Corrigendum: ESKAPEE pathogens newly released from biofilm residence by a targeted monoclonal are sensitized to killing by traditional antibiotics.

[This corrects the article DOI: 10.3389/fmicb.2023.1202215.].

Oral and middle ear delivery of otitis media standard of care antibiotics, but not biofilm-targeted antibodies, alter chinchilla nasopharyngeal and fecal microbiomes.

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.

Disruption of nontuberculous mycobacteria biofilms induces a highly vulnerable to antibiotic killing phenotype.

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.

Superior performance of biofilm versus planktonic Limosilactobacillus reuteri in protection of the intestines and brain in a piglet model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related death in premature infants. Its etiology is multifactorial, with intestinal dysbiosis playing a major role. Probiotics are a logical preventative therapy for NEC, however their benefits have been inconsistent. We previously developed a novel probiotic delivery system in which planktonic (free-living) Limosilactobacillus reuteri (Lr) is incubated with biocompatible dextranomer microspheres (DM) loaded with maltose (Lr-DM-maltose) to induce biofilm formation. Here we have investigated the effects of Lr-DM-maltose in an enteral feed-only piglet model of NEC. We found a significant decrease in the incidence of Definitive NEC (D-NEC), death associated with D-NEC, and activated microglia in the brains of piglets treated with Lr-DM-maltose compared to non-treated piglets. Microbiome analyses using 16S rRNA sequencing of colonic contents revealed a significantly different microbial community composition between piglets treated with Lr-DM-maltose compared to non-treated piglets, with an increase in Lactobacillaceae and a decrease in Clostridiaceae in Lr-DM-maltose-treated piglets. Furthermore, there was a significant decrease in the incidence of D-NEC between piglets treated with Lr-DM-maltose compared to planktonic Lr. These findings validate our previous results in rodents, and support future clinical trials of Lr in its biofilm state for the prevention of NEC in premature neonates.

ESKAPEE pathogens newly released from biofilm residence by a targeted monoclonal are sensitized to killing by traditional antibiotics.

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.

Z-Form Extracellular DNA in Pediatric CRS May Provide a Mechanism for Recalcitrance to Treatment.

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: Not applicable Laryngoscope, 2023.

Nontypeable Haemophilus influenzae released from biofilm residence by monoclonal antibody directed against a biofilm matrix component display a vulnerable phenotype.

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 ß-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 ß-lactam showed transient up-regulated expression of penicillin binding proteins by α-DNABII NTHI NRel, whereas there was limited expression of the ß-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.

Development of a novel definitive scoring system for an enteral feed-only model of necrotizing enterocolitis in piglets.

Introduction: Necrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity. Methods: Premature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome. Results: Compared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae. Conclusions: We have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

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

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.

Next-Generation Probiotic Therapy to Protect the Intestines From Injury.

Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host. Some strains of the probiotic Lactobacillus reuteri (L. reuteri) have both antimicrobial and anti-inflammatory properties that may be exploited for the treatment and prevention of different gastrointestinal diseases, including necrotizing enterocolitis (NEC) and Clostridioides difficile (C. difficile) infection. Our laboratory has developed a new delivery system for L. reuteri in which the probiotic is incubated with biocompatible, semipermeable, porous dextranomer microspheres (DM) that can be loaded with beneficial and diffusible cargo. L. reuteri can be induced to form a biofilm by incubating the bacteria on the surface of these microspheres, which enhances the efficacy of the probiotic. Loading the DM with sucrose or maltose induces L. reuteri to produce more biofilm, further increasing the efficacy of the probiotic. Using a rat model of NEC, L. reuteri administered in its biofilm state significantly increases animal survival, reduces the incidence of NEC, preserves gut barrier function, and decreases intestinal inflammation. In a murine model of Clostridiodes difficile infection, L. reuteri administered in its biofilm state decreases colitis when administered either before or after C. difficile induction, demonstrating both prophylactic and therapeutic efficacy. There are currently no FDA-approved probiotic preparations for human use. An FDA-approved phase I clinical trial of L. reuteri in its biofilm state in healthy adults is currently underway. The results of this trial will be used to support a phase 1 clinical trial in neonates, with the goal of utilizing L. reuteri in its biofilm state to prevent NEC in premature neonates in the future.

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

Bacterial Biofilms Utilize an Underlying Extracellular DNA Matrix Structure That Can Be Targeted for Biofilm Resolution.

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.

A Humanized Monoclonal Antibody Potentiates Killing of Diverse Biofilm-Forming Respiratory Tract Pathogens by Antibiotics.

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. We sought to demonstrate the breadth of the ability of a recently humanized monoclonal antibody against an essential biofilm structural element (DNABII protein) to disrupt biofilms formed by respiratory tract pathogens and potentiate antibiotic-mediated killing of bacteria released from biofilm residence. Biofilms formed by six respiratory tract pathogens were significantly disrupted by the humanized monoclonal antibody in a dose- and time-dependent manner, as corroborated by confocal laser scanning microscopy (CLSM) imaging. Bacteria newly released from the biofilms of 3 of 6 species were significantly more sensitive than their planktonic counterparts to killing by 2 of 3 antibiotics currently used clinically and were now also equally as sensitive to killing by the 3rd antibiotic. The remaining 3 pathogens were significantly more susceptible to killing by all 3 antibiotics. A humanized monoclonal antibody directed against protective epitopes of a DNABII protein effectively released six diverse respiratory tract pathogens from biofilm residence in a phenotypic state that was now as, or significantly more, sensitive to killing by three antibiotics currently indicated for use clinically. These data support this targeted, combinatorial, species-agnostic therapy to mitigate chronic bacterial diseases.

Antibacterial and anti-inflammatory effects of Lactobacillus reuteri in its biofilm state contribute to its beneficial effects in a rat model of experimental necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) remains a significant surgical emergency in neonates. We have demonstrated the efficacy of Lactobacillus reuteri (Lr) in protecting against experimental NEC when administered as a biofilm by incubation with maltose loaded dextranomer microspheres. Lr possesses antimicrobial and anti-inflammatory properties. We developed mutant strains of Lr to examine the importance of its antimicrobial and anti-inflammatory properties in protecting the intestines from NEC. METHODS: Premature rat pups were exposed to hypoxia/hypothermia/hypertonic feeds to induce NEC. To examine the importance of antimicrobial reuterin and anti-inflammatory histamine, pups received either native or mutant forms of Lr, in either its planktonic or biofilm states, prior to induction of NEC. Intestinal histology was examined upon sacrifice. RESULTS: Compared to no treatment, administration of a single dose of Lr in its biofilm state significantly decreased the incidence of NEC (67% vs. 18%, p < 0.0001), whereas Lr in its planktonic state had no significant effect. Administration of reuterin-deficient or histamine-deficient forms of Lr, in either planktonic or biofilm states, resulted in significant loss of efficacy. CONCLUSION: Antimicrobial and anti-inflammatory effects of Lr contribute to its beneficial effects against NEC. This suggests that both infectious and inflammatory components contribute to the etiology of NEC.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

Lactobacillus reuteri in its biofilm state promotes neurodevelopment after experimental necrotizing enterocolitis in rats.

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature newborns with no known cure. Up to half of survivors subsequently exhibit cognitive impairment and neurodevelopmental defects. We created a novel probiotics delivery system in which the probiotic Lactobacillus reuteri (Lr) was induced to form a biofilm [Lr (biofilm)] by incubation with dextranomer microspheres loaded with maltose (Lr-DM-maltose). We have previously demonstrated that a single dose of the probiotic Lr administered in its biofilm state significantly reduces the incidence of NEC and decreases inflammatory cytokine production in an animal model of the disease. The aim of our current study was to determine whether a single dose of the probiotic Lr administered in its biofilm state protects the brain after experimental NEC. We found that rat pups exposed to NEC reached developmental milestones significantly slower than breast fed pups, with mild improvement with Lr (biofilm) treatment. Exposure to NEC had a negative effect on cognitive behavior, which was prevented by Lr (biofilm) treatment. Lr administration also reduced anxiety-like behavior in NEC-exposed rats. The behavioral effects of NEC were associated with increased numbers of activated microglia, decreased myelin basic protein (MBP), and decreased neurotrophic gene expression, which were prevented by administration of Lr (biofilm). Our data indicate early enteral treatment with Lr in its biofilm state prevented the deleterious effects of NEC on developmental impairments.

Lactobacillus reuteri in Its Biofilm State Improves Protection from Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease predominately found in premature infants that is associated with significant morbidity and mortality. Despite decades of research, medical management with broad spectrum antibiotics and bowel rest has remained relatively unchanged, with no significant improvement in patient outcomes. The etiology of NEC is multi-factorial; however, gastrointestinal dysbiosis plays a prominent role in a neonate's vulnerability to and development of NEC. Probiotics have recently emerged as a new avenue for NEC therapy. However, current delivery methods are associated with potential limitations, including the need for at least daily administration in order to obtain any improvement in outcomes. We present a novel formulation of enterally delivered probiotics that addresses the current limitations. A single enteral dose of Lactobacillus reuteri delivered in a biofilm formulation increases probiotic survival in acidic gastric conditions, increases probiotic adherence to gastrointestinal epithelial cells, and reduces the incidence, severity, and neurocognitive sequelae of NEC in experimental models.

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

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-a-day, 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. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2698-E2704, 2021.

The dental plaque biofilm matrix.

The extracellular matrix is a critical component of microbial biofilms, such as dental plaque, maintaining the spatial arrangement of cells and coordinating cellular functions throughout the structure. The extracellular polymeric substances that comprise the matrix include carbohydrates, nucleic acids, proteins, and lipids, which are frequently organized into macromolecular complexes and/or are associated with the surfaces of microbial cells within the biofilm. Cariogenic dental plaque is rich in glucan and fructan polysaccharides derived from extracellular microbial metabolism of dietary sucrose. By contrast, the matrix of subgingival dental plaque is a complex mixture of macromolecules that is still not well understood. Components of the matrix escape from microbial cells during lysis by active secretion or through the shedding of vesicles and serve to anchor microbial cells to the tooth surface. By maintaining the biofilm in close association with host tissues, the matrix facilitates interactions between microorganisms and the host. The outcome of these interactions may be the maintenance of health or the development of dental disease, such as caries or periodontitis. The matrix affords microbial cells protection against chemical and physical insults and hinders the eradication of pathogenic dental plaque. Therefore, strategies to control the matrix are critical to maintain oral health. This review discusses recent advances in our understanding of the composition, origins, and function of the dental plaque matrix, with a focus on subgingival dental plaque. New strategies to control subgingival dental plaque based on targeting the biofilm matrix are also considered.