. The molecular and biochemical basis of resistance to high concentrations (MIC ± 1000 mg/L) of trimethoprim (Tp ) was examined in Australian isolates of and coagulase-negative staphylococci. The Tp determinant () was located within a 2.75-Kb /II fragment on the aminoglycoside-resistance plasmids pSK1 and pSK16 as judged by comparative restriction mapping with two naturally-occurring variants, pSK9 and pSK14, which did not encode trimethoprim resistance. This was confirmed in DNA-DNA hybridisation experiments in which a 0.9-Kb sequence of pSK1 DNA was used as a specific probe for the Tp gene. Plasmid DNA from three strains of coagulase-negative staphylococci, and the chromosomal material of one other isolate, were found to share homology with the probe DNA. Dihydrofolate reductases produced by these strains were virtually identical to the type S1 enzyme encoded by the plasmid pSK1. Interspecies transfer may have been responsible for the conservation of Tp gene sequences among staphylococci.


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