Genetics of resistance to trimethoprim in cotrimoxazole resistant uropathogenic Escherichia coli: integrons, transposons, and single gene cassettes

Abstract

Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic Escherichia coli strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole (sul) and trimethoprim (dfrA). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, dfrA14 or dfrA1, integrated in two different points of a conserved cluster sul2-strA-strB, carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.

Keywords: Escherichia coli; antibiotic resistance; cotrimoxazole; gene cassettes; integrons; plasmid transfer; transposons; trimethoprim.

Figure 1

Class 1 integrons or its remnants in the cotrimoxazole resistant strains. (A) General structure of an Int1: genes are indicated with thick arrows; the genetic content of the variable region is not specified due to its variability. Below, bars indicate the generated amplicons to detect different parts of Int1 in previous (*) and present determinations. Small arrows, new primers used in this work. (B) Int1 structures detected in the 69 strains with Int1 sequences. At left, number of strains carrying each type of structure: in dark gray squares, previously determined; in clear squares, determined in this work. At right, presence of sul2, sul3, Int2, and ISCR1, with the number of strains in brackets. ?, unknown genetic content in the right end of the variable region. **, TMP^R is encoded by the dfrA1 gene from the Int2. Previous determinations were described in Poey and Laviña (2014, 2018) and de los Santos et al. (2021).

Figure 2

PCR amplifications for the detection of dfrA genes in the cotrimoxazole resistant strains. The number of strains with or without integron markers is shown above. Attached to vertical arrows, primer pairs that gave rise to amplicons. Below, type and number of dfrA genes identified; dfrAx generically designates several types of dfrA genes. In gray boxes, dfrA genes previously identified in amplicons with primer pair HS458 and HS459 (Poey and Laviña, ; de los Santos et al., 2021). ?, strains in which the gene responsible for the TMP^R phenotype was not found.

Figure 3

Single dfrA gene-cassettes inserted into the sul2 strA strB locus in plasmids p31_CH-1 and p61_CH-1m. (A) Structure of the locus in p31_CH-1 compared with the same region in large and small plasmids with and without the dfrA14 gene cassette insertion. The site of a Tn3 insertion (4.957 bp) is indicated with a thin arrow. (B) The same locus with the dfrA1 gene cassette insertion in p61_CH-1m. At left, plasmids name, size and GenBank accession number. Plasmids from this work are boxed.

Figure 4

The four identified genetic contexts responsible for the trimethoprim resistance in the SXT^R strains. The genetic maps of the canonical integrons and transposons are depicted, although some strains may have deletions of parts of them. *, stop codon inactivating the intI2 gene. At left, number of strains carrying each type of structure. #, four of the 15 strains with an Int2 also have an Int1, and in one of them the dfrA1 gene of Int2 is the only responsible for the TMP^R of the strain.