Nontypeable Haemophilus influenzae newly released (NRel) from biofilms by antibody-mediated dispersal versus antibody-mediated disruption are phenotypically distinct

Biofilms contribute significantly to the chronicity and recurrence of bacterial diseases due to the fact that biofilm-resident bacteria are highly recalcitrant to killing by host immune effectors and antibiotics. Thus, antibody-mediated release of bacteria from biofilm residence into the surrounding milieu supports a powerful strategy to resolve otherwise difficult-to-treat biofilm-associated diseases. In our prior work, we revealed that antibodies directed against two unique determinants of nontypeable Haemophilus influenzae (NTHI) [e.g. the Type IV pilus (T4P) or a bacterial DNABII DNA-binding protein, a species-independent target that provides structural integrity to bacterial biofilms] release biofilm-resident bacteria via discrete mechanisms. Herein, we now show that the phenotype of the resultant newly released (or NRel) NTHI is dependent upon the specific mechanism of release. We used flow cytometry, proteomic profiles, and targeted transcriptomics to demonstrate that the two NRel populations were significantly different not only from planktonically grown NTHI, but importantly, from each other despite genetic identity. Moreover, each NRel population had a distinct, significantly increased susceptibility to killing by either a sulfonamide or β-lactam antibiotic compared to planktonic NTHI, an observation consistent with their individual proteomes and further supported by relative differences in targeted gene expression. The distinct phenotypes of NTHI released from biofilms by antibodies directed against specific epitopes of T4P or DNABII binding proteins provide new opportunities to develop targeted therapeutic strategies for biofilm eradication and disease resolution.