Regional dissemination of a trimethoprim-resistance gene cassette via a successful transposable element

Abstract

Background: Antimicrobial resistance is a growing international problem. We observed a 50% increase in the prevalence of trimethoprim resistance among fecal Escherichia coli from healthy Nigerian students between 1998 and 2005, a trend to increase that continued in 2009.

Methods and findings: A PCR-based screen revealed that 131 (43.1%) of isolates obtained in Nigeria in 2005 and 2009 carried integron-borne dfrA cassettes. In the case of 67 (51.1%) of these isolates, the cassette was a class 1-integron-borne dfrA7 gene, which has been reported at high prevalence from E. coli isolates from other parts of Africa. Complete sequencing of a 27 Kb dfrA7-bearing plasmid from one isolate located the dfrA7 gene within a Tn21-type transposon. The transposon also contained an IS26-derived bla/sul/str element, encoding resistance to β-lactams, sulphonamides and streptomycin, and mercury resistance genes. Although the plasmid backbone was only found in 12 (5.8%) of trimethoprim-resistant isolates, dfrA7 and other transposon-borne genes were detected in 14 (16.3%) and 32 (26.3%) of trimethoprim resistant isolates collected in Nigeria in 2005 and 2009, respectively. Additionally, 37 (19.3%) of trimethoprim-resistant E. coli isolates collected between 2006 and 2008 from Ghana were positive for the dfrA7 and a transposon marker, but only 4 (2.1%) harbored the plasmid backbone.

Conclusions: Our data point to transposition as a principal mechanism for disseminating dfrA7 among E. coli from Nigeria and Ghana. On-going intensive use of the affordable broad-spectrum antibacterials is likely to promote selective success of a highly prevalent transposable element in West Africa.

Figure 1. Structure of generic integron.

Each integron is comprised of 5′ and 3′ conserved ends separated by a variable region (black), which contains zero to several cassettes. The figure illustrates an integron bearing a single cassette. Cassette incorporation occurs by recombination between an attC attachment site at the 3′ end of the the gene cassette (hatched) at the integron’s attachment site (attI), located at the 5′ conserved end. This reaction is catalyzed by a site-specific recombinase encoded by the 5′ intI gene. Class 1 and Class 2 integrons, which are the sub-categories most commonly associated with drug resistance contain a promoter at the 5′ conserved end, from which cassette genes are transcribed.

Figure 2. Trimethoprim resistance trends among E. coli isolates from healthy adults in Ile-Ife Nigeria 1988–2009.

Figure 3. Plasmid profiles of E. coli strain 05-01a and its plasmid transformants.

Lane 2: Plasmid-free DH5α; Lane 3: 05-01a, dfrA7-positive 2005 isolate from Nigeria; Lanes 4–7: four independent transformants produced by electroporating DH5α with a plasmid preparation from 05-01a and selecting on plates containing 50 µg/ml trimethoprim. The arrow indicates the position of chromosomal DNA, as inferred from the DH5α profile. Lane 1: Hyperladder 1 marker.

Figure 4. Circular map of 27,072bp pASL01a.

Circles display (from the inside) (a) GC skew ([G + C]/[G - C]) in a 1,000-bp window (b) GC content in a 1,000-bp window (purple representing above average and yellow below average in both cases) (c) plasmid backbone in beige (d) predicted coding sequences and (e) kilobase scale. Open reading frames predicted to encode drug resistance genes are colored red and those that encode resistance to other chemical entities are gold. All other predicted open reading frames are marked in blue. Dark gray lines on the open-reading frame and backbone tracks mark the position of inverted repeats.