Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons

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Abstract
The 36-megadalton H. influenzae R plasmid pHK539 specified resistance to tetracycline (Tc) and ampicillin (AP). By molecular hybridization studies and by EM showed that the plasmid pHK539 contained the Tc translocation DNA segment (TnTc) and the Ap translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both Tc and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a Cm resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from diferent countries. The TcCm resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing Tc. The presence of the 2 copies of the transposon was correlated with higher minimum inhibitory concentrations against Tc and against Cm. After its growth in medium containing Tc, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained 1, 2, 3, or even 4 copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against Tc.