Molecular Characterization of a Plasmid Specifying Ampicillin Resistance and its Relationship to other R Factors from Haemophilus influenzae

Abstract

The ampicillin-resistant isolate Haemophilus influenzae KRE5367 which produced a TEM type β-lactamase contained a 30 megadalton (Mdal) plasmid (pKRE5367) with a copy number of approximately 2 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. Ampicillin resistance was transferred by cell-to-cell contact, and the transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pKRE5367 DNA proved that the structural gene for the TEM type β-lactamase resided on the plasmid genome. It was shown by molecular hybridization that pKRE5367 probably contained the ampicillin translocation DNA segment (TnA) found on some R factors of enteric origin. The H. influenzae plasmid pKRE5367 had most of its polynucleotide sequences in common with other R factors recently isolated from H. influenzae. DNA-DNA hybridization studies indicated that pKRE5367 shared about 70% base sequence homology with the two tetracycline-resistance R plasmids pLU 121 (31.5 Mdal) and pFR 16017 (33 Mdal) recently isolated in W. Germany. In addition, pKRE5367 had nearly 100% of its polynucleotide sequences in common with the 30 Mdal ampicillin-resistance R plasmid RSF007, isolated in the U.S.A.; it shared 70% base sequence homology with the 31.5 Mdal tetracycline-resistance plasmid pUB701, isolated in the U.K.; and it had 65% of its polynucleotide sequences in common with the 38 Mdal tetracycline- and chloramphenicol-resistance R plasmid pR1234 isolated in the Netherlands. Despite the broad geographical range, the R plasmids of H. influenzae appeared to be closely related.