Humanized Anti‐DNABII Fab Fragments Plus Ofloxacin Eradicated Biofilms in Experimental Otitis Media

Abstract

Objectives/Hypothesis: To evaluate the ability of humanized monoclonal antibody fragments directed against a bacterial DNABII protein plus ofloxacin delivered directly into the chinchilla middle ear via tympanostomy tube (TT) to enhance the ability of ofloxacin to eradicate biofilms formed by nontypeable Haemophilus influenzae (NTHI).Study Design: A blinded pre-clinical study of comparative efficacy of single versus combinatorial treatment strategies. Methods: NTHI was allowed to form biofilms in the middle ears of chinchillas prior to TT placement. Ofloxacin, humanized Fab fragments against a bacterial DNABII protein that disrupts biofilms or Fab fragments plus ofloxacin were instilled into the middle ear via TT. For two consecutive days, ofloxacin was delivered twice-a-day, Fab fragments were delivered once-aday, or these treatments were combined. Relative biofilm resolution (as determined via two outcome measures) and eradication of viable NTHI were assessed 1-day later.Results: Whereas ofloxacin alone did not resolve biofilms or eradicate NTHI from the middle ear, delivery of Fab fragments significantly reduced both biofilms and NTHI burden over this short course of treatment. Notably, co-delivery of ofloxacin plus humanized Fab fragments eradicated both NTHI and biofilms from the middle ear, an enhanced outcome compared to receipt of either treatment alone.Conclusion: This study demonstrated a powerful combinatorial approach to release bacteria from their protective biofilms and rapidly render them vulnerable to killing by a previously ineffective antibiotic. An approach to combine ofloxacin with humanized Fab fragments that disrupt biofilms has tremendous potential to quickly resolve chronic otorrhea suffered by children with chronic suppurative otitis media or chronic post-tympanostomy tube otorrhea and thereby improve their quality of life.

Crumbling the Castle: Targeting DNABII Proteins for Collapsing Bacterial Biofilms as a Therapeutic Approach to Treat Disease and Combat Antimicrobial Resistance

“…Using an acute otitis media model in chinchillas challenged with nontypeable Haemophilus influenzae (NTHI), NRel bacteria generated upon rapid biofilm collapse showed an increased expression of genes associated with cell envelope biogenesis, translation, and ribosomal structure and biogenesis, consistent with the observed sensitization to amoxicillin-clavulanate [ 54 ]. The transient and unique phenotype of NRel bacteria from rapidly collapsed biofilms accounts for previous in vitro [ 39 , 45 , 49 , 51 , 53 , 54 , 61 ] and in vivo [ 50 , 59 ] observations in multiple bacterial species of enhanced killing by common first-line antibiotics when used in combination with anti-DNABII antibodies. Although the degree of sensitization of NRel bacteria to different classes of antibiotics may differ, we have observed no instances where NRel bacteria are less sensitive than planktonic bacteria.…”

“…The rapid collapse of biofilms using anti-DNABII antibodies has been demonstrated in more than 20 Gram-positive and -negative bacterial species in vitro [ 39 , 43 , 45 , 48 , 49 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ] and in vivo [ 48 , 50 , 55 , 58 , 59 ], as well as in polymicrobial biofilms from clinical specimens [ 46 , 60 ]. Table 2 lists the bacterial species and strains tested to date.…”

“…Sensitization to antibiotics was also observed in a murine pulmonary infection model with P. aeruginosa treated with anti-DNABII antibodies and tobramycin; anti-DNABII antibody treatment was not only effective in reducing lung CFU burden when administered by itself, but also further reduced CFU burden when co-administered with tobramycin [ 50 ]. Furthermore, Fab fragments generated from a humanized anti-DNABII monoclonal antibody in combination with ofloxacin eradicated NTHI and cleared biofilms from the middle ear after a shortened course of treatment [ 59 ].…”

“…Bacteria in biofilms are generally protected from clearance mediated by the innate immune system (neutrophils, macrophages). The anti-DNABII antibody treatment of biofilm-mediated infections in murine lung, chinchilla ear, and rat oral cavity enables complete or nearly complete resolution in the absence of antibiotic co-administration [ 50 , 55 , 58 , 59 ], demonstrating that antibody-mediated biofilm collapse enables more effective neutrophil/macrophage-mediated bacterial clearance. An analysis of lung tissue after antibody treatment also showed a decrease in the number of bacterial aggregates and the recruitment of neutrophils to sites of bacterial aggregates in the lung [ 50 ].…”

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice

“…To overcome this limitation, pathogens are injected directly into their middle ears to establish infections ( Park and Lee, 2013 ). Despite the biological caveats and technical challenges, direct middle ear inoculations have been productively used to establish and study complex aspects of middle ear infections ( Babl et al., 2002 ; Bouchet et al., 2003 ; Novotny et al., 2021 ) and remain the leading approach to gain insight into the innate immune mechanisms of host response and pathogen-specific aspects of middle ear infection ( Schilder et al., 2016 ).…”