New Gene Cassettes for Trimethoprim Resistance, dfr13 , and Streptomycin-Spectinomycin Resistance, aadA4 , Inserted on a Class 1 Integron

Abstract

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 IS 26 , which was contiguous with a truncated form of Tn 3 . On the same plasmid, pUK2381, a second copy of IS 26 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 .