Use of Southern Hybridization to Detect the Haemophilus Influenzae β-lactamase Gene

　The most common cause of ampicillin resistance in Haemophilus influenzae is due to the production of β-lactamase. In most strains, the β-lactamase produced is TEM-1. Since 1981, ROB-1 β-lactamase appears to be responsible for ampicillin resistance in H. influenzae and several animal pathogens. Forty-five ampicillin-resistant, β-lactamase-producing clinical isolates of Haemophilus influenzae were collected. We used Southern hybridization to determine whether they produced TEM-1 or ROB-1 β-lactamase. Plasmids were obtained from a ROB-1-producing strain of H. influenzae, and a plasmid (pBR322) known to encode TEM-1. Both β-lactamase genes were labeled with non-isotope digoxigenin as probes, and then hybridized on with PCR amplified β-lactamase gene product from H. influenzae strains. The results revealed that β-lactamase-mediated ampicillin resistance type in H. influenzae could be detected by the Southern hybridization method.