The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined. The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons. A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region. The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1. Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa. This segment includes a 59 base element previously found flanking the Tn7 aadA gene. A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.

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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.

Minimal bactericidal concentrations (MBCs) of nine antimicrobial agents were determined for clinical isolates by a replica plating method. Membranes were placed on the antibiotic-containing plates and the organisms replicated onto the membranes. After 18 h incubation the minimal inhibitory concentrations (MICs) were determined, the membranes were transferred to antibiotic-free plates and incubated a further 18 h and the MBCs determined. MICs and MBCs were also determined in broth. The reproducibility of the 'membrane' method and the agreement of these results for MIC and MBC with the agar and/or broth methods was satisfactory for most antibiotics, within one two-fold dilution. With sulphamethoxazole, trimethoprim and co-trimoxazole the results were less satisfactory, especially with Gram-negative rods, but agreement within two dilutions could be achieved.

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