Bacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

ABSTRACT:Human infections caused by pathogens transmi�ed from fish or the aquatic environment are quite common and depend on the season, patients' contact with fish and related environment, dietary habits and the immune system status of the exposed individual. They are o�en bacterial species facultatively pathogenic for both fish and human beings and may be isolated from fish without apparent symptoms of the disease. The infection source may be fish kept for both for food and as a hobby.

Summary The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice-like network wherein a member of the DNABII family of DNA-binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using nontypeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF-specific and mediated by binding of transiently dissociated IHF by anti-IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA-binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.

Haemophilus influenzae is a gram-negative pleiomorphic bacterium that is a common commensal/mutualist within the human airways (30). Encapsulated H. influenzae strains are overt pathogens causing invasive disease (3) and have largely been contained by a vaccine effective against the predominant capsular serotype b strains (32). In contrast, the so-called nontypeable H. influenzae (NTHi) strains lacking capsular polysaccharides remain predominant in asymptomatic carriage and localized airway infections (14, 29). These infections are mostly opportunistic in nature and include bronchiopneumonia, sinusitis, and otitis media (OM). OM is among the most common pediatric infections, causing an estimated ϳ$5 billion in costs of treatment and parents' missed work days per year (20). OM infections include chronic OM that is difficult to resolve with antibiotic therapy, and it has long been postulated that chronic OM involves the formation of bacterial biofilm communities (5, 35). In support of that hypothesis, biofilms have been visualized in tympanostomy drain tubes removed from patients with OM and on middle ear tissue from experimentally infected chinchillas (7,18,33). More recent evidence shows that NTHi and other bacterial agents are present within biofilms on tissue specimens obtained from patients with chronic and recurrent OM (13).The H. influenzae surface is covered with lipooligosaccharide (LOS) endotoxins that lack a repeating O side chain. Instead, the H. influenzae LOS features a diverse collection of LOS glycoforms that differ in the length, content, and nature of the chemical linkages found in the oligosaccharide portion. These LOS oligosaccharides include structures that are antigenically similar to host cell-surface glycolipids and may also contain the host membrane constituents sialic acid (NeuAc) and phosphorylcholine (PCho) (41). LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55). In this study, we compared the virulence

SummaryWe recently described the expression of type IV pili (Tfp) by non-typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase-producing NTHI in infected chinchillas. Luciferase-producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

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.

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.