OBJECTIVE:Chronic and recurrent cystic fibrosis lung infections, commonly due to the presence of recalcitrant Pseudomonas aeruginosa biofilms, are not effectively cleared with traditional antibiotics, largely due to a structurally sound and protective extracellular DNA matrix that envelopes biofilm-resident bacteria. We recently showed that when extracellular DNA architecture is disrupted by a humanized monoclonal antibody (HuTipMab) directed against the protective epitopes of universal bacterial proteins that serve as linchpins within the lattice (DNABII proteins), resident bacteria are released in a highly sensitive phenotype that renders them vulnerable to killing by host immune effectors and antibiotics. METHODS:P. aeruginosa biofilms formed by 3 clinical isolates were incubated with media alone or HuTipMab to generate non-biofilm-associated bacteria (NBfA) or bacteria newly released from biofilm residence (NRel), respectively. Antibiotic susceptibility testing at sub-MIC concentrations was then performed on each bacterial population to determine the sensitivity of NRel to the action of antibiotics compared to their non-biofilm-associated bacteria counterparts. RESULTS:Biofilms formed by three P. aeruginosa clinical isolates were significantly disrupted by HuTipMab prior to treatment with three commonly prescribed antibiotics for people with cystic fibrosis: colistin, aztreonam, or tobramycin. Bacteria newly released from HuTipMab-treated biofilms were significantly more susceptible to killing by each tested antibiotic compared with both their non-biofilm-associated counterparts and those resident within a biofilm. CONCLUSIONS:Whereas antibiotics alone do not successfully clear P. aeruginosa from lungs with cystic fibrosis, we herein provide support for a novel approach toward the aforementioned goal that both independently targets biofilm infrastructure and empowers the use of traditional antibiotics.

Biofilms are structured multicellular bacterial communities encased within an extracellular matrix comprised of exopolysaccharides, proteins, extracellular DNA (eDNA), and other biopolymers that provide protection against environmental stressors. We and others have shown that eDNA serves as a fundamental structural element common to even multispecies biofilms. During biofilm maturation, ubiquitous DNABII proteins facilitate the conversion and stabilization of eDNA into the rare and rigid Z-DNA conformation, thereby enhancing matrix integrity and rendering the underlying eDNA resistant to nucleases. We have previously shown that the removal of positively charged molecules, such as DNABII proteins, results in rapid, significant disruption of diverse biofilms. Here, we identify the polyamine spermidine as another essential positively charged molecule that, together with DNABII proteins, contributes to the development and maintenance of the eDNA-dependent extracellular matrix. We also provide evidence that SPD is present within the biofilm matrix alongside DNABII proteins in multiple bacterial pathogens. Our findings indicate that SPD and DNABII proteins cooperate to promote Z-DNA formation. Depletion of SPD and DNABII using cation exchanger P11-phosphocellulose or inhibition of SPD synthesis via dicyclohexylamine impaired biofilm formation and destabilized preformed biofilms. These results suggest that polyamine synthesis or accumulation represents a potential target for biofilm disruption and control.

IntroductionBiofilms are communities of microorganisms encased in a self-produced matrix, a structure that makes resident bacteria up to 1,000 times more resistant to antibiotics than their free-living, or planktonic, counterparts. Intriguingly, new methods that use reagents to release the biofilm-resident bacteria result in a transitory yet highly antibiotic sensitive phenotype. These newly released (NRel) bacteria are at least, if not more, sensitive to antibiotics than those in their planktonic form. Here, we sought to determine if the production of the NRel phenotype is reagent- dependent or can be accomplished by alternative means.MethodsAcross four pathogenic bacteria: non-typeable Haemophilus influenzae (NTHI), methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumoniae, we investigated whether rapid mechanical disruption of biofilms, or the use of a novel cationic depletion method in NTHI, could similarly induce this NRel phenotype. The presence of NRel was assessed by comparing the antibiotic sensitivity of the released bacteria to that of their planktonic counterparts. For NTHI specifically, we further characterized the phenotype by measuring the kinetics of antibiotic sensitivity via comparative plate counts over time. We also analyzed the relative expression levels of known NRel-associated genes using quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsWe show that using either intense mechanical disruption or a cationic depletion method not only facilitate rapid biofilm disruption but produce the NRel phenotype. In each case, both NRel signature gene expression and the transient antibiotic sensitivity phenotypes were observed compared to planktonic cells.DiscussionSimilar to methods using reagents, we found that mechanical or cationic depletion disruption of pathogenic bacterial biofilms were sufficient to trigger the NRel phenotype. These results are consistent with the NRel state potentially being a rate-dependent physiological response rather than being induced by specific chemical or biological agents. We discuss the changes in gene expression permissive to NRel and the possibility that rapid and premature release of bacteria from a biofilm fails to allow the resident bacteria to be sufficiently prepared for their biofilm free state. This new insight both expands our understanding of the NRel phenotype and provides further validation for our rapid-release therapeutic strategy.

Biofilm-mediated infections are highly recalcitrant to antibiotics and host immune system clearance. The matrix that envelopes biofilm-resident bacteria is stabilized by extracellular DNA as well as integrated ubiquitous bacterial DNABII proteins that exhibit potential as a common therapeutic target. We’ve shown that a monoclonal antibody directed against the immunoprotective DNA-binding ‘tips’ of DNABII proteins disrupts diverse biofilms, prevents their formation, and augments disease resolution in four distinct pre-clinical models. As an immunogen, a synthetic peptide that mimics the immunoprotective domains induces a response with equivalent effectiveness. Confoundingly, however, sera from both children with chronic otitis media (OM) and healthy adults preferentially recognize non-protective ‘tail’ domains of DNABII proteins. Thus, we wondered if this is a universal, natural immune response that contributes to biofilm recalcitrance. Here, we used both surface plasmon resonance and ELISA assays to assess sera from 16 healthy children, 16 additional children with chronic OM, 15 adults with chronic obstructive pulmonary disease, and 30 people with cystic fibrosis for relative recognition of synthetic peptides that mimicked either the protective or the non-protective DNABII protein domains. In 74 of 77 sera assessed (96%), we found significant IgG-specific preferential immune recognition of non-protective domains. These new data support continued development of our DNABII-directed vaccine candidate designed to re-direct the immune response toward the protective domains to mediate biofilm eradication.

Bacterial biofilms mediate chronic and recurrent bacterial infections that are extremely difficult to treat by currently available standards of care. In nature, these encased bacterial communities are typically comprised of more than one genus or species. Specifically, in the airway, nontypeable Haemophilus influenzae (NTHI) predominates and is commonly isolated with one or more of the following co-pathogens with which it forms unique relationships: methicillin-resistant Staphylococcus aureus, Burkholderia cenocepacia, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Moraxella catarrhalis. We recently showed that dual-genera biofilms comprised of NTHI plus a co-pathogen are disrupted when the biofilm matrix is destabilized by a pathogen-directed strategy that uses a humanized monoclonal antibody directed against the protective domains of bacterial DNABII proteins found at vertices of crossed strands of eDNA within the biofilm matrix. We also recently showed that a peptide synthesized from the host innate immune effector High Mobility Group Box 1 (HMGB1), called mB Box-97syn, competitively inhibits binding of the bacterial DNABII proteins to eDNA, which thereby also destabilizes single-species biofilms to release biofilm-resident bacteria into a transient yet highly vulnerable state that is more effectively cleared by the host innate immune system and/or antibiotics. Here, we expanded upon these studies to assess the ability of host-augmenting mB Box-97syn to both disrupt two-genera biofilms formed by NTHI plus a common co-pathogen, and prevent their formation. Disruption of established two-genera biofilms ranged from 57% to 88%, whereas prevention of two-genera biofilm formation ranged from 65% to 80% (P = .002 to P < .0001). The sobering recalcitrance of chronic and recurrent respiratory tract infections, combined with growing global concern of antimicrobial resistance (AMR), demands development of more effective management and prevention options. Ideally, novel treatment strategies would both target the pathogens and augment the host's natural abilities to eradicate them. Herein, we provide additional data to support continued development of the latter concept via demonstration of mB Box-97syn's efficacy against polymicrobial biofilms.

IntroductionNeutrophil Extracellular Traps (NETs) are vital for innate immunity, playing a key role in controlling pathogen and biofilm proliferation. However, excessive NETosis is implicated in autoimmunity, inflammatory and neoplastic diseases, as well as thrombosis, stroke, and post-COVID-19 complications. Managing NETosis, therefore is a significant area of ongoing research. Herein, we have identified a peptide derived from HMGB1 that we have modified via a point mutation that is referred to as mB Box-97. In our recent study in a murine lung infection model, mB Box-97 was shown to be safe and effective at disrupting biofilms without eliciting an inflammatory response typically associated with HMGB1. Here we show that the lack of an inflammatory response of mB Box-97 is in part due to the inhibition of NETosis of which we investigated the mechanism of action.MethodsmB Box-97’s anti-NETosis activity was assessed using human neutrophils with known NET inducers PMA, LPS, or Ionomycin. Additionally, mB Box-97’s binding to Protein Kinase C (PKC), in addition to downstream effects on NADPH oxidase (NOX) activation, Reactive Oxygen Species (ROS) generation and thereby NETosis were assessed.ResultsmB Box-97 significantly inhibited NETosis regardless of the type of induction pathway. Mechanistically, mB Box-97 inhibits PKC activity likely through direct binding and thereby reduced downstream activities including NOX activation, ROS production and NETosis.ConclusionsmB Box-97 is a promising dual acting therapeutic candidate for managing NET-mediated pathologies and resolving biofilm infections. Our results reveal that PKC is a viable target for NETosis inhibition independent of NET inducer and worthy of further study. These findings pave the way for a novel class of therapeutics aimed at controlling excessive NETosis, potentially offering new treatments for a range of inflammatory and immune-related diseases.

Biofilm-resident bacteria exhibit diverse mechanisms to evade eradication, including the highly protective self-produced matrix in which they are embedded. Thus, releasing bacteria from biofilm residence affords antibiotics and immune effectors greater access. We developed a monoclonal antibody directed against an essential biofilm matrix protein that induces rapid collapse of the biofilm matrix with release of bacteria that are in a transient but highly vulnerable phenotype. Bacteria that are newly released (“NRel”) due to this monoclonal are significantly more sensitive to antibiotic-, antimicrobial peptide- or human PMN-mediated killing in vitro and are rapidly eradicated in four animal models without adjunct antibiotic treatment, the speed of which highlights the role of innate immune effectors. Here, we characterized the reactivity of human PMNs to three distinct bacterial populations derived from three clinical isolates of the predominant respiratory tract pathogen nontypeable Haemophilus influenzae (NTHI). Via timelapse microscopy and flow cytometry, PMN engulfment, NETosis (e.g., programmed neutrophil cell death accompanied by release of web-like condensed DNA with associated antimicrobial proteins), and migratory activity were assessed when PMNs were incubated with NTHI that were dual-fluorescently labeled with green-fluorescent FM 1–43 and pH-sensitive red-fluorescent pHrodo™ Red, SE, which fluoresces in acidic environments such as within a phagolysosome. Relative concentrations of endotoxin and DNA, as well as cytokines/chemokines potentially responsible for observed PMN activities were also assessed. PMN-mediated engulfment, NETosis, and migratory activity were all greatest when incubated with NTHI NRel compared to both NTHI that had been grown planktonically in rich medium or to NTHI that were present in the culture medium that overlayed the biofilm. Whereas neither NTHI endotoxin nor DNA played a role in the observed relative activities, PMNs incubated with NTHI NRel released significantly more IL-8 which likely served to enhance the migration of additional PMNs. These data contribute to our understanding of mechanisms anticipated to be operational in the DNABII protein-targeted monoclonal antibody-based treatment regimen we developed to facilitate host-mediated contribution to biofilm eradication after induced release of formerly biofilm-resident bacteria into the highly vulnerable NRel state.

Background Pulmonary infections with multidrug-resistant nontuberculous mycobacteria (NTM), particularly Mycobacterium abscessus (MAB), are increasingly more prevalent in individuals with lung disease such as cystic fibrosis and are extremely difficult to treat. Protracted antibiotic therapies consist of multidrug regimens that last for months to years. Despite these intense protocols, failure rates are high with 50%-60% of patients not achieving a sustained culture-negative status. A major contributor to the difficult medical management of NTM infections is formation of pulmonary aggregate MAB biofilms which protect the resident bacteria from antimicrobials and host immune effectors. Thereby, novel and more effective approaches to combat recalcitrant NTM infections are urgently needed. Methods We developed an epitope-targeted monoclonal antibody-based technology to rapidly disrupt biofilms and release resident bacteria into a transient yet highly vulnerable phenotype that is significantly more sensitive to killing by both antibiotics and host innate immune effectors (e.g., PMNs and antimicrobial peptides). Herein, we tested this technology in a pre-clinical murine lung infection model to determine whether this treatment would mediate clearance of MAB from the lungs and speed return to homeostasis. Results As early as 48 h after a single treatment, bacterial loads were reduced to below the level of detection and histopathologic analysis showed markedly decreased inflammation and rapid eradication of aggregate biofilms compared to controls. Conclusions These new data add to those from multiple prior published studies which show the significant efficacy of this novel therapeutic approach to resolve recalcitrant bacterial biofilm diseases, now potentially including those induced by NTM.

Bacteria newly released (NRel) from biofilm residence via multiple methodologies are commonly significantly more sensitive to antibiotics. We've induced NRel with this phenotype after incubation of biofilms formed by diverse human pathogens with an epitope-targeted monoclonal antibody directed at protective domains within bacterial DNABII proteins that provide structural support to the eDNA-dependent biofilm matrix. The observed heightened sensitivity was due, in part, to increased NRel membrane permeability. In three animal models of human biofilm-mediated infections, this monoclonal induced biofilm disruption with rapid concomitant bacterial clearance and disease resolution in the absence of any co-delivered antibiotic, which suggested a key role of innate immune effectors. Recently, we showed that NRel of the respiratory pathogen nontypeable Haemophilus influenzae (NTHI), as mediated by the DNABII-directed monoclonal, are also highly vulnerable to killing by human polymorphonuclear neutrophils (PMNs). Here, we extended these observations to show that the transient, yet highly vulnerable anti-DNABII NRel phenotype of three isolates of both NTHI and methicillin-resistant Staphylococcus aureus (MRSA) included significant sensitivity to killing by three antimicrobial peptides commonly expressed within the respiratory tract or by PMNs (e.g., human β-defensins 1 and 3 as well as the cathelicidin, LL-37). We envision induction of the NRel phenotype by delivery of this monoclonal antibody to patients with recalcitrant biofilm-mediated diseases to provide greatly improved medical management. Ideally, clearance of NRel will be mediated by innate immune effectors of an immunocompetent host or, if needed, by co-delivered traditional antibiotics, which are canonically ineffective against biofilm-resident bacteria but would be highly effective against NRel. IMPORTANCE:Pathogenesis of most common chronic and/or recurrent bacterial diseases (e.g., middle ear infections, urinary tract infections, rhinosinusitis, among others) can be attributed to biofilms that are canonically highly resistant to both immune effectors and antibiotics. If we treat biofilms formed by diverse human pathogens with a targeted monoclonal antibody directed at protective domains of bacterial DNA-binding proteins integral to the structural stability of the eDNA-rich biofilm matrix, they are rapidly disrupted with concomitant release of the resident bacteria. These newly released (NRel) bacteria are transiently significantly more sensitive to killing by both traditional antibiotics and human PMNs, and herein, we showed that they are also more readily killed by antimicrobial peptides. Clinically, we hope to leverage this understanding of the NRel phenotype for better medical management of these challenging infections, as well as perhaps even limit or eliminate further contribution to the global antimicrobial resistance 'pandemic'.

Otitis media (OM) is one of the most globally pervasive pediatric conditions. Translocation of nasopharynx-resident opportunistic pathogens like nontypeable Haemophilus influenzae (NTHi) assimilates into polymicrobial middle ear biofilms, which promote OM pathogenesis and substantially diminish antibiotic efficacy. Oral or tympanostomy tube (TT)-delivered antibiotics remain the standard of care (SOC) despite consequences including secondary infection, dysbiosis, and antimicrobial resistance. Monoclonal antibodies (mAb) against two biofilm-associated structural proteins, NTHi-specific type IV pilus PilA (anti-rsPilA) and protective tip-region epitopes of NTHi integration host factor (anti-tip-chimer), were previously shown to disrupt biofilms and restore antibiotic sensitivity in vitro. However, the additional criterion for clinical relevance includes the absence of consequential microbiome alterations. Here, nine chinchilla cohorts (n = 3/cohort) without disease were established to evaluate whether TT delivery of mAbs disrupted nasopharyngeal or fecal microbiomes relative to SOC-OM antibiotics. Cohort treatments included a 7d regimen of oral amoxicillin-clavulanate (AC) or 2d regimen of TT-delivered mAb, AC, Trimethoprim-sulfamethoxazole (TS), ofloxacin, or saline. Fecal and nasopharyngeal lavage (NPL) samples were collected before and several days post treatment (DPT) for 16S sequencing. While antibiotic-treated cohorts displayed beta-diversity shifts (PERMANOVA, P < 0.05) and reductions in alpha diversity (q < 0.20) relative to baseline, mAb antibodies failed to affect diversity, indicating maintenance of a eubiotic state. Taxonomic and longitudinal analyses showed blooms in opportunistic pathogens (ANCOM) and greater magnitudes of compositional change (P < 0.05) following broad-spectrum antibiotic but not mAb treatments. Collectively, results showed broad-spectrum antibiotics induced significant fecal and nasopharyngeal microbiome disruption regardless of delivery route. Excitingly, biofilm-targeting antibodies had little effect on fecal and nasopharyngeal microbiomes.

Background Bacterial biofilm communities are embedded in a protective extracellular matrix comprised of various components, with its' integrity largely owed to a 3-dimensional lattice of extracellular DNA (eDNA) interconnected by Holliday Junction (HJ)-like structures and stabilised by the ubiquitous eubacterial DNABII family of DNA-binding architectural proteins. We recently showed that the host innate immune effector High Mobility Group Box 1 (HMGB1) protein possesses extracellular anti-biofilm activity by destabilising these HJ-like structures, resulting in release of biofilm-resident bacteria into a vulnerable state. Herein, we showed that HMGB1's anti-biofilm activity was completely contained within a contiguous 97 amino acid region that retained DNA-binding activity, called ‘mB Box-97’. Methods We engineered a synthetic version of this 97-mer and introduced a single amino acid change which lacked any post-translational modifications, and tested its activity independently and in combination with a humanised monoclonal antibody that disrupts biofilms by the distinct mechanism of DNABII protein sequestration. Findings mB Box-97 disrupted and prevented biofilms, including those formed by the ESKAPEE pathogens, and importantly reduced measurable proinflammatory activity normally associated with HMGB1 in a murine lung infection model. Interpretation Herein, we discuss the value of targeting the ubiquitous eDNA-dependent matrix of biofilms via mB Box-97 used singly or in a dual host-augmenting/pathogen-targeted cocktail to resolve bacterial biofilm infections. Funding This work was supported by NIH/NIDCD R01DC011818 to L.O.B. and S.D.G. and NIH/NIAID R01AI155501 to S.D.G.

Introduction The “silent” antimicrobial resistance (AMR) pandemic is responsible for nearly five million deaths annually, with a group of seven biofilm-forming pathogens, known as the ESKAPEE pathogens, responsible for 70% of these fatalities. Biofilm-resident bacteria, as they exist within the disease site, are canonically highly resistant to antibiotics. One strategy to counter AMR and improve disease resolution involves developing methods to disrupt biofilms. These methods aim to release bacteria from the protective biofilm matrix to facilitate their killing by antibiotics or immune effectors. Several laboratories working on such strategies have demonstrated that bacteria newly released from a biofilm display a transient phenotype of significantly increased susceptibility to antibiotics. Similarly, we developed an antibody-based approach for biofilm disruption directed against the two-membered DNABII family of bacterial DNA-binding proteins, which serve as linchpins to stabilize the biofilm matrix. The incubation of biofilms with α-DNABII antibodies rapidly collapses them to induce a population of newly released bacteria (NRel). Methods In this study, we used a humanized monoclonal antibody (HuTipMab) directed against protective epitopes of a DNABII protein to determine if we could disrupt biofilms formed by the high-priority ESKAPEE pathogens as visualized by confocal laser scanning microscopy (CLSM) and COMSTAT2 analysis. Then, we demonstrated the potentiated killing of the induced NRel by seven diverse classes of traditional antibiotics by comparative plate count. Results To this end, ESKAPEE biofilms were disrupted by 50%−79% using a single tested dose and treatment period with HuTipMab. The NRel of each biofilm were significantly more sensitive to killing than their planktonically grown counterparts (heretofore, considered to be the most sensitive to antibiotic-mediated killing), even when tested at a fraction of the MIC (1/250–1/2 MIC). Moreover, the bacteria that remained within the biofilms of two representative ESKAPEE pathogens after HuTipMab disruption were also significantly more susceptible to killing by antibiotics. Discussion New data presented in this study support our continued development of a combinatorial therapy wherein HuTipMab is delivered to a patient with recalcitrant disease due to an ESKAPEE pathogen to disrupt a pathogenic biofilm, along with a co-delivered dose of an antibiotic whose ability to rapidly kill the induced NRel has been demonstrated. This novel regimen could provide a more successful clinical outcome to those with chronic, recurrent, or recalcitrant diseases, while limiting further contribution to AMR.

Bacterial biofilms contribute significantly to pathogenesis, recurrence and/or chronicity of the majority of bacterial diseases due to their notable recalcitrance to clearance. Herein, we examined kinetics of the enhanced sensitivity of nontypeable Haemophilus influenzae (NTHI) newly released (NRel) from biofilm residence by a monoclonal antibody against a bacterial DNABII protein (α-DNABII) to preferential killing by a β-lactam antibiotic. This phenotype was detected within 5 min and lasted for ~ 6 h. Relative expression of genes selected due to their known involvement in sensitivity to a β-lactam showed transient up-regulated expression of penicillin binding proteins by α-DNABII NTHI NRel, whereas there was limited expression of the β-lactamase precursor. Transient down-regulated expression of mediators of oxidative stress supported similarly timed vulnerability to NADPH-oxidase sensitive intracellular killing by activated human PMNs. Further, transient up-regulated expression of the major NTHI porin aligned well with observed increased membrane permeability of α-DNABII NTHI NRel, a characteristic also shown by NRel of three additional pathogens. These data provide mechanistic insights as to the transient, yet highly vulnerable, α-DNABII NRel phenotype. This heightened understanding supports continued validation of this novel therapeutic approach designed to leverage knowledge of the α-DNABII NRel phenotype for more effective eradication of recalcitrant biofilm-related diseases.

Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of the human airway. We report high-depth coverage RNA-Seq data from prototype NTHi strains 723 and R2866, encoding two of the most common phase-variable ModA alleles found in NTHi strains, ModA2 and ModA10, respectively.

Objectives: Structural or mucus hypersecretory pulmonary diseases such as cystic fibrosis (CF), wherein viscous mucus accumulates and clearance functions are impaired, predispose people to lung infection by inhaled bacteria that form biofilm aggregates. Nontuberculous mycobacteria (NTM), primarily Mycobacterium abscessus and Mycobacterium avium, are the growing cause of these lung infections and are extremely challenging to treat due to antibiotic recalcitrance. Better therapeutic approaches are urgently needed. We developed a humanized monoclonal antibody (HuTipMab) directed against a biofilm structural linchpin, the bacterial DNABII proteins, that rapidly disrupts biofilms and generates highly vulnerable newly released bacteria (NRel). Methods: HuTipMab's ability to recognize HupB, NTM's DNABII homologue was determined by ELISA. Relative ability of HuTipMab to disrupt biofilms formed by lab-passaged and clinical isolates of NTM was assessed by CLSM. Relative sensitivity of NTM NRel to antibiotic killing compared to when grown planktonically was evaluated by plate count. Results: HuTipMab recognized HupB and significantly disrupted NTM biofilms in a time- and dose-dependent manner. Importantly, NTM NRel of lab-passaged and clinical isolates were now highly sensitive to killing by amikacin and azithromycin. Conclusions: If successful, this combinatorial treatment strategy would empower existing antibiotics to more effectively kill NTM newly released from a biofilm by HuTipMab and thereby both improve clinical outcomes and perhaps decrease length of antibiotic treatment for people that are NTM culture-positive.

OBJECTIVES:We examined sinus mucosal samples recovered from pediatric chronic rhinosinusitis (CRS) patients for the presence of Z-form extracellular DNA (eDNA) due to its recently elucidated role in pathogenesis of disease. Further, we immunolabeled these specimens for the presence of both members of the bacterial DNA-binding DNABII protein family, integration host factor (IHF) and histone-like protein (HU), due to their known role in converting common B-DNA to the rare Z-form. METHODS:Sinus mucosa samples recovered from 20 patients during functional endoscopic sinus surgery (FESS) were immunolabelled for B- and Z-DNA, as well as for both bacterial DNABII proteins. RESULTS:Nineteen of 20 samples (95%) included areas rich in eDNA, with the majority in the Z-form. Areas positive for B-DNA were restricted to the most distal regions of the mucosal specimen. Labeling for both DNABII proteins was observed on B- and Z-DNA, which aligned with the role of these proteins in the B-to-Z DNA conversion. CONCLUSIONS:Abundant Z-form eDNA in culture-positive pediatric CRS samples suggested that bacterial DNABII proteins were responsible for the conversion of eukaryotic B-DNA that had been released into the luminal space by PMNs during NETosis, to the Z-form. The presence of both DNABII proteins on B-DNA and Z-DNA supported the known role of these bacterial proteins in the B-to-Z DNA conversion. Given that Z-form DNA both stabilizes the bacterial biofilm and inactivates PMN NET-mediated killing of trapped bacteria, we hypothesize that this conversion may be contributing to the chronicity and recalcitrance of CRS to treatment. LEVEL OF EVIDENCE:NA Laryngoscope, 134:1564-1571, 2024.

BackgroundNontypeable Haemophilus influenzae (NTHi) is the most common bacterial otopathogen associated with otitis media (OM). NTHi persists in biofilms within the middle ears of children with chronic and recurrent OM. Australian Aboriginal children suffer exceptionally high rates of chronic and recurrent OM compared to non-Aboriginal children. NTHi protein vaccines comprised of antigens associated with both adhesion and persistence in a biofilm are under development and could be beneficial for children with chronic and recurrent OM. Understanding the ontogeny of natural antibody development to these antigens provides insight into the value of vaccinating with particular antigens.MethodsAn in-house multiplex fluorescent bead immunoassay was used to measure serum IgG titres and avidity for three putative vaccine antigens: recombinant soluble PilA (rsPilA), ChimV4, and outer membrane protein 26 (OMP26) in sera from Australian Aboriginal otitis-prone children (n=77), non-Aboriginal otitis-prone children (n=70) and non-otitis-prone children (n=36). Serum IgG titres were adjusted for age, and geometric mean concentrations (GMCs) were compared between groups using a univariate analysis model. Antibody avidity was calculated as a relative avidity index and compared between groups using ANOVA.ResultsAustralian Aboriginal otitis-prone children had lower serum IgG titres to rsPilA and ChimV4 than non-Aboriginal otitis-prone children (p<0.001), and non-otitis-prone children (p<0.020). No differences were observed between serum IgG titres from non-Aboriginal otitis-prone children and non-otitis-prone children. There were also no differences in the proportion of high avidity IgG specific for these antigens between these groups. Serum IgG titres to OMP26 were similar between all groups (p>0.670) although otitis-prone children had a higher proportion of high avidity antibodies to this antigen.ConclusionsAustralian Aboriginal otitis-prone children had lower serum IgG titres to 2/3 major NTHi vaccine candidate antigens, suggesting these children are unable to develop persistent IgG responses due to repeated NTHi exposure. These reduced IgG titres may relate to earlier and more frequent exposure to diverse NTHi strains in Aboriginal children through carriage or infection. These data suggest that Aboriginal children may benefit from immunisation with vaccines containing these antigens to increase titres of protective antibodies.

New strategies to treat diseases in which biofilms contribute significantly to pathogenesis are needed, as biofilm-resident bacteria are highly recalcitrant to antibiotics due to physical biofilm architecture and a canonically quiescent metabolism, among many additional attributes. We, and others, have shown that when biofilms are dispersed or disrupted, bacteria released from biofilm residence are in a distinct physiologic state that, in part, renders these bacteria highly sensitive to killing by specific antibiotics.

Bacterial biofilms contribute significantly to the antibiotic resistance, pathogenesis, chronicity and recurrence of bacterial infections. Critical to the stability and survival of extant biofilms is the extracellular DNA (eDNA)-dependent matrix which shields the resident bacteria from hostile environments, allows a sessile metabolic state, but also encourages productive interactions with biofilm-inclusive bacteria. Given the importance of the eDNA, approaches to this area of research have been to target not just the eDNA, but also the additional constituent structural components which appear to be widespread. Chief among these is a ubiquitous two-member family of bacterial nucleoid associated proteins (the DNABII proteins) responsible for providing structural integrity to the eDNA and thereby the biofilm. Moreover, this resultant novel eDNA-rich secondary structure can also be targeted for disruption. Here, we provide an overview of both what is known about the eDNA-dependent matrix, as well as the resultant means that have resulted in biofilm resolution. Results obtained to date have been highly supportive of continued development of DNABII-targeted approaches, which is encouraging given the great global need for improved methods to medically manage, or ideally prevent biofilm-dependent infections, which remains a highly prevalent burden worldwide.