Genotypically defined β-lactamase-negative ampicillin-resistant isolates of non-typable Haemophilus influenzae are associated with increased invasion of bronchial epithelial cells in vitro

Abstract

The aim of the study was to investigate the association between the presence of altered penicillin-binding protein 3 (PBP3) in non-typable Haemophilus influenzae (NTHi) and an increased capacity to invade bronchial epithelial cells in vitro. A collection of 40 clinical isolates of NTHi comprised of 20 with normal PBP3 and 20 with altered PBP3 (defined by an N526K substitution) was established. The isolates were tested for the ability to invade bronchial epithelial cells in vitro using a 4 h gentamicin survival assay. Invasion was measured as the percentage of intracellular organisms relative to the initial inoculum. The mean invasion rate was 0.00–14.79 % in the normal PBP3 isolates and 0.02–36.69 % in the altered PBP3 isolates. The altered PBP3 isolates had a higher (P = 0.003) mean invasion rate (6.86 %, n = 20) than the normal PBP3 isolates (1.31 %, n = 20). Subsequently, two variants of altered PBP3 (transformant 1, N526K; transformant 2, M377I, S385T, L389F and N526K) were cloned into three of the initial isolates (parents) with normal PBP3 and relatively low invasive ability, and the parents and transformants tested for invasion as above. There was no difference (P = 0.89) in the mean invasion rates for the parents (0.81 %, n = 3), transformants 1 (0.90 %, n = 3) and transformants 2 (1.38 %, n = 3). There was an association between the presence of altered PBP3 in NTHi and an increased capacity to invade BEAS-2B cells in vitro, but cloning experiments suggested that the altered PBP3 was not involved directly in enhanced invasion.