Construction of a gentamicin resistance gene probe for epidemiological studies

Abstract

A 7.7-kilobase BamHI fragment was cloned from the transconjugant of a clinical isolate of Escherichia coli containing a 120-kilobase multiresistance IncC plasmid. The recombinant plasmid conferred resistance to kanamycin, gentamicin, tobramycin, sulfamethoxazole, and trimethoprim. This clone was used to generate a series of subclones from which a 2.0-kilobase BamHI-HindIII probe containing a gentamicin 2''-O-adenylyltransferase [AAD(2'')] gene was obtained. This probe hybridized specifically in both colony and Southern hybridizations with the AAD(2'') gene but not with other resistance genes, including other aminoglycoside-modifying genes, or with a reference IncC plasmid lacking the AAD(2'') gene. The AAD(2'') gene may be part of a transposon, since hybridization occurred with both nonconjugative plasmids and the chromosome in some isolates.

In34, a Complex In5 Family Class 1 Integron Containing orf513 and dfrA10

“…The plasmid pRMH760 studied here confers resistance to the same set of antibiotics as pDGO100, which is the source of In7 (17), and the overlapping portions of the sequenced regions of In34 and In7 are identical. The identification of two closely related integrons on different plasmids from E. coli and K. pneumoniae strains, isolated almost 20 years apart in 1978 and 1997 at the same hospital, respectively, illustrates how complex structures containing antibiotic resistance genes can persist in a local environment.…”

“…In7 is part of multiresistance plasmid pDGO100 from an E. coli strain isolated in 1978 at the same Sydney hospital as pRMH760 (17). pDGO100 is also transferable and confers resistance to the same antibiotics as pRMH760.…”

A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100

“…A DNA segment of 2.8 kb is located between the partial 3'-conserved segment and a second 3'-conserved segment (9), and only one gene, which determines resistance to trimethoprim, has been identified in this intervening segment (6). The …”

“…The trimethoprim resistance determinant studied here was fortuitously coisolated with the gentamicin resistance determinant aadB on a 7.7-kb BamHI fragment (6) (2,8). A DNA segment of 2.8 kb is located between the partial 3'-conserved segment and a second 3'-conserved segment (9), and only one gene, which determines resistance to trimethoprim, has been identified in this intervening segment (6).…”

“…Among the wide variety of antibiotic resistance genes found as integron inserts, four distinct isolated during an outbreak of gentamicin resistance at Royal North Shore Hospital, Sydney (6). The plasmid pDGO101, which contains the cloned 7.7-kb BamHI fragment, confers resistance to gentamicin, tobramycin, and kanamycin (aadB gene) and also to trimethoprim and sulfonamides (6). A detailed map of the BamHI fragment compiled from sequence and restriction mapping data (2, 9; this study) is shown in Fig.…”