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.Class 1 integrons are DNA elements that encode a sitespecific recombinase (intI1) of the family of integrases and are capable of catalyzing the insertion and recombination of mobile DNA elements (gene cassettes) at specific sites (core ite) with a consensus sequence, GTTRRRY. The integron also provides a 5Ј conserved core site, attI1, into which gene cassettes are preferentially inserted, and the promoters responsible for expression of the cassette-encoded genes (10,19). Gene cassettes are identified by a 59-base element which occurs at the 3Ј end of the cassette and which consists of an inverted imperfect repeat of between 50 and 150 bp which has an inverse core site at the 5Ј end of the inverted repeat and a core site at the 3Ј end. The insertion of a gene cassette into the attI1 site results in the formation of a secondary site (attC) downstream of the cassette. The attI1 site differs from attC sites in that the inverse core site and secondary structure are missing 5Ј of the attI1 core site (21). The majority of published cassettes contain genes for resistance to various antimicrobial agents including trimethoprim and aminoglycosides (41). Trimethoprim selectively inhibits the bacterial dihydrofolate reductase (DHFR), thus preventing reduction of dihydrofolate to tetrahydrofolate (11). The most common mechanism of resistance to trimethoprim in enterobacteria is the production of an additional plasmid-mediated DHFR which, unlike the chromosomal enzyme, i...

CodY ofStreptococcus pneumoniae: Link between Nutritional Gene Regulation and Colonization

“…The gene that encodes CodY (spd1412) was deleted from strain D39 by allelic replacement with the dfr13 cassette, which confers trimethoprim resistance (2). To this end, codY with 1,000 bp of upstream and downstream flanking sequences was amplified from chromosomal D39 DNA with primers CodSacFwd and CodKpnRv (Table 2).…”

The Streptococcal Lipoprotein Rotamase A (SlrA) Is a Functional Peptidyl-prolyl Isomerase Involved in Pneumococcal Colonization

“…The genes encoding slrA (accession number spr0679) and ppmA (accession number spr0884) were deleted from the parental strains D39 and NCTC10319 by inframe insertion deletion mutagenesis using the resistance cassettes ermAM (32), and dfr13 (33), encoding resistance to erythromycin and trimethoprim, respectively. SlrA and ppmA with 1000 bp of upstream and downstream flanking sequences were amplified from chromosomal DNA from the D39 strain.…”

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains

“…1 kb integron. According to an alternative nomenclature this corresponds to the ant(3")-Ib gene (Adrian et al, 2000). In the 2 kb integron of a DT104 strain the aadA1a gene was found by Guerra et al (2000).…”

“…Sm/Spc resistance is mediated by the adenylating enzymes designated AAD(3") or ANT(3") and encoded by the aadA gene cassette family (Shaw et al, 1993;Fluit and Schmitz, 1999;Naas et al, 1999;Sandvang, 1999;Adrian et al, 2000;White and Rawlinson, 2001). In S. Typhimurium DT104 the aadA2 gene is re-sponsible for Sm/Spc resistance (Arcangioli et al, 1999;Briggs and Fratamico, 1999;Casin et al, 1999;Ng et al, 1999;Poirel et al, 1999;Guerra et al, 2000;Boyd et al, 2001;Frana et al, 2001), which is located in an approx.…”

“…Its variants constitute a gene family the two most important members of which are aadA1 sequenced by Hollingshead and Vapnek (1985), Fling et al (1985) and Sundström et al (1988), and aadA2 sequenced by Tait et al (1985) and Bito and Susani (1994). Further variants (aadA3-A8, aadAsc) have been described from E. coli, Pseudomonas aeruginosa and Salmonella choleraesuis isolates (Leung et al, 1992;Naas et al, 1999;Adrian et al, 2000;Mazel et al, 2000;White et al, 2000;Peters et al, 2001). The integron nature of the aadA gene and the plasmid-and/or transposon-associated location of the integrons may have played a role in the excessive horizontal spread of aadA.…”