Nontypeable Haemophilus Influenzae Biofilms Research Articles

Molecular characteristics and biofilm formation capacity of nontypeable Haemophilus influenza strains isolated from lower respiratory tract in children

With the widespread introduction of the Hib conjugate vaccine, Nontypeable Haemophilus influenzae (NTHi) has emerged as the predominant strain globally. NTHi presents a significant challenge as a causative agent of chronic clinical infections due to its high rates of drug resistance and biofilm formation. While current research on NTHi biofilms in children has primarily focused on upper respiratory diseases, investigations into lower respiratory sources remain limited. In this study, we collected 54 clinical strains of lower respiratory tract origin from children. Molecular information and drug resistance features were obtained through whole gene sequencing and the disk diffusion method, respectively. Additionally, an in vitro biofilm model was established. All clinical strains were identified as NTHi and demonstrated the ability to form biofilms in vitro. Based on scanning electron microscopy and crystal violet staining, the strains were categorized into weak and strong biofilm-forming groups. We explored the correlation between biofilm formation ability and drug resistance patterns, as well as clinical characteristics. Stronger biofilm formation was associated with a longer cough duration and a higher proportion of abnormal lung imaging findings. Frequent intake of β-lactam antibiotics might be associated with strong biofilm formation. While a complementary relationship between biofilm-forming capacity and drug resistance may exist, further comprehensive studies are warranted. This study confirms the in vitro biofilm formation of clinical NTHi strains and establishes correlations with clinical characteristics, offering valuable insights for combating NTHi infections.

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.

Respiratory quinolones can eradicate amoxicillin-induced mature biofilms and nontypeable Haemophilus influenzae in biofilms

ObjectivesBiofilm is thought to be involved in the persistent bacterial infections caused by nontypeable Haemophilus influenzae (NTHi). This study aims to evaluate the efficacy of antibiotics against NTHi biofilms. MethodsA 96-wells pin replicator assay was applied for evaluation of antimicrobial efficacies against NTHi biofilms. The NTHi IH-202 strain for the standard and 10 clinical strains were evaluated, as well as the viability of NTHi in biofilms after antimicrobial exposures. ResultsBiofilms formed by IH-202 strain accumulated during incubation. AMPC if not high concentrations, neither reduce or inhibit biofilm formation, nor eradicate matured NTHi biofilms. The NTHi in matured biofilm were alive after exposure to amoxicillin (AMPC). Even high concentration of AMPC produced live NTHi after suspension of exposure, while tosufloxacin and garenoxacin inhibited biofilm formation of NTHi and eradicated matured biofilms. The respiratory quinolones, but not AMPC, killed NTHi in biofilms even at sub-MIC. ConclusionsNTHi persists in biofilms, even after exposure to AMPC. These findings may eventually lead to a better understanding of effective use of antibiotics to eradicate NTHi growing as biofilms, or even to the development of novel therapeutic agents for treating patients with mucosal NTHi biofilm infections. Meanwhile, respiratory quinolones are attractive agents in reducing NTHi biofilm formation and destroying established biofilm.

Induction of Mucosal IgA-Mediated Protective Immunity Against Nontypeable Haemophilus influenzae Infection by a Cationic Nanogel-Based P6 Nasal Vaccine.

Nontypeable Haemophilus influenzae (NTHi) strains form a major group of pathogenic bacteria that colonizes the nasopharynx and causes otitis media in young children. At present, there is no licensed vaccine for NTHi. Because NTHi colonizes the upper respiratory tract and forms biofilms that cause subsequent infectious events, a nasal vaccine that induces NTHi-specific secretory IgA capable of preventing biofilm formation in the respiratory tract is desirable. Here, we developed a cationic cholesteryl pullulan–based (cCHP nanogel) nasal vaccine containing the NTHi surface antigen P6 (cCHP-P6) as a universal vaccine antigen, because P6 expression is conserved among 90% of NTHi strains. Nasal immunization of mice with cCHP-P6 effectively induced P6-specific IgA in mucosal fluids, including nasal and middle ear washes. The vaccine-induced P6-specific IgA showed direct binding to the NTHi via the surface P6 proteins, resulting in the inhibition of NTHi biofilm formation. cCHP-P6 nasal vaccine thus protected mice from intranasal NTHi challenge by reducing NTHi colonization of nasal tissues and eventually eliminated the bacteria. In addition, the vaccine-induced IgA bound to different NTHi clinical isolates from patients with otitis media and inhibited NTHi attachment in a three-dimensional in vitro model of the human nasal epithelial surface. Therefore, the cCHP-P6 nanogel nasal vaccine induced effective protection in the airway mucosa, making it a strong vaccine candidate for preventing NTHi-induced infectious diseases, such as otitis media, sinusitis, and pneumonia.

Non-typeable Haemophilus influenzae biofilm formation on primary human airway epithelia cultured at air-liquid interface

Introduction Primary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia on airway epithelia, resulting in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to biofilm-associated infection. Aim The development of a biologically representative co-culture model to investigate the interactions between PCD airway epithelia and NTHi biofilms. Methods Air-liquid interface (ALI)-cultured non-PCD primary nasal cells were grown in transwells. Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junction staining. Ciliary function was assessed by high-speed video microscopy at 37°C. ALI cultures were infected with a PCD NTHi isolate at multiplicity of infections (MOIs) 10, 50 or 100 and co-cultured to form biofilms over 3 days. TEER was measured pre- and post-infection, NTHi recoverability assessed by conventional culture, and biofilm formation confirmed by scanning electron microscopy (SEM). Results Epithelial culture at ALI was successful, with tight barriers being formed and normal ciliary beat pattern and frequency recorded (mean 14Hz). Following co-culture, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50 (median colony forming units, 6.8x106per cm2). SEM confirmed NTHi biofilm formation at MOI 50. Conclusions A successful NTHi biofilm co-culture model on human primary epithelia was established. This will be used with PCD epithelia to investigate host-pathogen interactions during NTHi biofilm colonisation.

The Role of Non-Typeable Haemophilus influenzae Biofilms in Chronic Obstructive Pulmonary Disease.

Non-typeable Haemophilus influenzae (NTHi) is an ubiquitous commensal-turned-pathogen that colonises the respiratory mucosa in airways diseases including Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive inflammatory syndrome of the lungs, encompassing chronic bronchitis that is characterised by mucus hypersecretion and impaired mucociliary clearance and creates a static, protective, humid, and nutrient-rich environment, with dysregulated mucosal immunity; a favourable environment for NTHi colonisation. Several recent large COPD cohort studies have reported NTHi as a significant and recurrent aetiological pathogen in acute exacerbations of COPD. NTHi proliferation has been associated with increased hospitalisation, disease severity, morbidity and significant lung microbiome shifts. However, some cohorts with patients at different severities of COPD do not report that NTHi is a significant aetiological pathogen in their COPD patients, indicating other obligate pathogens including Moraxella catarrhalis, Streptococcus pneumoniae and Pseudomonas aeruginosa as the cause. NTHi is an ubiquitous organism across healthy non-smokers, healthy smokers and COPD patients from childhood to adulthood, but it currently remains unclear why NTHi becomes pathogenic in only some cohorts of COPD patients, and what behaviours, interactions and adaptations are driving this susceptibility. There is emerging evidence that biofilm-phase NTHi may play a significant role in COPD. NTHi displays many hallmarks of the biofilm lifestyle and expresses key biofilm formation-promoting genes. These include the autoinducer-mediated quorum sensing system, epithelial- and mucus-binding adhesins and expression of a protective, self-produced polymeric substance matrix. These NTHi biofilms exhibit extreme tolerance to antimicrobial treatments and the immune system as well as expressing synergistic interspecific interactions with other lung pathogens including S. pneumoniae and M. catarrhalis. Whilst the majority of our understanding surrounding NTHi as a biofilm arises from otitis media or in-vitro bacterial monoculture models, the role of NTHi biofilms in the COPD lung is now being studied. This review explores the evidence for the existence of NTHi biofilms and their impact in the COPD lung. Understanding the nature of chronic and recurrent NTHi infections in acute exacerbations of COPD could have important implications for clinical treatment and identification of novel bactericidal targets.

Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

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

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. 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. 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. 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. NA Laryngoscope, 130:1364-1371, 2020.

Tosufloxacin for Eradicating Biofilm-Forming Nontypeable Haemophilus influenzae Isolated from Intractable Acute Otitis Media

Background: Nontypeable Haemophilus influenzae (NTHi)-caused acute otitis media persists even after antimicrobial treatment, presumably as a result of biofilm formation. It is urgent to establish an alternative antimicrobial treatment against acute otitis media that effectively deals with NTHi biofilm. Methods: An otopathogenic clinical strain of NTHi IH-202 isolated from the middle ear fluid of intractable pediatric cases of acute otitis media was used in the study. Antimicrobial susceptibility and biofilm assay using antimicrobial agent tosufloxacin were performed. Results: Exposure to tosufloxacin at 0.96 µg/mL for 20 minutes reduced the amount of NTHi biofilm formation and killed viable NTHi in biofilm as compared with the control. A clinically attainable concentration of tosufloxacin could destroy NTHi biofilm and showed bactericidal activity against NTHi in biofilm in shorter exposure periods. Conclusions: The current findings suggest an alternative antimicrobial treatment strategy against intractable cases of acute otitis media caused by NTHi biofilms. Tosufloxacin will be an effective alternative for the treatment of intractable acute otitis media caused by NTHi biofilm. In addition, our in vitro bacteria-epithelial cell co-culture model is a relatively simple and static method for studying NTHi biofilms.

Autoinducer 2 (AI-2) Production by Nontypeable Haemophilus influenzae 86-028NP Promotes Expression of a Predicted Glycosyltransferase That Is a Determinant of Biofilm Maturation, Prevention of Dispersal, and Persistence In Vivo.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.

Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae.

ABSTRACTBiofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling.

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

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.

Primary ciliary dyskinesia ciliated airwaycells show increased susceptibility to Haemophilus influenzae biofilm formation.

Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development.We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air-liquid interface culture and then co-cultured with NTHi.NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells (p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone.Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.

D-methionine interferes with non-typeable Haemophilus influenzae peptidoglycan synthesis during growth and biofilm formation.

Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that plays a major role in a number of respiratory tract infections, including otitis media, cystic fibrosis and chronic obstructive pulmonary disease. Biofilm formation has been implicated in both NTHi colonization and disease, and is responsible for the increased tolerance of this pathogen towards antibiotic treatment. Targeting metabolic pathways that are important in NTHi biofilm formation represents a potential strategy to combat this antibiotic recalcitrance. A previous investigation demonstrated increased expression of a putative d-methionine uptake protein following exposure of NTHi biofilms to the ubiquitous signalling molecule, nitric oxide. We therefore hypothesized that treatment with exogenous d-methionine would impact on NTHi biofilm formation and increase antibiotic sensitivity. Treatment of NTHi during the process of biofilm formation resulted in a reduction in biofilm viability, increased biomass, changes in the overall biofilm architecture and the adoption of an amorphous cellular morphology. Quantitative proteomic analyses identified 124 proteins that were differentially expressed following d-methionine treatment, of which 51 (41 %) were involved in metabolic and transport processes. Nine proteins involved in peptidoglycan synthesis and cell division showed significantly increased expression. Furthermore, d-methionine treatment augmented the efficacy of azithromycin treatment and highlighted the potential of d-methionine as an adjunctive therapeutic approach for NTHi biofilm-associated infections.

Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms.

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that frequently colonizes the human nasopharynx; it is a common cause of chronic and recurrent otitis media in children and of exacerbations of chronic obstructive pulmonary disease. To date, no exopolysaccharide clearly contributing to NTHi biofilms has been identified. Consequently, there is some debate as to whether NTHi forms biofilms during colonization and infection. The present work shows that NTHi can form biofilms in vitro, producing an extracellular matrix composed of proteins, nucleic acids, and a β-glucan. Extracellular DNA, visualized by immunostaining and using fluorochromes, is an important component of this matrix and appears to be essential in biofilm maintenance. Extracellular RNA appears to be required only in the first steps of biofilm formation. Evidence of a matrix polysaccharide was obtained by staining with Calcofluor white M2R and by disaggregating biofilms with cellulase. Using strain 54997, residues of Glcp(1→4) in the NTHi biofilm were confirmed by gas-liquid chromatography-mass spectrometry. Evidence that N-acetyl-L-cysteine shows notable killing activity towards in vitro NTHi biofilm-forming bacteria is also provided.

In vitro interference of cefotaxime at subinhibitory concentrations on biofilm formation by nontypeable Haemophilus influenzae

ObjectiveTo investigate the in vitro interference of cefotaxime at subinhibitory concentrations [sub-minimal inhibitory concentrations (MIC)] on biofilm formation by nontypeable Haemophilus influenzae (NTHi). MethodsThe interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong-biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay. The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test. Additionally, the effects on bacterial adherence to human fibronectin and expression of bacterial adhesins were also investigated. ResultsSubinhibitory concentrations of cefotaxime, both at 0.1× and 0.5× MIC levels, efficiently reduced the NTHi biofilm formation, and this effect was independent of decreasing bacterial viability. Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronectin. Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted. ConclusionsTaken together, our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm, and this effect is feasibly related to the interference with cell-surface hydrophobicity, fibronectin-binding activity as well as alteration of the P2 and P6 gene expression. The findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases.

Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production.

Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18-20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at -80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

Peroxiredoxin-glutaredoxin and catalase promote resistance of nontypeable Haemophilus influenzae 86-028NP to oxidants and survival within neutrophil extracellular traps.

Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) in resistance of NTHI to oxidants and persistence in vivo. Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.

A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, frequently causes biofilm infections. Currently, resistance of bacteria within the biofilm to conventional antimicrobials poses a major obstacle to effective medical treatment on a global scale. Novel agents that are effective against NTHi biofilm are therefore urgently required. In this study, a series of natural and synthetic chalcones with various chemical substituents were evaluated in vitro for their antibiofilm activities against strong biofilm-forming strains of NTHi. Of the test chalcones, 3-hydroxychalcone (chalcone 8) exhibited the most potent inhibitory activity, its mean minimum biofilm inhibitory concentration (MBIC50 ) being 16 μg/mL (71.35 μM), or approximately sixfold more active than the reference drug, azithromycin (MBIC50 419.68 μM). The inhibitory activity of chalcone 8, which is a chemically modified chalcone, appeared to be superior to those of the natural chalcones tested. Significantly, chalcone 8 inhibited biofilm formation by all studied NTHi strains, indicating that the antibiofilm activities of this compound occur across multiple strong-biofilm forming NTHi isolates of different clinical origins. According to antimicrobial and growth curve assays, chalcone 8 at concentrations that decreased biofilm formation did not affect growth of NTHi, suggesting the biofilm inhibitory effect of chalcone 8 is non-antimicrobial. In terms of structure-activity relationship, the possible substituent on the chalcone backbone required for antibiofilm activity is discussed. These findings indicate that 3-hydroxychalcone (chalcone 8) has powerful antibiofilm activity and suggest the potential application of chalcone 8 as a new therapeutic agent for control of NTHi biofilm-associated infections.