Chinchilla Middle Ear Research Articles

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

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). A blinded pre-clinical study of comparative efficacy of single versus combinatorial treatment strategies. 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. 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. 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. NA Laryngoscope, 131:E2698-E2704, 2021.

Patent evaluation of US2019338018 (A1) 2019-11-07 (antibody fragments for the treatment of biofilm-related disorders)

ABSTRACT Introduction To date, microbial infections are also difficult to eradicate due to the increasing capability of bacteria to form a biofilm. In the era of antibiotic resistance, antibody-based approaches represent great promise in curing infective pathogens. The authors of US2019338018 patent propose a method for the treatment of biofilm-related disorders by using specific antibody fragments. Areas covered The US2019338018 patent reports antibody fragments, pharmaceutical composition that contains it, and their application for the treatment of biofilm-linked disorders. Proof concept and preclinical results show that mAb mIhfB4NTHI Fab caused robust eradication of the biofilm in the middle ear lumen of chinchillas affected by Hemophilus influenzae infection. Expert opinion Fab fragments of the US2019338018 patent are new in a general concept to treat bacterial biofilms and biofilm-linked disorders. However, pre-clinical data are only shown for the treatment with Fab fragments of infections caused by H. influenzae in the middle ear of chinchillas. There are no clinical trials that demonstrate that the treatment with Fab fragments may induce a disruption of biofilm produced by H. influenzae or other pathogens and an anti-inflammatory response in infected patients.

Significance of middle ear model for prediction of metabolic exhaustion of the auditory system

Modern day military noise is complex, involving irregular impulses riding on top of moderate continuous background noise. An accurate prediction of injury from complex noise exposure requires both a cochlear metabolic exhaustion (ME) model and an accurate middle ear model (MEM), accounting for acoustic reflex (AR) and annular ligament (AL) nonlinearities. An end-to-end biomechanical model including a ME model was developed that established a good correlation between the outer hair cell energy deficit (OHC-D) outcomes and the temporary threshold shift (TTS) data from chinchilla complex noise exposure, explained the data collected, and provided insights on the effect of complex noise exposure on outcomes, including the effect of inter-pulse Interval (IPI), shot sequence, and background noise. Literature data were used to derive the open ear and middle ear transfer functions (TF). In this paper, the model results are discussed with an emphasis on the MEM component. The differences in the end-to-end model results are compared between the new MEM and a MEM adopted from the improved AHAAH-ICE model that includes the AR and AL nonlinearities, with model parameters adjusted for chinchilla. The results show that the TFs from the two MEMs cannot be matched using mere parametric optimization, due mainly to the fact that the chinchilla middle ear is rather tuned to the higher frequencies compared to that of human. There is a need for an improved MEM for the chinchilla middle ear TF including AR effects.

Optical coherence tomographic measurements of the sound-induced motion of the ossicular chain in chinchillas: Additional modes of ossicular motion enhance the mechanical response of the chinchilla middle ear at higher frequencies

Wavelength-swept optical coherence tomography (OCT) was used to scan the structure of cadaveric chinchilla ears in three dimensions with high spatial resolution and measure the sound-induced displacements of the entire OCT-visible lateral surfaces of the ossicles in the lateral-to-medial direction. The simultaneous measurement of structure and displacement allowed a precise match between the observed motion and its structural origin. The structure and measured displacements are consistent with previously published data. The coincident detailed structural and motion measurements demonstrate the presence of several frequency-dependent modes of ossicular motion, including: (i) rotation about an anteriorly-to-posteriorly directed axis positioned near the commonly defined anatomical axis of rotation that dominates at frequencies below 8 kHz, (ii) a lateral-to-medial translational component that is visible at frequencies from 2 to greater than 10 kHz, and (iii) a newly described rotational mode around an inferiorly-to-superiorly directed axis that parallels the manubrium of the malleus and dominates ossicular motion between 10 and 16 kHz. This new axis of rotation is located near the posterior edge of the manubrium. The onset of the second rotational mode leads to a boost in the magnitude of sound-induced stapes displacement near 14 kHz, and adds a half-cycle to the accumulating phase in middle-ear sound transmission. Similar measurements in one ear after interruption of the incudostapedial joint suggest the load of the cochlea and stapes annular ligament is important to the presence of the second rotational mode, and acts to limit simple ossicular translation.

A lumped-element model of the chinchilla middle ear.

An air-conduction circuit model was developed for the chinchilla middle ear and cochlea. The lumped-element model is based on the classic Zwislocki model of the same structures in human. Model parameters were fit to various measurements of chinchilla middle-ear transfer functions and impedances, using a combination of error-minimization-driven computer-automated and manual fitting methods. The measurements used to fit the model comprise a newer, more-extensive data set than previously used, and include measurements of stapes velocity and inner-ear sound pressure within the vestibule and the scala tympani near the round window. The model is in agreement with studies of the effects of middle-ear cavity holes in experiments that require access to the middle-ear air space. The structure of the model allows easy addition of other sources of auditory stimulation, e.g., the multiple sources of bone-conducted sound-the long-term goal for the model's development-and mechanical stimulation of the ossicles and round window.

Dornase Alfa Ototoxic Effects in Animals and Efficacy in the Treatment of Clogged Tympanostomy Tubes in Children

Many treatments for clogged tympanostomy tubes (TTs) have been proposed, but none have met scientific rigor for safety and efficacy, including the popular empirical use of ototopical antibiotic drops. Dornase alfa, a recombinant molecule with the unique property of cleaving DNA, may be ideal in treating clogged TTs because both middle-ear effusion and the plug are abundant with DNA. To investigate the ototoxic effects of dornase alfa in a chinchilla model and its efficacy in a clinical trial in children with clogged TTs. The safety profiles of dornase alfa (full-strength and 1:10 strength) were evaluated in chinchilla middle ears using serial auditory brainstem response. The efficacy of ototopical dornase alfa (full-strength) was evaluated in children with clogged TTs in a prospective, single-blind randomized clinical trial. The animal study included 21 chinchillas and was conducted at Loma Linda University, Loma Linda, California, and the clinical trial was conducted at Children's Hospital Colorado, Aurora. A total of 40 children (50 ears with tubes) were enrolled. In the animal study, chinchillas were assigned to 3 groups: controls (saline), full-strength dornase alfa, or 1:10 dornase alfa dilution. Children were randomly assigned to receive either topical dornase alfa or ofloxacin for clogged TT, 5 drops each ear twice a day for 7 days. Animal study: Auditory brainstem responses. Randomized trial of children participants: The primary outcome was patency of TT at day 14 assessed by otoscopy and tympanometry. The chinchilla study showed similar auditory brainstem response degradation during a 6-hour period between the control (n = 5) and treatment groups (n = 21). In the clinical trial, a total of 40 clogged TTs (in 33 children, including 25 boys [76%]; mean age, 4.3 years; median [range] age, 3.4 [1.0-14.3] years) were analyzed. The number of unclogged TTs was higher in the dornase alfa group (13 [59%]) compared with the ofloxacin group (8 [44%]), with a difference of 15% (odds ratio, 1.8; 95% CI, 0.54-6.72). The chinchilla model suggests that dornase alfa is likely nonototoxic. The pilot clinical trial failed to show efficacy of dornase alfa to unclog TTs. With the difference seen between the treatment groups, a sample size estimate could be calculated for a future large-scale trial. ClinicalTrials.gov identifier: NCT00419380.

Neutrophil Extracellular Traps and Fibrin in Otitis Media

Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

Chinchilla middle ear transmission matrix model and middle-ear flexibility.

The function of the middle ear (ME) in transforming ME acoustic inputs and outputs (sound pressures and volume velocities) can be described with an acoustic two-port transmission matrix. This description is independent of the load on the ME (cochlea or ear canal) and holds in either direction: forward (from ear canal to cochlea) or reverse (from cochlea to ear canal). A transmission matrix describing ME function in chinchilla, an animal commonly used in auditory research, is presented, computed from measurements of forward ME function: input admittance YTM, ME pressure gain GMEP, ME velocity transfer function HV, and cochlear input admittance YC, in the same set of ears [Ravicz and Rosowski (2012b). J. Acoust. Soc. Am. 132, 2437-2454; (2013a). J. Acoust. Soc. Am. 133, 2208-2223; (2013b). J. Acoust. Soc. Am. 134, 2852-2865]. Unlike previous estimates, these computations require no assumptions about the state of the inner ear, effectiveness of ME manipulations, or measurements of sound transmission in the reverse direction. These element values are generally consistent with physical constraints and the anatomical ME "transformer ratio." Differences from a previous estimate in chinchilla [Songer and Rosowski (2007). J. Acoust. Soc. Am. 122, 932-942] may be due to a difference in ME flexibility between the two subject groups.

Middle-ear function in the chinchilla: Circuit models and comparison with other mammalian species

The middle ear efficiently transmits sound from the ear canal into the inner ear through a broad range of frequencies. Thus, understanding middle-ear transmission characteristics is essential in the study of hearing mechanics. Two models of the chinchilla middle ear are presented. In the first model, the middle ear is modeled as a lumped parameter system with elements that represent the ossicular chain and the middle-ear cavity. Parameters of this model are fit using available experimental data of two-port transmission matrix parameters. In an effort to improve agreement between model simulations and the phase of published experimental measurements for the forward pressure transfer function at high frequencies, a second model in which a lossless transmission line model of the tympanic membrane is appended to the original model is proposed. Two-port transmission matrix parameter results from this second model were compared with results from previously developed models of the guinea pig, cat, and human middle ears. Model results and published experimental data for the two-port transmission matrix parameters are found to be qualitatively similar between species. Quantitative differences in the two-port transmission matrix parameters suggest that the ossicular chains of chinchillas, cats, and guinea pigs are less flexible than in humans.

3D finite element model of the chinchilla ear for characterizing middle ear functions

Chinchilla is a commonly used animal model for research of sound transmission through the ear. Experimental measurements of the middle ear transfer function in chinchillas have shown that the middle ear cavity greatly affects the tympanic membrane (TM) and stapes footplate (FP) displacements. However, there is no finite element (FE) model of the chinchilla ear available in the literature to characterize the middle ear functions with the anatomical features of the chinchilla ear. This paper reports a recently completed 3D FE model of the chinchilla ear based on X-ray micro-computed tomography images of a chinchilla bulla. The model consisted of the ear canal, TM, middle ear ossicles and suspensory ligaments, and the middle ear cavity. Two boundary conditions of the middle ear cavity wall were simulated in the model as the rigid structure and the partially flexible surface, and the acoustic-mechanical coupled analysis was conducted with these two conditions to characterize the middle ear function. The model results were compared with experimental measurements reported in the literature including the TM and FP displacements and the middle ear input admittance in chinchilla ear. An application of this model was presented to identify the acoustic role of the middle ear septa-a unique feature of chinchilla middle ear cavity. This study provides the first 3D FE model of the chinchilla ear for characterizing the middle ear functions through the acoustic-mechanical coupled FE analysis.

Stapes Vibration in the Chinchilla Middle Ear: Relation to Behavioral and Auditory-Nerve Thresholds.

The vibratory responses to tones of the stapes and incus were measured in the middle ears of deeply anesthetized chinchillas using a wide-band acoustic-stimulus system and a laser velocimeter coupled to a microscope. With the laser beam at an angle of about 40 ° relative to the axis of stapes piston-like motion, the sensitivity-vs.-frequency curves of vibrations at the head of the stapes and the incus lenticular process were very similar to each other but larger, in the range 15-30 kHz, than the vibrations of the incus just peripheral to the pedicle. With the laser beam aligned with the axis of piston-like stapes motion, vibrations of the incus just peripheral to its pedicle were very similar to the vibrations of the lenticular process or the stapes head measured at the 40 ° angle. Thus, the pedicle prevents transmission to the stapes of components of incus vibration not aligned with the axis of stapes piston-like motion. The mean magnitude curve of stapes velocities is fairly flat over a wide frequency range, with a mean value of about 0.19 mm(.)(s Pa(-1)), has a high-frequency cutoff of 25 kHz (measured at -3 dB re the mean value), and decreases with a slope of about -60 dB/octave at higher frequencies. According to our measurements, the chinchilla middle ear transmits acoustic signals into the cochlea at frequencies exceeding both the bandwidth of responses of auditory-nerve fibers and the upper cutoff of hearing. The phase lags of stapes velocity relative to ear-canal pressure increase approximately linearly, with slopes equivalent to pure delays of about 57-76 μs.

Pneumococcal PspA and PspC proteins: Potential vaccine candidates for experimental otitis media

ObjectiveOtitis media is the most commonly diagnosed disease in ambulatory care and Streptococcuspneumoniae continues to be the most common bacterial agent. Bacterial resistance to antibiotics underscores the need for better vaccines. Current pneumococcal conjugate vaccines are modestly protective against otitis media; however, limited serotype coverage and serotype replacement have led to the investigation of pneumococcal proteins as potential vaccine candidates. Two proteins, pneumococcal surface proteins A (PspA) and C (PspC) are important virulence factors, expressed by virtually all strains. Although a number of pneumococcal proteins have been investigated in other infection sites, these proteins can have diverse organ-specific effects. In this study, we investigated the viability and virulence of single (PspA– and PspC–) and double (PspA–/PspC–) mutants of pneumococcal PspA and PspC proteins in the chinchilla middle ear. MethodsBullae of 24 chinchillas were inoculated with 0.5ml of 106 colony forming units (CFUs)/ml bacteria: 6 with wild-type D39 strain; 6 with PspA–; 6 with PspC–; and 6 with PspA–/PspC– isogenic mutant strains. Bacterial CFU levels in middle ear effusions and light microscopic analysis of the number of inflammatory cells in the round window membrane (RWM) were compared 48h after inoculation. ResultsAt 48h, CFUs in middle ears were increased for wild-type and PspC– strains compared to inoculum levels; however, they were significantly less for the group inoculated with the PspC– strain compared to wild-type strain. No bacteria were detected in the PspA– and PspA–/PspC– groups. The number of inflammatory cells in the RWM was significantly higher in wild-type compared to the PspA–, PspC–, and PspA–/PspC– groups. No significant difference in number of inflammatory cells was observed between any pairs of groups inoculated with mutant strains. ConclusionViability and virulence of the PspC– strain were similar to the wild-type strain. The single PspA– and double PspA–/PspC– mutants were highly attenuated in the ear. Bacterial clearance of the PspA–/PspC– double mutant was indistinguishable from that of the PspA mutant. These studies provide no reason to exclude PspC from a multi-component protein vaccine containing PspA.

Deletion of arcD in Streptococcus pneumoniae D39 Impairs Its Capsule and Attenuates Virulence

The arginine deiminase system (ADS) is associated with arginine catabolism and plays a role in virulence of several pathogenic bacteria. In Streptococcus pneumoniae, the ADS genes exist as a locus consisting of arcABCDT. A recent genome-wide mutagenesis approach revealed that both arcD and arcT are potentially essential in a chinchilla otitis media (OM) model. In the present study, we generated ΔarcD, ΔarcT, and ΔarcDT mutants by homologous recombination and evaluated their infectivity. Our results showed that only arcD, and not arcT, of an OM isolate is required during chinchilla middle ear infection. Additionally, D39 ΔarcD exhibited enhanced nasopharyngeal colonization and was attenuated in both mouse pneumonia and bacteremia models. In vitro, D39 ΔarcD displayed enhanced adherence to A549 epithelial cells and increased phagocytosis by J774A.1 macrophages compared to those with the parental strain. This mutant also exhibited an impaired capsule, as detected using electron microscopy, immunofluorescence, and a capsule assay. We demonstrated that the capsule defect in the D39 ΔarcD mutant may not be associated with a deficiency in arginine but rather is likely caused by a loss of interaction between the capsule and the transmembrane protein ArcD.

Bioluminescent Imaging of Pneumococcal Otitis Media in Chinchillas

Bioluminescent imaging has emerged as a powerful tool for monitoring the pathological process of infections in animals. The purpose of this study was to harness this new tool for objective assessment of acute otitis media (AOM) in animals with and without antibiotic interventions. Thirty-six healthy chinchillas, free of middle ear infections, were randomly divided into a control group and a group that received amoxicillin treatment. Bioluminescent Streptococcus pneumoniae (Xen 10) was injected into the epitympanic bullae of chinchillas (50 colony-forming units each) for induction of AOM. The infectious process of Xen 10 in the bullae of living animals with and without antibiotic interventions was monitored in real time with bioluminescence equipment. A dynamic change of bioluminescent signals in the bullae of chinchillas from days 1 to 14 was observed after Xen 10 injection. Amoxicillin treatment reduced the bioluminescent signals in the bullae of chinchillas compared with controls. The AOM persisted for 14 days, and middle ear effusion for 6 weeks, in the control animals, whereas AOM lasted for 2 days, and effusion for 6 to 12 days, in the antibiotic-treated animals. Bioluminescent imaging provides an innovative method for assessment of the bacterial loads in the middle ear of chinchillas in a real-time manner and is very useful for objective evaluation of the efficacy of therapeutic interventions.

The Vitamin B 6 Biosynthesis Pathway in Streptococcus pneumoniae Is Controlled by Pyridoxal 5′-Phosphate and the Transcription Factor PdxR and Has an Impact on Ear Infection

Vitamin B₆ is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5'-phosphate (PLP) biosynthesis pathway in Streptococcus pneumoniae. Our results revealed that S. pneumoniae possesses a de novo vitamin B₆ biosynthesis pathway encoded by the pdxST genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity in vitro. Deletion of pdxS, but not pdxT, resulted in a vitamin B₆ auxotrophic mutant. The defective growth of the ΔpdxS mutant in a vitamin B₆-depleted medium could be chemically restored in the presence of the B₆ vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of pdxS was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal ΔpdxR mutant also exhibited as a vitamin B₆ auxotroph. In addition, we found that disruption of the vitamin B₆ biosynthesis pathway in S. pneumoniae caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B₆ synthesis on virulence depends upon the bacterial infection niche.

Haemophilus influenzae OxyR: Characterization of Its Regulation, Regulon and Role in Fitness

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

The Haemophilus influenzae Sap Transporter Mediates Bacterium-Epithelial Cell Homeostasis

Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or the sapA mutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, the sapA mutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to the sapA mutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with the sapA mutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.

RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP

Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2.

Primary Culture of Chinchilla Middle Ear Epithelium

Primary Culture of Chinchilla Middle Ear Epithelium