Monoclonal antibodies that target extracellular DNABII proteins or the type IV pilus of nontypeable Haemophilus influenzae (NTHI) worked additively to disrupt 2-genera biofilms

Abstract

The biofilm state is the preferred lifestyle of bacteria in nature. Within a biofilm, the resident bacteria are protected from environmental stresses, antibiotics and other antimicrobials, including those due to multiple immune effectors of their host during conditions of disease. Thereby, biofilms contribute significantly to pathogenicity, recalcitrance to clearance and chronicity/recurrence of bacterial diseases, including diseases of the respiratory tract. In the absence of highly effective, biofilm-targeted therapeutics, antibiotics are commonly prescribed to attempt to treat these diseases, however, in light of the canonical resistance of biofilm-resident bacteria to antibiotic-mediated killing, this ineffectual practice often fails to resolve the diseased condition and contributes significantly to the global threat of rising antimicrobial resistance.Nontypeable Haemophilus influenzae is a common respiratory tract disease co-pathogen, often present in partnership with other airway pathogens. Herein we aspired to determine whether either of two monoclonal antibodies we developed, one specific for NTHI [directed against the majority subunit (PilA) of the type IV pilus (T4P) of NTHI] and the other able to act agnostically on all bacteria tested to date (directed against a structural protein of the biofilm matrix, a DNABII protein), were able to disrupt 2-genera biofilms wherein NTHI co-partnered with another respiratory tract pathogen. These monoclonals were tested singly as well as when within an antibody cocktail.The monoclonal directed against the NTHI antigen PilA was only effective on single species NTHI biofilms and not on single species biofilms formed by other unrelated species. However, when NTHI co-partnered with any of 5 respiratory tract pathogens tested here (Burkholderia cenocepacia, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae or Moraxella catarrhalis), this exclusively NTHI-directed monoclonal was able to disrupt these 2-genera biofilms. Conversely, the monoclonal antibody directed against protective epitopes of a DNABII protein, significantly disrupted all single species and 2-genera biofilms, which reflected the universal presence of this structural protein in all tested biofilm matrices. However, greatest release of both pathogens from a 2-genera biofilm was uniformly achieved by incubation with a 1:1 cocktail of both monoclonals. These data support the use of an approach wherein patients with respiratory tract disease could be treated with a therapeutic monoclonal antibody cocktail to release NTHI and its common co-pathogens from the protective biofilm to be killed by either traditional antibiotics and/or host immune effectors.