Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media.

Otitis media (OM) is a polymicrobial disease wherein prior or concurrent infection with an upper respiratory tract virus plays an essential role, predisposing the middle ear to bacterial invasion. In episodes of acute bacterial OM, respiratory syncytial virus (RSV) is the most commonly isolated virus and thus serves as an important co-pathogen. Of the predominant bacterial agents of OM, the pathogenesis of disease due to Moraxella catarrhalis is the least well understood. Rigorous study of M. catarrhalis in the context of OM has been significantly hindered by lack of an animal model. To bridge this gap, we assessed whether co-infection of chinchillas with M. catarrhalis and RSV would facilitate ascension of M. catarrhalis from the nasopharynx into the middle ear. Chinchillas were challenged intranasally with M. catarrhalis followed 48 hours later by intranasal challenge with RSV. Within 7 days, 100% of nasopharynges were colonized with M. catarrhalis and homogenates of middle ear mucosa were also culture-positive. Moreover, within the middle ear space, the mucosa exhibited hemorrhagic foci, and a small volume of serosanguinous effusion was present in one of six ears. To improve upon this model, and based on epidemiologic data, nontypeable Haemophilus influenzae (NTHI) was included as an additional bacterial co-pathogen via intranasal administration four days before M. catarrhalis challenge. With this latter protocol, M. catarrhalis was cultured from the nasopharynx and middle ear homogenates of a maximum of 88% and 79% animals, respectively, for up to 17 days after intranasal challenge with M. catarrhalis. Additionally, hemorrhagic foci were observed in 79% of middle ears upon sacrifice. Thus, these data demonstrated that co-infection with RSV and NTHI predisposed to M. catarrhalis-induced ascending experimental OM. This model can be used both in studies of pathogenesis as well as to investigate strategies to prevent or treat OM due to M. catarrhalis

Disruption of pre-formed <i>B. cenocepacia</i> biofilms by incubation with antiserum directed against IHF.

<p><i>B. cenocepacia</i> biofilms after 24 hr. growth in a chamber slide, then treated for 16 hrs with: <b>Panel A</b>-sterile medium. <b>Panel B</b> – naive rabbit serum. <b>Panel C</b> –rabbit antiserum directed against isolated IHF. <b>Panel D –</b>IgG enriched anti-IHF. <b>Panel E</b> –serum effluent from enrichment column. <b>Panel F</b> – Western blot showing recognition of antibody directed against IHF as well as IgG-enriched anti-IHF to purified IHF and IHF within <i>B. cenocepacia</i> whole cell lysate. Red arrows indicate recognition of the monomer form of IHF in each blot. Moreover, each serum fraction recognized the di- and tri-meric forms of IHF within whole cell lysates of <i>B. cenocepacia</i> strain K56-2. <b>Panel G</b> – plot of changes in average biofilm thickness ± SEM following each of the indicated treatments. <b>Panel H</b> – plot of changes in biofilm biomass ± SEM following each of the indicated treatments. Note that statistically significant disruption of pre-formed <i>B. cenocepacia</i> biofilms is mediated only by rabbit anti-IHF serum as well as a fraction of that serum that was enriched for IgG when compared to treatment with sterile medium, naive rabbit serum or serum effluent from IgG enrichment column. Asterisks indicate statistical significance (<i>p</i> < 0.05) compared to sterile medium, naive serum and enrichment column effluent.</p

Expression of a robust <i>B. cenocepacia</i> biofilm that incorporates IHF was dependent upon an active T6SS.

<p><b>Panel A</b> - Biofilm formed by the parental isolate (<i>B. cenocepacia</i> strain K56-2). <b>Panel B</b> - biofilm formed by the type III secretion system mutant (strain JRL2) stained with propidium iodide. <b>Panel C</b> – biofilm formed by the type VI secretion system mutant (strain DFA2) stained with propidium iodide. All biofilms were labeled for the presence of a DNABII protein (IHF) – see yellow color in images. Whereas biofilms formed by either secretion system mutant were notably less robust than that formed by the parental isolate, note marked reduction in yellow labeling of biofilm formed by the T6SS mutant (see Panel C), which suggested that the T6SS mutant was compromised in its ability to incorporate IHF into biofilms formed under these conditions. <b>Panel</b> D-IHF bound DNase footprint of the intergenic space (386 bp) between BCAL0339 and BCAL0340, part of the T6SS gene cluster for <i>B. cenocepacia</i>. The IHF footprint covers the region from 25 bp to 52 bp upstream of the BACL0340 start codon while a putative promoter was found 75 bp to 104 bp upstream of the start codon. HSS: hyper-sensitive site indicative of DNA bending. This observation suggested that IHF might self-regulate its own release as well as perhaps that of eDNA that is incorporated into the biofilm matrix.</p

Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits.

Uropathogenic Escherichia coli (UPEC) utilizes a complex community-based developmental pathway for growth within superficial epithelial cells of the bladder during cystitis. Extracellular DNA (eDNA) is a common matrix component of organized bacterial communities. Integration host factor (IHF) is a heterodimeric protein that binds to double-stranded DNA and produces a hairpin bend. IHF-dependent DNA architectural changes act both intrabacterially and extrabacterially to regulate gene expression and community stability, respectively. We demonstrate that both IHF subunits are required for efficient colonization of the bladder, but are dispensable for early colonization of the kidney. The community architecture of the intracellular bacterial communities (IBCs) is quantitatively different in the absence of either IhfA or IhfB in the murine model for human urinary tract infection (UTI). Restoration of Type 1 pili by ectopic production does not restore colonization in the absence of IhfA, but partially compensates in the absence of IhfB. Furthermore, we describe a binding site for IHF that is upstream of the operon that encodes for the P-pilus. Taken together, these data suggest that both IHF and its constituent subunits (independent of the heterodimer), are able to participate in multiple aspects of the UPEC pathogenic lifestyle, and may have utility as a target for treatment of bacterial cystitis

Demonstration of the synergistic behavior of antibodies directed against IHF in combination with antibiotics.

<p><b>Panels A-C</b> - An untreated <i>B. cenocepacia</i> biofilm. <b>Panels D–F</b> – a <i>B. cenocepacia</i> biofilm after treatment with a 1:50 dilution of anti-IHF. <b>Panels G-I</b> – a <i>B. cenocepacia</i> biofilm after treatment with the MIC of ceftazidime (16 µg/ml). <b>Panels J-L</b> – a <i>B. cenocepacia</i> biofilm after treatment with a combination of anti-IHF plus ceftazidime at the noted MIC. Note marked reduction of biofilm height and notably increased killing of <i>B. cenocepacia</i> when treated with both anti-IHF and ceftazidime compared to treatment with antibiotic alone (indicated by red/orange color in second row of images; compare Panels H and K). Panel M – average thickness of biofilms after treatment with antibiotic or antibiotic plus anti-IHF ± SEM. Panel N – Biofilm biomass following incubation with antibiotic or antibiotic plus anti-IHF ± SEM. Asterisks indicate statistical significance between designated pairs (<i>p</i>< 0.05). Note significant reduction in both average biofilm thickness and biomass as mediated by a combination of anti-IHF serum plus ceftazidime, ciprofloxacin, imipenem and minocycline.</p

Induction of a more robust<i>B. cenocepacia</i> biofilm following exposure to Pulmozyme (DNase).

<p><b>Panel A</b> – Treatment of a 24 hr <i>B. cenocepacia</i> biofilm with saline diluent alone. <b>Panel B</b> - Treatment of a 24 hr. <i>B. cenocepacia</i> biofilm with Pulmozyme (DNase) induced the formation of a markedly denser and thicker biofilm than that treated with saline diluent alone (compare panels A and B). <b>Panel C</b> – treatment of a 24 hr <i>B. cenocepacia</i> biofilm with both Pulmozyme and anti-IHF. Biofilms were stained for viability and pseudocolored white (live cells) and blue (dead cells) and demonstrate minimal bacterial death upon any treatment. Mean relative biofilm thickness ± SD and biomass ± SEM are depicted graphically in <b>Panels E & F</b>. Asterisk indicates significantly thicker biofilm after exposure to Pulmozyme compared to treatment with saline diluent (<i>p</i>< 0.05).</p

Labeling of IHF and eDNA within sputum collected from a CF patient culture-positive for <i>B. cenocepacia</i>.

<p>Immunolabeled light micrograph demonstrates heavy labeling of both eDNA (white strands) and the DNABII protein IHF (punctate red labeling; see yellow arrows) within a sputum sample recovered from a CF patient infected with <i>B. cenocepacia</i>. As previously shown with nontypeable <i>Haemophilus influenzae</i>-formed biofilms, in those formed by <i>B. cenocepacia</i>, junctions where strands of bacterial eDNA cross label strongly for the presence of IHF, suggesting their role in maintaining the structural scaffolding of these biofilms. Scale bar equals 5 µm.</p

Structural Stability of <i>Burkholderia cenocepacia</i> Biofilms Is Reliant on eDNA Structure and Presence of a Bacterial Nucleic Acid Binding Protein

<div><p>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 <i>Burkholderia cenocepacia</i> 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 <i>B. cenocepacia</i> biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of <i>B. cenocepacia</i> 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 <i>B. cenocepacia</i> biofilm was combined with exposure to traditional antibiotics, <i>B. cenocepacia</i> resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of <i>B. cenocepacia</i> with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested <i>B. cenocepacia</i>, resulted in a statistically significant increase in killing of phagocytized <i>B. cenocepacia</i>. 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 <i>B. cenocepacia</i>.</p> </div

Challenge protocol time table.

<p>Protocols to assess the ability of <i>M. catarrhalis</i> to induce OM in RSV-compromised juvenile chinchillas. For both protocols, nasopharyngeal mucosae, Eustachian tubes and middle ear mucosae were collected for culture at the time of sacrifice. Bullar washes were performed upon bisection of the bullae to recover planktonic <i>M. catarrhalis</i>.</p

<i>M. catarrhalis</i> was detected adherent to multiple mucosal tissues.

<p>Expanded study: Percentage of mucosal sites that were culture-positive for <i>M. catarrhalis. M. catarrhalis</i> was detected in the middle ear as early as seven days and up to 17 days after intranasal challenge with <i>M. catarrhalis</i>.</p