PENICILLIN BINDING PROTEINS (PBPs) IN HAEMOPHILUS INFLUENZAE (HI)

Abstract

We previously described different PBP patterns of 3 non-typeable ampicillin-resistant (AmpR)-β-lactamase-neg. HI. The resistance determinant could be mobilized by transformation permitting assessment and comparison of the PBPs in the AmpR transformants (TFs) and the isogenic AmpS recipient. PBPs were detected by incubation of crude membrane preparations with radiolabelled penicillin (PCN) followed by selective solubilization with sarkosyl, electrophoresis in SDS-PAGE and fluorography. Ten major PBPs ranging in mol. wt. from 88 to 27 Kd. were defined in the AmpS recipient. Two TF strains showed apparent decreases in PBP 4 and 9. To measure the affinity of the PBPs for Amp, we assessed 3H-PCN binding of the AmpS recipient and 1 of these AmpR TFs in the presence of Amp at serial ten-fold conc. ranging from 10−3 to 102 times the MIC. The affinity of PBP 1 and 3 was identical for both the AmpR TF and the AmpS strain. However, affinity of PBP 5 of the AmpR TF was decreased 10,000 fold. We conclude that the mechanism of resistance in this TF is alteration in the apparent amount and/or affinity of PBPs. We compared 6 additional clinical non-typeable AmpR-β-lactamase-neg. HI (5 from England, 1 USA) to this AmpS isolate. All 6 revealed differences in their PBP profile; there was strain-to-strain variation in the apparent amount of PBP 3, 4, 5, or 6. We conclude that the mechanism of resistance in these 6 isolates may be altered PBPs.