arrows indicate the direction of transcription. Terminal inverted repeats (IR) of transposons and insertion sequences (IS) (left IR, IR_L and right IR, IR_R) are indicated: 38 bp IR of a Tn<i>3</i> family transposon/IS remnant adjacent to the Tn<i>3-</i>like Δ<i>tnpA</i> gene (IR<i>_tnp</i>_-Tn<i>3</i>) and <i>orf14</i> (IR<i>_orf14</i>_-Tn<i>3</i>) (red), 23 bp IR IS<i>1R </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034718#pone.0034718-Ohtsubo1" target="_blank">[43]</a>, 8 bp imperfect terminal IRs carried by IS<i>Ec37</i>, 8 bp IR at the left end of Tn<i>7</i> (IR_Tn<i>7</i>). Tn<i>7</i>L denotes 150 bp at the left end of Tn<i>7</i> containing multiple TnsB binding sites (open box). The location of the three origins of replication are indicated (α and β, unfilled arrows; and γ, unfilled box). The arrows mark the <i>in vivo</i> direction of the initial replication from the α and β origins. The origins of conjugal transfer (<i>oriT</i>α and <i>oriT</i>β) are indicated by a green filled box. Colour-coded functional categories of predicted CDSs include insertion sequence/transposon transposases (red); conjugal transfer (<i>ddp1</i>, <i>ddp2</i> and <i>ddp3</i> genes: green); plasmid maintenance and stability including replication initiation (<i>bis</i> and <i>repB</i> genes), plasmid partitioning (<i>orf14</i> and <i>parA</i> genes) and plasmid stability (<i>stbD</i> gene: toxin and <i>stbE</i> gene: anti-toxin) (blue); gene expression modulation (<i>mdbA</i> gene: light blue); and hypothetical proteins of unknown function (grey). Integron features indicated include <i>intI2</i> and cassette genes (yellow arrows), <i>attI2</i> (closed box) and <i>attC</i> (grey boxes).</p

Comparison of pECTm80 with the <i>E. coli</i> plasmid pE001.

<p>Pairwise BLASTn comparisons between the plasmids pECTm80 and pE001 were visualized using the Easyfig program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034718#pone.0034718-Finan1" target="_blank">[23]</a>. Regions of nucleotide identity are connected by yellow blocks. The yellow color gradient indicates the extent of similarity as shown in the color scale on the right. Functions of CDS in pE001 have been taken from BLAST matches and existing annotation. Functional categories of predicted CDSs include: insertion sequence/transposon transposases (red); conjugal transfer (green); plasmid maintenance and stability (blue); gene expression modulation (light blue); class 2 integron <i>intI2</i> gene and gene cassettes (yellow); type IV secretion system (mauve); bla_TEM-52 (yellow) and hypothetical proteins of unknown function (grey). Scale bar represents 3000 base pairs.</p

Identification of CDS in the nucleotide sequence of pECTm80.

<p>AGenBank accession numbers provided represent the results of BLAST searches (NCBI and IS BLAST server) showing the highest identity to the query sequence. The accession number for pE001 is given when there is greater than one BLAST hit at 100% ID to pECTM80. BPlasmids showing 100% ID to pECTM80 are represented as follows: 1, pE001 (JF776874.1); 2, R485 (HE577112.1); 3, pMAS2027 (FJ666132.1); 4, pOLA52 (EU370913.1); 5, pOU1114 (DQ115387.2); 6, pSE34 (EU219533.1); and 7, pMccC7-H22 (EF536825.1). Boundaries of mobile elements in the nucleotide sequence of pECTm80 are as follows: CISEc37, 7355–9182; DTn3-like, 12671–18314; FIS1R, 17240–18007. ETn3-like ΔtnpA gene showed 64% to 76% identity to transposase genes of the Tn3 family of transposons (subgroups Tn501 and Tn3) and the IS elements ISSba14, and ISSod9 of the Tn3 family.</p