Molecular Epidemiology of Plasmid-Mediated Ampicillin Resistance in Haemophilus influenzae Type b Isolates from Alaska

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Abstract
Native Alaskans have a high incidence of disease caused by invasive Haemophilus influenzae type b and represent an isolated population for epidemiological study. We used plasmid DNA analysis and subtyping of outer membrane proteins as markers to characterize 29 ampicillin-resistant, invasive strains and seven ampicillin-resistant, noninvasive strains of this organism from distinct geographic regions. All 36 strains produced β-lactamase; 34 strains transferred resistance by conjugation. Seven of the 36 strains harbored detectable plasmid DNA: four had a molecular mass of 40 MDa, and three had a molecular mass of 3 MDa. Furthermore, 20 transconjugants had a similar large plasmid, and four had a similar small plasmid. Ten of 12 transconjugants with either the large, small, or undetectable plasmid DNA were able to retransfer resistance. Transformation of resistance was successful with two large plasmids. DNA-DNA hybridization studies revealed that 33 of 36 strains had DNA homology. Restriction endonuclease digestion of 10 large plasmids revealed five patterns; identity was evident within a geographic region, and similarity existed between regions. Seven restricted plasmids demonstrated an identical pattern with a small β-lactamase probe. Ampicillin resistance in these isolates from Alaska is primarily due to a common, 40-MDa conjugative plasmid that mediates β-lactamase production, a finding which differs from that for ampicillin-resistant plasmids isolated elsewhere in the United States. Despite variable outer membrane protein profiles of the distinct strains of H. influenzae type b, the plasmids shared significant DNA homology. It appears that a common genetic element was responsible for the dissemination of this phenotype for resistance in Alaska.