Haemophilus influenzae luxS mutants form a biofilm and have increased virulence

To gain insight into the role of luxS(Hi) in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxS(Hi) mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB 170 bioassay. Culture filtrates from Escherichia coli strain DH5 alpha expressing a cloned luxS(Hi) contained 350-fold more AI-2 activity per cell than E. coli DH5 alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxS(Hi) mutants. Two clinical H. influenzae and their luxS(Hi) mutants produced an identical biofilm in a flow system. Invasion Of human cells by the luxS(Hi) mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxS(Hi) Mutants tended to be more invasive. The luxS(Hi) mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits. (C) 2005 Elsevier Ltd. All rights reserved.