Specific growth conditions induce a Streptococcus pneumoniae non-mucoidal, small colony variant and determine the outcome of its co-culture with Haemophilus influenzae

: Haemophilus influenzae and Streptococcus pneumoniae are known aetiologic agents of chronic otitis media, frequently as a multispecies infection. In this study, we show that the outcome of H. influenzae/S. pneumoniae interactions is dependent on the nutrient source. In continuous culture containing chemically defined media with lactose, S. pneumoniae was non-viable in mono-culture, and in co-culture remained non-viable until 288 h. With glucose, S. pneumoniae became non-viable in mono-culture, but uniquely existed in 3 distinct states in co-culture: parental cells (until 24 h), a dormant state until 336 h and its re-emergence as a non-mucoidal, small colony variant (SCV). The S. pneumoniae SCV was stable and whole genome sequencing showed three major single nucleotide polymorphisms in the SCV cells-cap3A (capsule biosynthesis pathway), fpg (DNA glycosylase of the DNA repair mechanism) and glutamate-5-kinase. Previously, fpg mutants have shown increased mutator rates, permitting bacterial survival against host-generated stresses. Transcriptomics showed these SCV cells up-regulated sugar transporters and toxin/antitoxin systems. An animal model revealed a reduced survival in the lungs and ear by SCV cells. This is the first study documenting the effect of carbon source and the development of a distinct S. pneumoniae cell type during H. influenzae/S. pneumoniae interactions.