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

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

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

<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