New gene cassettes for trimethoprim resistance, dfr13, and Streptomycin-spectinomycin resistance, aadA4, inserted on a class 1 integron

Abstract

In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA ¿ANT(3")(9)-I, which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3' end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2.

FIG. 1

Restriction map of the cloned fragments of pUK2408 and pUK2418 showing the organization and direction of transcription of intI1, dfr13, and aadA4, the positions of insertion sequences IS1 and IS26, and the truncated sections of transposons Tn21 and Tn3. The core elements of the gene cassettes and the inverted repeats (IRs) of the insertion sequence elements are indicated.

FIG. 2

Nucleotide sequence and translated peptides of the PvuII-BamHI fragment of pUK2381 showing the class I integron context of gene cassettes dfr13 and aadA4 and the position and flanking sequences of IS26. The −35 and −10 promoter regions of the 5′CS are underlined, the oligonucleotide probe for dfr13 is underlined, the points of insertion of the gene cassettes are marked (↓), the imperfect inverted repeat of the 59-base element of the dfr13 cassette is underlined and the direction is marked (><), the inverted repeats of IS26 are in boldface type, and asterisks indicate stop codons.

FIG. 3

Comparison of the flanking sequences of aadA1 (17), aadA2 (9), aadA4, and the aadA gene from S. enterica serovar Choleraesuis (S. chol) (32). The GTT triplet of the core sequences is marked in boldface type, and the inverted repeats of the 59-base element are underlined. Shaded areas mark the start and stop codons.