Publications by authors named "Steven D Goodman"

62 Publications

in Its Biofilm State Improves Protection from Experimental Necrotizing Enterocolitis.

Nutrients 2021 Mar 12;13(3). Epub 2021 Mar 12.

Department of Pediatric Surgery, Nationwide Children's Hospital, The Ohio State University College of Medicine, Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43205, USA.

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 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.
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http://dx.doi.org/10.3390/nu13030918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000340PMC
March 2021

The dental plaque biofilm matrix.

Periodontol 2000 2021 Mar 10. Epub 2021 Mar 10.

Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany.

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.
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http://dx.doi.org/10.1111/prd.12361DOI Listing
March 2021

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

Laryngoscope 2021 Mar 5. Epub 2021 Mar 5.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, U.S.A.

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, 2021.
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http://dx.doi.org/10.1002/lary.29497DOI Listing
March 2021

Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota.

PLoS One 2021 16;16(2):e0246193. Epub 2021 Feb 16.

Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America.

Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246193PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886176PMC
February 2021

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

PLoS Pathog 2021 01 19;17(1):e1009209. Epub 2021 Jan 19.

Center for Microbial Pathogenesis, Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.
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http://dx.doi.org/10.1371/journal.ppat.1009209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815147PMC
January 2021

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

Biofilm 2020 Dec 18;2:100039. Epub 2020 Nov 18.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.

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 (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.
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http://dx.doi.org/10.1016/j.bioflm.2020.100039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798465PMC
December 2020

Measurement of uracil-DNA glycosylase activity by matrix assisted laser desorption/ionization time-of-flight mass spectrometry technique.

DNA Repair (Amst) 2021 Jan 24;97:103028. Epub 2020 Nov 24.

Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, ROC. Electronic address:

Uracil-DNA glycosylase (UDG) is a highly conserved DNA repair enzyme that acts as a key component in the base excision repair pathway to correct hydrolytic deamination of cytosine making it critical to genome integrity in living organisms. We report here a non-labeled, non-radio-isotopic and very specific method to measure UDG activity. Oligodeoxyribonucleotide duplex containing a site-specific G:U mismatch that is hydrolyzed by UDG then subjected to Matrix Assisted Laser Desorption/Ionization time-of-flight mass spectrometry analysis. A protocol was developed to maintain the AP product in DNA without strand break then the cleavage of uracil was identified by the mass change from uracil substrate to AP product. From UDG kinetic analysis, for G:U substrate the K is 50 nM, V is 0.98 nM/s and K = 9.31 s. The method was applied to uracil glycosylase inhibitor measurement with an IC value of 7.6 pM. Single-stranded and double-stranded DNAs with uracil at various positions of the substrates were also tested for UDG activity albeit with different efficiencies. The simple, rapid, quantifiable, scalable and versatile method has potential to be the reference method for monofunctional glycosylase measurement, and can also be used as a tool for glycosylase inhibitors screening.
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http://dx.doi.org/10.1016/j.dnarep.2020.103028DOI Listing
January 2021

A novel probiotic therapeutic in a murine model of colitis.

Gut Microbes 2020 11;12(1):1814119

Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Department of Pediatric Surgery, Nationwide Children's Hospital , Columbus, OH, USA.

For prophylactic therapy, mice received an oral antibiotic cocktail followed by clindamycin injection, followed by probiotic administration (planktonic . biofilm state), followed by oral gavage. For treatment therapy, mice received antibiotics and first, followed by probiotic administration. Clinical sickness scores (CSS) and intestinal histologic injury scores (HIS) were assigned.

In the model, CSS: 67% of untreated mice exposed to demonstrated CSS ≥ 6, which is consistent with infection (< .001 compared to unexposed mice). In mice treated with planktonic , 55% had a CSS ≥ 6, but only 19% of mice treated with in its biofilm state had CSS ≥ 6 (< .001). Mice receiving + DM-Maltose lost the least amount of weight compared to mice receiving saline ( = .004676) or to mice receiving (= .003185). HIS: 77% of untreated mice exposed to had HIS scores ≥4, which is consistent with infection. In mice treated with planktonic , 62% had HIS ≥4, but only 19% of mice treated with in its biofilm state had HIS ≥4. (< .001). Additionally, mice treated with in its biofilm state had better survival compared to untreated mice and to mice treated with planktonic ( ≤ 0.05). Similar findings for weight loss, CSS, HIS and survival were obtained for .

A single dose of in its biofilm state reduces the severity and incidence of experimental infection when administered as both prophylactic and treatment therapy.
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http://dx.doi.org/10.1080/19490976.2020.1814119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524353PMC
November 2020

Targeting a bacterial DNABII protein with a chimeric peptide immunogen or humanised monoclonal antibody to prevent or treat recalcitrant biofilm-mediated infections.

EBioMedicine 2020 Sep 7;59:102867. Epub 2020 Jul 7.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, 700 Children's Drive, W591, Columbus, OH 43205 USA; The Ohio State University College of Medicine, 700 Children's Drive, Columbus, OH 43210 USA. Electronic address:

Background: Chronic and recurrent bacterial diseases are recalcitrant to treatment due to the ability of the causative agents to establish biofilms, thus development of means to prevent or resolve these structures are greatly needed. Our approach targets the DNABII family of bacterial DNA-binding proteins, which serve as critical structural components within the extracellular DNA scaffold of biofilms formed by all bacterial species tested to date. DNABII-directed antibodies rapidly disrupt biofilms and release the resident bacteria which promote their subsequent clearance by either host immune effectors or antibiotics that are now effective at a notably reduced concentration.

Methods: First, as a therapeutic approach, we used intact IgG or Fab fragments against a chimeric peptide immunogen designed to target protective epitopes within the DNA-binding tip domains of integration host factor to disrupt established biofilms in vitro and to mediate resolution of existing disease in vivo. Second, we performed preventative active immunisation with the chimeric peptide to induce the formation of antibody that blocks biofilm formation and disease development in a model of viral-bacterial superinfection. Further, toward the path for clinical use, we humanised a monoclonal antibody against the chimeric peptide immunogen, then characterised and validated that it maintained therapeutic efficacy.

Findings: We demonstrated efficacy of each approach in two well-established pre-clinical models of otitis media induced by the prevalent respiratory tract pathogen nontypeable Haemophilus influenzae, a common biofilm disease.

Interpretation: Collectively, our data revealed two approaches with substantive efficacy and potential for broad application to combat diseases with a biofilm component.

Funding: Supported by R01 DC011818 to LOB and SDG.
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http://dx.doi.org/10.1016/j.ebiom.2020.102867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502671PMC
September 2020

Immunization with a Biofilm-Disrupting Nontypeable Vaccine Antigen Did Not Alter the Gut Microbiome in Chinchillas, Unlike Oral Delivery of a Broad-Spectrum Antibiotic Commonly Used for Otitis Media.

mSphere 2020 04 15;5(2). Epub 2020 Apr 15.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA

The use of broad-spectrum antibiotics to treat diseases, such as the highly prevalent pediatric disease otitis media (OM), contributes significantly to the worldwide emergence of multiple-antibiotic-resistant microbes, and gut dysbiosis with diarrhea is a common adverse sequela. Moreover, for many diseases, like OM, biofilms contribute significantly to chronicity and recurrence, yet biofilm-resident bacteria are characteristically highly resistant to antibiotics. The most cost-effective way to both prevent and resolve diseases like OM, as well as begin to address the problem of growing antibiotic resistance, would be via the development of novel approaches to eradicate bacterial biofilms. Toward this goal, we designed a vaccine antigen that induces the formation of antibodies that prevent biofilm formation and, thereby, experimental OM in the middle ears of chinchillas by the predominant Gram-negative pathogen responsible for this disease, nontypeable These antibodies also significantly disrupt preexisting biofilms formed by diverse pathogens. Whereas preclinical data strongly support the continued development of this vaccine antigen, which targets an essential structural element of bacterial biofilms, a concern has been whether active immunization would also lead to unintended collateral damage in the form of an altered gut microbiome. To address this concern, we assessed changes in the microbiome of the chinchilla gut over time after the delivery of either amoxicillin-clavulanate, the standard of care for OM, or after immunization with our biofilm-targeted vaccine antigen either via a traditional subcutaneous route or via a novel noninvasive transcutaneous route. We show that differences in the abundance of specific taxa were found only in the stools of antibiotic-treated animals. The prevalence of chronic and recurrent diseases, combined with the overuse/abuse of antibiotics that has led to the sobering emergence of bacteria resistant to multiple antibiotics, has mandated that we develop novel approaches to better manage these diseases or, ideally, prevent them. Biofilms play a key role in the pathogenesis of chronic and recurrent bacterial diseases but are difficult, if not impossible, to eradicate with antibiotics. We developed a vaccine antigen designed to mediate biofilm disruption; however, it is also important that delivery of this vaccine does not induce collateral damage to the microbiome. The studies described here validated a vaccine approach that targets biofilms without the consequences of an altered gut microbiome. While delivery of the antibiotic most commonly given to children with ear infections did indeed alter the gut microbiome, as expected, immunization via traditional injection or by noninvasive delivery to the skin did not result in changes to the chinchilla gut microbiome.
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http://dx.doi.org/10.1128/mSphere.00296-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160684PMC
April 2020

Proofreading of single nucleotide insertion/deletion replication errors analyzed by MALDI-TOF mass spectrometry assay.

DNA Repair (Amst) 2020 04 30;88:102810. Epub 2020 Jan 30.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, 10002, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, 10063, Taipei, Taiwan, ROC. Electronic address:

Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1-5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3' end are partially corrected and extended. Indels located 7-9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.
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http://dx.doi.org/10.1016/j.dnarep.2020.102810DOI Listing
April 2020

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Proc Natl Acad Sci U S A 2019 12 25;116(50):25068-25077. Epub 2019 Nov 25.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH 43205;

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic , nontypeable influenzae, and Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.
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http://dx.doi.org/10.1073/pnas.1909017116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911203PMC
December 2019

Antibodies against the DNABII protein integration host factor (IHF) inhibit sinus implant biofilms.

Laryngoscope 2020 06 17;130(6):1364-1371. Epub 2019 Jul 17.

Center for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, U.S.A.

Objectives: Chronic rhinosinusitis is a common, costly condition often treated with endoscopic sinus surgery and intraoperative placement of intranasal sinus implant materials. Whereas these materials aid in postoperative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pretreatment of sinus implant materials with antibody against an essential bacterial biofilm structural component, the DNABII family of DNA-binding proteins, as a strategy to prevent biofilm formation.

Methods: Sinus implant materials were equilibrated in immunoglobulin G (IgG)-enriched antiserum against the DNABII protein integration host factor (IHF), individually or in combination with amoxicillin-clavulanate prior to inoculation with nontypeable Haemophilus influenzae (NTHI), a predominant pathogen of chronic rhinosinusitis. After 16 hours, the bacterial burden was quantitated and compared to pretreatment with saline, IgG-enriched naive serum, or amoxicillin-clavulanate alone.

Results: NTHI readily formed biofilms on all three materials in vitro. However, pretreatment of each material with IgG-enriched anti-IHF resulted in a significant decrease in bacterial burden compared to controls (P ≤ 0.05). Moreover, a significant and synergistic outcome was achieved with a cocktail of anti-IHF plus amoxicillin-clavulanate (P ≤ 0.05) with complete inhibition of NTHI biofilm formation on all three materials.

Conclusions: Biofilm formation was well supported in vitro on three sinus implant materials that vary in composition and resorption characteristics; however, pretreatment of each with DNABII protein targeted antibodies in combination with a previously ineffective antibiotic was highly effective to prevent the formation NTHI biofilms. These data demonstrate the potential for clinical utility of pretreatment of sinus implant and additional surgical materials with anti-DNABII antibodies.

Level Of Evidence: NA Laryngoscope, 130:1364-1371, 2020.
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http://dx.doi.org/10.1002/lary.28188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980231PMC
June 2020

Identification of essential biofilm proteins in middle ear fluids of otitis media with effusion patients.

Laryngoscope 2020 03 25;130(3):806-811. Epub 2019 Apr 25.

Center for Microbial Pathogenesis, Nationwide Children's Hospital, Columbus, Ohio, U.S.A.

Objectives: Otitis media with effusion (OME) is a common disease of childhood that is largely asymptomatic. However, middle ear fluid can persist for months and negatively impact a child's quality of life. Many cases of OME remain chronic and require surgical intervention. Because biofilms are known to contribute to the persistence of many diseases, this study examined effusions collected from children with chronic OME for the presence of essential biofilm structural components, members of the DNABII family of bacterial DNA-binding proteins.

Methods: Middle ear effusions were recovered from 38 children with chronic OME at the time of tympanostomy tube insertion. A portion of each specimen was submitted for microbiology culture. The remaining material was assessed by immunoblot to quantitate individual DNABII proteins, integration host factor (IHF), and histone-like protein (HU).

Results: Sixty-five percent of effusions (24 of 37) were culture-positive for bacterial species or yeast, whereas 35% (13 of 37) were culture-negative. IHF was detected in 95% (36 of 38) at concentrations from 2 to 481 ng/μL effusion. HU was detected in 95% (36 of 38) and quantitated from 13 to 5,264 ng/μL effusion (P ≤ 0.05 compared to IHF).

Conclusion: Because DNABII proteins are essential structural components of bacterial biofilms, these data lend further support to our understanding that biofilms are present in the vast majority of chronic middle ear effusions, despite negative culture results. The presence and ubiquity of DNABII proteins in OME specimens indicated that these proteins can serve as an important clinical target for our novel DNABII-directed strategy to treat biofilm diseases such as chronic OME.

Level Of Evidence: NA Laryngoscope, 130:806-811, 2020.
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http://dx.doi.org/10.1002/lary.28011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814449PMC
March 2020

Development of a Standardized Scoring System to Assess a Murine Model of Colitis.

J Invest Surg 2020 Dec 20;33(10):887-895. Epub 2019 Mar 20.

Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Department of Pediatric Surgery, Nationwide Children's Hospital, Columbus, OH, USA.

infection is the most common cause of antimicrobial-associated diarrhea. Our aim was to introduce a novel and efficient clinical sickness score (CSS), and to define a detailed histologic injury score (HIS) in a murine model of colitis. Mice received an antibiotic cocktail (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 96 h. After 48 h, mice received an intraperitoneal injection of clindamycin, followed by oral (1.5 × 10 CFU). Signs of sickness were scored using a novel CSS (range 0-12) with scores ≥6 consistent with colitis. Intestinal tissue was analyzed utilizing an adapted HIS (range 0-9) with scores ≥4 consistent with colitis. Stool was analyzed for and survival evaluated. No control mice showed signs of sickness, whereas 23% of mice receiving antibiotics alone and 65% of mice exposed to antibiotics and subsequently demonstrated signs of sickness ( = 0.0134). No control mice had histologic injury, whereas 8% of mice receiving antibiotics alone and 75% of mice exposed to antibiotics followed by had evidence of histologic injury ( = 0.0001). Mice exposed to lost more weight, although not significant ( = 0.070). Mice that received had decreased survival compared to control mice and mice receiving antibiotics only ( = 0.03). We have developed a novel clinical scoring system, and detailed histological grading system, that enables the objective evaluation of a murine colitis model. This model allows the study of this disease in a host that demonstrates clinical and histologic signs comparable to human infection. This will allow for improved study of therapeutics for this disease in the future.
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http://dx.doi.org/10.1080/08941939.2019.1571129DOI Listing
December 2020

The conserved mosaic prophage protein paratox inhibits the natural competence regulator ComR in Streptococcus.

Sci Rep 2018 11 8;8(1):16535. Epub 2018 Nov 8.

Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.

Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become "competent" and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus.
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http://dx.doi.org/10.1038/s41598-018-34816-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224593PMC
November 2018

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis.

J Vis Exp 2018 06 19(136). Epub 2018 Jun 19.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University; Department of Laboratory Medicine, National Taiwan University Hospital;

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.
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http://dx.doi.org/10.3791/57862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101880PMC
June 2018

An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis.

Am J Physiol Gastrointest Liver Physiol 2018 09 31;315(3):G408-G419. Epub 2018 May 31.

Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Department of Pediatric Surgery, Nationwide Children's Hospital , Columbus, Ohio.

One significant drawback of current probiotic therapy for the prevention of necrotizing enterocolitis (NEC) is the need for at least daily administration because of poor probiotic persistence after enteral administration, increasing the risk of the probiotic bacteria causing bacteremia or sepsis if the intestines are already compromised. We previously showed that the effectiveness of Lactobacillus reuteri ( Lr) in preventing NEC is enhanced when Lr is grown as a biofilm on the surface of dextranomer microspheres (DM). Here we sought to test the efficacy of Lr administration by manipulating the Lr biofilm state with the addition of biofilm-promoting substances (sucrose and maltose) to DM or by mutating the Lr gtfW gene (encoding an enzyme central to biofilm production). Using an animal model of NEC, we determined that Lr adhered to sucrose- or maltose-loaded DM significantly reduced histologic injury, improved host survival, decreased intestinal permeability, reduced intestinal inflammation, and altered the gut microbiome compared with Lr adhered to unloaded DM. These effects were abolished when DM or GtfW were absent from the Lr inoculum. This demonstrates that a single dose of Lr in its biofilm state decreases NEC incidence. Importantly, preloading DM with sucrose or maltose further enhances Lr protection against NEC in a GtfW-dependent fashion, demonstrating the tunability of the approach and the potential to use other cargos to enhance future probiotic formulations. NEW & NOTEWORTHY Previous clinical trials of probiotics to prevent necrotizing enterocolitis have had variable results. In these studies, probiotics were delivered in their planktonic, free-living form. We have developed a novel probiotic delivery system in which Lactobacillus reuteri (Lr) is delivered in its biofilm state. In a model of experimental necrotizing enterocolitis, this formulation significantly reduces intestinal inflammation and permeability, improves survival, and preserves the natural gut microflora compared with the administration of Lr in its free-living form.
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http://dx.doi.org/10.1152/ajpgi.00078.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415713PMC
September 2018

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo.

DNA Repair (Amst) 2018 04 17;64:59-67. Epub 2018 Feb 17.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, 100-02, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 100-02, Taiwan, ROC. Electronic address:

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3'-5' exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA- strain, a pol I mutant defective in 3'-5' exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C-C mismatch as strand discrimination marker we found that the high level of dI removal in polA- was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5' upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.
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http://dx.doi.org/10.1016/j.dnarep.2018.02.005DOI Listing
April 2018

Targeting the HUβ Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms.

J Bacteriol 2018 06 9;200(11). Epub 2018 May 9.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA

The oral cavity is home to a wide variety of bacterial species, both commensal, such as various streptococcal species, and pathogenic, such as , one of the main etiological agents of periodontal disease. Our understanding of how these bacteria ultimately cause disease is highly dependent upon understanding how they coexist and interact with one another in biofilm communities and the mechanisms by which biofilms are formed. Our research has demonstrated that the DNABII family of DNA-binding proteins are important components of the extracellular DNA (eDNA)-dependent matrix of bacterial biofilms and that sequestering these proteins via protein-specific antibodies results in the collapse of the biofilm structure and release of the resident bacteria. While the high degree of similarity among the DNABII family of proteins has allowed antibodies derived against specific DNABII proteins to disrupt biofilms formed by a wide range of bacterial pathogens, the DNABII proteins of have proven to be antigenically distinct, allowing us to determine if we can use anti- HUβ antibodies to specifically target this species for removal from a mixed-species biofilm. Importantly, despite forming homotypic biofilms , must enter preexisting biofilms in order to persist within the oral cavity. The data presented here indicate that antibodies derived against the DNABII protein, HUβ, reduce by half the amount of organisms entering into preexisting biofilm formed by four oral streptococcal species. These results support our efforts to develop methods for preventing and treating periodontal disease. Periodontitis is one of the most prevalent chronic infections, affecting 40 to 50% of the population of the United States. The root cause of periodontitis is the presence of bacterial biofilms within the gingival space, with being strongly associated with the development of the disease. Periodontitis also increases the risk of secondary conditions and infections such as atherosclerosis and infective endocarditis caused by oral streptococci. To induce periodontitis, needs to incorporate into preformed biofilms, with oral streptococci being important binding partners. Our research demonstrates that targeting DNABII proteins with an antibody disperses oral streptococcus biofilm and prevents entry into oral streptococcus biofilm. These results suggest potential therapeutic treatments for endocarditis caused by streptococci as well as periodontitis.
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http://dx.doi.org/10.1128/JB.00790-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952391PMC
June 2018

The DNABII family of proteins is comprised of the only nucleoid associated proteins required for nontypeable Haemophilus influenzae biofilm structure.

Microbiologyopen 2018 06 12;7(3):e00563. Epub 2017 Dec 12.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital and The Ohio State University College of Medicine, Columbus, OH, USA.

Biofilms play a central role in the pathobiology of otitis media (OM), bronchitis, sinusitis, conjunctivitis, and pneumonia caused by nontypeable Haemophilus influenzae (NTHI). Our previous studies show that extracellular DNA (eDNA) and DNABII proteins are essential components of biofilms formed by NTHI. The DNABII protein family includes integration host factor (IHF) and the histone-like protein HU and plays a central role in NTHI biofilm structural integrity. We demonstrated that immunological targeting of these proteins during NTHI-induced experimental OM in a chinchilla model caused rapid clearance of biofilms from the middle ear. Given the essential role of DNABII proteins in maintaining the structure of an NTHI biofilm, we investigated whether any of the other nucleoid associated proteins (NAPs) expressed by NTHI might play a similar role, thereby serving as additional target(s) for intervention. We demonstrated that although several NAPs including H-NS, CbpA, HfQ and Dps are present within the biofilm extracellular matrix, only the DNABII family of proteins is critical for the structural integrity of the biofilms formed by NTHI. We have also demonstrated that IHF and HU are located at distinct regions within the extracellular matrix of NTHI biofilms formed in vitro, indicative of independent functions of these two proteins.
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http://dx.doi.org/10.1002/mbo3.563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011942PMC
June 2018

Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay.

DNA Repair (Amst) 2018 01 2;61:63-75. Epub 2017 Dec 2.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 10002, Taiwan, ROC; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 100-63, Taiwan, ROC. Electronic address:

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2-4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6-9-nucleotides from the primer terminus. We also subjected primers containing 3' penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.
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http://dx.doi.org/10.1016/j.dnarep.2017.11.011DOI Listing
January 2018

Nontypeable releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery.

Proc Natl Acad Sci U S A 2017 08 10;114(32):E6632-E6641. Epub 2017 Jul 10.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205;

Biofilms formed by nontypeable (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.
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http://dx.doi.org/10.1073/pnas.1705508114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559034PMC
August 2017

Enhanced Probiotic Potential of When Delivered as a Biofilm on Dextranomer Microspheres That Contain Beneficial Cargo.

Front Microbiol 2017 27;8:489. Epub 2017 Mar 27.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital Columbus, OH, USA.

As with all orally consumed probiotics, the Gram-positive bacterium encounters numerous challenges as it transits through the gastrointestinal tract of the host, including low pH, effectors of the host immune system, as well as competition with commensal and pathogenic bacteria, all of which can greatly reduce the availability of live bacteria for therapeutic purposes. Recently we showed that , when adhered in the form of a biofilm to a semi-permeable biocompatible dextranomer microsphere, reduces the incidence of necrotizing enterocolitis by 50% in a well-defined animal model following delivery of a single prophylactic dose. Herein, using the same semi-permeable microspheres, we showed that providing compounds beneficial to as diffusible cargo within the microsphere lumen resulted in further advantageous effects including glucosyltransferase-dependent bacterial adherence to the microsphere surface, resistance of bound bacteria against acidic conditions, enhanced adherence of to human intestinal epithelial cells , and facilitated production of the antimicrobial compound reuterin and the anti-inflammatory molecule histamine. These data support continued development of this novel probiotic formulation as an adaptable and effective means for targeted delivery of cargo beneficial to the probiotic bacterium.
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http://dx.doi.org/10.3389/fmicb.2017.00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366311PMC
March 2017

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model.

Clin Vaccine Immunol 2017 Jun 5;24(6). Epub 2017 Jun 5.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's and The Ohio State University College of Medicine, Columbus, Ohio, USA

Otitis media (OM) is a common pediatric disease, and nontypeable (NTHI) is the predominant pathogen in chronic OM, recurrent OM, and OM associated with treatment failure. OM is also a polymicrobial disease, wherein an upper respiratory tract viral infection predisposes to ascension of NTHI from the nasopharynx, the site of colonization, to the normally sterile middle ear, resulting in disease. Using a clinically relevant viral-bacterial coinfection model of NTHI-induced OM, we performed transcutaneous immunization (TCI) via a band-aid delivery system to administer each of three promising NTHI vaccine candidates derived from bacterial adhesive proteins and biofilm mediators: recombinant soluble PilA (rsPilA), chimV4, and integration host factor. Each immunogen was admixed with the adjuvant LT(R192G/L211A), a double mutant of heat-labile enterotoxin, and assessed for relative ability to prevent the onset of experimental OM. For each cohort, the presence of circulating immunogen-specific antibody-secreting cells and serum antibody was confirmed prior to intranasal NTHI challenge. After bacterial challenge, blinded video otoscopy and tympanometry revealed a significant reduction in the proportion of animals with signs of OM compared to levels in animals receiving adjuvant only, with an overall vaccine efficacy of 64 to 77%. These data are the first to demonstrate the efficacy afforded by TCI with a band-aid vaccine delivery system in a clinically relevant polymicrobial model of OM. The simplicity of TCI with a band-aid and the significant efficacy observed here hold great promise for reducing the global burden of OM in the pediatric population.
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http://dx.doi.org/10.1128/CVI.00563-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461379PMC
June 2017

Identification of biofilms in post-tympanostomy tube otorrhea.

Laryngoscope 2016 08 4;126(8):1946-51. Epub 2016 Jan 4.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital.

Objectives/hypothesis: Otitis media is a common problem in the pediatric population. Despite antibiotic therapy, post-tympanostomy otorrhea can be difficult to treat. Biofilms have been shown to play a role in chronic and recurrent otitis media and are implicated in otorrhea. This study investigated both the microbial composition and the presence of biofilm fragments rich in extracellular DNA (eDNA) and the bacterial DNA-binding protein, integration host factor (IHF), in post-tympanostomy tube otorrhea.

Study Design: Clinical samples.

Methods: Institutional review board approval was obtained, and samples were recovered from pediatric patients with tympanostomy tubes and persistent otorrhea for both microbial culture and biofilm analysis. For biofilm assessment, frozen samples were sectioned and then labeled using a rabbit anti-IHF, which was detected with goat anti-rabbit IgG conjugated to AlexaFluor 594. Samples were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) to detect DNA, and images were captured by inverted light microscopy.

Results: Of 15 pediatric otorrhea samples analyzed, nine (60%) contained solids that were positive for labeling of IHF in association with a lattice of eDNA, and 75% yielded positive bacterial cultures. Bacterial culture results included H. influenzae, Methicillin-resistant Staphylococcus aureus, S. pneumoniae, M. catarrhalis, and P. aeruginosa.

Conclusion: Positive labeling of otorrhea solids for eDNA and IHF, in combination with microbiological culture results, indicated that biofilms likely played a key role in chronic otorrhea. Moreover, as a known critical structural component of biofilms, these findings suggest that DNABII proteins in association with eDNA may serve as an important therapeutic target in post-tympanostomy tube otorrhea.

Level Of Evidence: NA. Laryngoscope, 126:1946-1951, 2016.
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http://dx.doi.org/10.1002/lary.25826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955598PMC
August 2016

Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo.

EBioMedicine 2016 Aug 16;10:33-44. Epub 2016 Jun 16.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH, 43205, USA; The Ohio State University College of Medicine, Department of Pediatrics, 700 Children's Drive, Columbus, OH, 43205, USA. Electronic address:

The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA) which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.
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http://dx.doi.org/10.1016/j.ebiom.2016.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006588PMC
August 2016

Harvesting the benefits of biofilms: A novel probiotic delivery system for the prevention of necrotizing enterocolitis.

J Pediatr Surg 2016 Jun 2;51(6):936-41. Epub 2016 Mar 2.

Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Department of Pediatric Surgery, Nationwide Children's Hospital, Columbus, OH. Electronic address:

Background/purpose: Probiotics reduce the incidence of necrotizing enterocolitis (NEC) albeit only when administered at high frequency (at least daily). We have developed a novel probiotic delivery system in which probiotics are grown as a biofilm on microspheres, allowing enhanced efficacy with only a single treatment.

Methods: Neonatal rats were subjected to experimental NEC. Pups received a single enteral dose of: (1) vehicle only, (2) unloaded microspheres, (3) MRS (broth)-loaded microspheres, (4) Lactobacillus reuteri, (5) L. reuteri grown on unloaded microspheres, or (6) L. reuteri grown on MRS-loaded microspheres. Intestinal injury was graded histologically and intestinal permeability determined by serum levels of enterally administered fluorescein isothiocyanate-labeled dextran.

Results: 69% of untreated pups developed NEC, whereas 32% of pups treated with L. reuteri grown as a biofilm on unloaded microspheres (p=0.009) and 33% of pups treated with L. reuteri grown as a biofilm on MRS-loaded microspheres (p=0.005) developed NEC. No other group had a significant reduction in NEC. Furthermore, pups treated with L. reuteri grown as a biofilm had significantly reduced intestinal permeability.

Conclusions: A single dose of Lactobacillus biofilm grown on biocompatible microspheres significantly reduces NEC incidence and severity. This novel probiotic delivery system may be beneficial in the prevention of NEC in the future.
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http://dx.doi.org/10.1016/j.jpedsurg.2016.02.062DOI Listing
June 2016

Deoxyinosine repair in nuclear extracts of human cells.

Cell Biosci 2015 8;5:52. Epub 2015 Sep 8.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung-Shan South Road, Taipei, 10002 Taiwan ROC ; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, 10002 Taiwan ROC.

Background: Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mammalian cells appears more complex and was not fully understood.

Results: All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofactors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair.

Conclusions: A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention.
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http://dx.doi.org/10.1186/s13578-015-0044-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563847PMC
September 2015

DNABII proteins play a central role in UPEC biofilm structure.

Mol Microbiol 2015 Jun 16;96(6):1119-35. Epub 2015 Apr 16.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, OH, 43205, USA.

Most chronic and recurrent bacterial infections involve a biofilm component, the foundation of which is the extracellular polymeric substance (EPS). Extracellular DNA (eDNA) is a conserved and key component of the EPS of pathogenic biofilms. The DNABII protein family includes integration host factor (IHF) and histone-like protein (HU); both are present in the extracellular milieu. We have shown previously that the DNABII proteins are often found in association with eDNA and are critical for the structural integrity of bacterial communities that utilize eDNA as a matrix component. Here, we demonstrate that uropathogenic Escherichia coli (UPEC) strain UTI89 incorporates eDNA within its biofilm matrix and that the DNABII proteins are not only important for biofilm growth, but are limiting; exogenous addition of these proteins promotes biofilm formation that is dependent on eDNA. In addition, we show that both subunits of IHF, yet only one subunit of HU (HupB), are critical for UPEC biofilm development. We discuss the roles of these proteins in context of the UPEC EPS.
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http://dx.doi.org/10.1111/mmi.12994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464964PMC
June 2015