Mechanical properties of the Papio anubis tympanic membrane: Change significantly from infancy to adulthood.

Mechanical properties of the tympanic membrane (TM) are important for studying the transfer function of the auditory system. However, nearly all reported human data are limited to adults because of the unavailability of temporal bones from children. In this study, we used the baboon (Papio anubis), a genetically close human relative, as a model to address the occurrence of age-dependent changes of the human TM. Forty-five baboon TMs were characterized in five age groups: <1 year, 1 to <2 years, 2 to <3 years, 3 to <5, and >5 years of age, comparable to human ages ranging from newborn to adult. The elastic properties of the baboon TMs were characterized by a micro-fringe projection technique. Volume displacement of the TM under quasi-static pressure was first determined from its micro-fringe pattern. Subsequently, these displacement values were used in a finite element model to derive mechanical properties. The Young's modulus of the baboon TM exhibited a modest decrease from 29.1 MPa to 26.0 MPa over the age groups. The average Young's modulus was ∼1.4 times higher than that of the adult human TM. This is the first time that age-related TM mechanical properties of high primate are reported. These new findings may help to explore the potential value of the baboon as a new primate model for future age-related hearing research on the normal and diseased ear.

Factors affecting sound energy absorbance in acute otitis media model of chinchilla.

Acute otitis media (AOM) is a rapid-onset infection of the middle ear which results in middle ear pressure (MEP), middle ear effusion (MEE), and structural changes in middle ear tissues. Previous studies from our laboratory have identified that MEP, MEE, and middle ear structural changes are three factors affecting tympanic membrane (TM) mobility and hearing levels (Guan et al., 2014, 2013). Sound energy reflectance or absorbance (EA) is a diagnostic tool increasingly used in clinical settings for the identification of middle ear diseases. However, it is unclear whether EA can differentiate these three factors in an AOM ear. Here we report wideband EA measurements in the AOM model of chinchilla at three experimental stages: unopened, pressure released, and effusion removed. These correspond to the combined and individual effects of the three factors on sound energy transmission. AOM was produced by transbullar injection of Haemophilus influenzae in two treatment groups: 4 days (4D) and 8 days (8D) post inoculation. These time points represent the relatively early and later phase of AOM. In each group of chinchillas, EA at 250-8000 Hz was measured using a wideband tympanometer at three experimental stages. Results show that the effects of MEP, MEE, and tissue structural changes over the frequency range varied with the disease time course. MEP was the primary contributor to reduction of EA in 4D AOM ears and had a smaller effect in 8D ears. MEE reduced the EA at 6-8 kHz in 4D ears and 2-8 kHz in 8D ears and was responsible for the EA peak in both 4D and 8D ears. The residual EA loss due to structural changes was observed over the frequency range in 8D ears and only at high frequencies in 4D ears. The EA measurements were also compared with the published TM mobility loss in chinchilla AOM ears.

Morphological changes in the round window membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla.

OBJECTIVE: The round window membrane (RWM) encloses the round window, the opening into the scala tympani (ST) from the middle ear. During the course of acute otitis media (AOM), structural changes of the RWM can occur that potentially affect sound transmission into and out of the cochlea. The relationship between such structural changes and conductive hearing loss during AOM has remained unclear. The focus of the current study was to compare the thickness distribution across the RWM surface between normal ears and those with AOM in the chinchilla. We assessed the occurrence of AOM-associated histological changes in this membrane compared to uninfected control animals after AOM of two relatively short durations. MATERIAL AND METHODS: AOM was induced by transbullar injection of the nontypeable Haemophilus influenzae strain 86-028NP into two groups of adult chinchillas (n = 3 each). Bullae were obtained from the two infected groups, at 4 days or 8 days post challenge. Structures and thickness of these RWMs were compared between the two infected treatment groups and to RWMs from uninfected control animals (n = 3) at seven different RWM locations. RESULTS: RWM thickness in infected chinchillas increased significantly at locations along the central line on the 4th day post bacterial challenge compared to values found for uninfected control animals. Lymphocyte infiltration and edema were the primary contributors to these thickness increases. No significant further increases in RWM thickness were observed when RWMs from chinchillas ears infected for 4 and 8 days were compared. Thickness and structural changes at the RWM lateral and medial areas were less visually obvious and not statistically significant among the three treatment groups. These latter RWM regions clearly were less affected during AOM than the central areas. CONCLUSIONS: This histological study establishes that H. influenzae-induced AOM causes significant acute changes in chinchilla RWM structure that are characterized by region-specific increases in thickness. Our new morphological findings comparing normal and diseased chinchilla RWMs identify yet another biomechanical mechanism by which nontypeable H. influenzae may contribute to hearing loss in AOM.

Draft Genome Sequences of Eight Nontypeable Haemophilus influenzae Strains Previously Characterized Using an Electrophoretic Typing Scheme.

Nontypeable Haemophilus influenzae is an important cause of human disease. Strains were selected for genome sequencing to represent the breadth of nontypeable strains within the species, as previously defined by the electrophoretic mobility of 16 metabolic enzymes.

Draft Genome Sequences of Six Nontypeable Haemophilus influenzae Strains That Establish Bacteremia in the Infant Rat Model of Invasive Disease.

Haemophilus influenzae is an important cause of invasive disease. The infant rat is the accepted model of invasive H. influenzae disease. Here, we report the genome sequences of six nontypeable H. influenzae strains that establish bacteremia in the infant rat.

Antisera Against Certain Conserved Surface-Exposed Peptides of Nontypeable Haemophilus influenzae Are Protective.

Nontypeable Haemophilus influenzae (NTHi) cause significant disease, including otitis media in children, exacerbations of chronic obstructive pulmonary disease, and invasive disease in susceptible populations. No vaccine is currently available to prevent NTHi disease. The interactions of NTHi and the human host are primarily mediated by lipooligosaccharide and a complex array of surface-exposed proteins (SEPs) that act as receptors, sensors and secretion systems. We hypothesized that certain SEPs are present in all NTHi strains and that a subset of these may be antibody accessible and represent protective epitopes. Initially we used 15 genomic sequences available in the GenBank database along with an additional 11 genomic sequences generated by ourselves to identify the core set of putative SEPs present in all strains. Using bioinformatics, 56 core SEPs were identified. Molecular modeling generated putative structures of the SEPs from which potential surface exposed regions were defined. Synthetic peptides corresponding to ten of these highly conserved surface-exposed regions were used to raise antisera in rats. These antisera were used to assess passive protection in the infant rat model of invasive NTHi infection. Five of the antisera were protective, thus demonstrating their in vivo antibody accessibility. These five peptide regions represent potential targets for peptide vaccine candidates to protect against NTHi infection.

Morphological changes in the tympanic membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla.

OBJECTIVE: The tympanic membrane (TM) couples sound waves entering the outer ear canal to mechanical vibrations of the ossicular chain in the middle ear. During acute otitis media (AOM), dynamic structural changes in the TM can occur, which potentially affect sound transmission. It has remained unclear whether TM changes contribute significantly to the conductive hearing loss associated with human AOM. Studies that systematically and quantitatively assess the impact of morphological and mechanical characteristics of the TM on hearing in animal models of AOM have been few in number and lack detail. Our current study focused on the identification of quantitative morphological changes in the TM of the adult chinchilla. METHOD: AOM was produced by transbullar injection of the nontypeable (acapsular) Haemophilus influenzae strain 86-028NP into two treatment groups of chinchillas: one 4 days (4D) post bacterial challenge, and a second treatment group after 8 days (8D) post challenge. Structure and thickness were examined histologically at nine locations over the TM in untreated controls and in animals from both AOM treatment groups. RESULTS: TM thickness was found to have increased significantly (110-150%) at all measured locations of H. influenzae-infected ears when compared with uninfected (normal) TMs at 4D post bacterial challenge. Cellular proliferation and infiltration in the outer epithelial layer were primary contributors to this thickening. In ears infected for 8D, the TM was substantially thicker, a 200-300% increase from uninfected control values, due to edema and cell proliferation in both the outer and inner epithelial layers. In both 4D and 8D ears, thickening of the TM was more prominent in the superior-anterior quadrant. CONCLUSION: This study provides unequivocal structural evidence that significant TM thickness increases are associated with AOM induced by a well characterized H. influenzae human clinical isolate of low passage number. These and additional thickness data from early and later stages in middle ear infection will be used to derive the mechanical properties of the TM in a future study from our laboratory.

Comparison of transcription of the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear.

BACKGROUND: Haemophilus influenzae is a significant cause of childhood otitis media, and also has an absolute growth requirement for heme. Recent microarray studies using three H. influenzae isolates were used to propose a putative core of genes responsive to iron and heme levels. Included in the core modulon were thirty seven genes that are preferentially expressed under iron/heme limitation, most of which are directly involved with iron and or heme acquisition. In this report, the core iron/heme modulon was further refined following microarray analysis of two additional nontypeable H. influenzae isolates from patients with otitis media. The transcriptional status of the genes comprising the refined iron/heme core modulon was then assessed in vivo, in a chinchilla model of otitis media. These in vivo experiments were performed to address the hypothesis that iron and heme regulated genes are both highly expressed in vivo and important, during clinical infection. RESULTS: Microarray analysis of two additional H. influenzae strains resulted in the definition of a core of iron/heme responsive genes. This core consisted of 35 genes maximally expressed under heme restriction and a further 20 genes maximally expressed in heme replete conditions. In vivo studies were performed with two nontypeable H. influenzae strains, 86-028NP and HI1722. The majority of operons identified as members of the core modulon by microarray were also actively upregulated in the chinchilla ear during otitis media. In 86-028NP, 70% of the operons were significantly upregulated while in HI1722 100% of the operons were upregulated in samples recovered from the chinchilla middle ear. CONCLUSION: This study elucidates a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and heme in the growth environment, and further assesses transcription of these genes in vivo. Elucidation of this modulon allows for identification of genes with unrecognized roles in iron/heme acquisition or homeostasis and/or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis.

BACKGROUND: The RNA binding protein Hfq of Haemophilus influenzae is highly homologous to Hfq from other bacterial species. In many of these other bacteria, Hfq affects the expression of a broad range of genes and enhances the ability to respond to stressful environments. However, the role of Hfq in H. influenzae is unknown. RESULTS: Deletion mutants of hfq were generated in the nontypeable H. influenzae strains R2866 and 86-028NP to assess the role of Hfq in these well characterized but genotypically and phenotypically divergent clinical isolates. A deletion mutation of hfq had no effect on growth of H. influenzae in nutrient rich media and had no effect on survival in several stressful conditions in vitro. However, the mutation resulted in a reduced ability to utilize heme from hemoglobin. The mutant and wild type strains were assessed for virulence and competitive fitness in models of invasive disease and otitis media. In the chinchilla model of otitis media, the hfq mutant of 86-028NP exhibited impaired competitive fitness when compared to its wild type progenitor but exhibited no apparent defect in virulence. In the infant rat model, deletion of hfq in R2866 resulted in reduced bacterial titers in blood and a shorter duration of infection when compared to the wild type strain in the competitive fitness study. CONCLUSION: We conclude that Hfq is involved in the utilization of essential nutrients and facilitates infection by H. influenzae.

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.

Signature-tagging of a bacterial isolate demonstrates phenotypic variability of the progeny in vivo in the absence of defined mutations.

Awareness of the high degree of redundancy that occurs in several nutrient uptake pathways of Haemophilus influenzae led us to attempt to develop a quantitative STM method that could identify both null mutants and mutants with decreased fitness that remain viable in vivo. To accomplish this task we designed a modified STM approach that utilized a set of signature tagged wild-type (STWT) strains (in a single genetic background) as carriers for mutations in genes of interest located elsewhere in the genome. Each STWT strain differed from the others by insertion of a unique, Q-PCR-detectable, seven base pair tag into the same redundant gene locus. Initially ten STWTs were created and characterized in vitro and in vivo. As anticipated, the STWT strains were not significantly different in their in vitro growth. However, in the chinchilla model of otitis media, certain STWTs outgrew others by several orders of magnitude in mixed infections. Removal of the predominant STWT resulted in its replacement by a different predominant STWT on retesting. Unexpectedly we observed that the STWT exhibiting the greatest proliferation was animal dependent. These findings identify an inherent inability of the signature tag methodologies to accurately elucidate fitness in this animal model of infection and underscore the subtleties of H. influenzae gene regulation.

A functional tonB gene is required for both virulence and competitive fitness in a chinchilla model of Haemophilus influenzae otitis media.

BACKGROUND: Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. METHODS: An insertional mutation in tonB was constructed and the impact of the mutation on virulence and fitness in a chinchilla model of otitis media was determined. The tonB insertion mutant strain was significantly impacted in both virulence and fitness as compared to the wildtype strain in this model. CONCLUSIONS: The tonB gene of H. influenzae is required for the establishment and maintenance of middle ear infection in this chinchilla model of bacterial disease.

Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae. RESULTS: Comparative genomics revealed a putative four gene operon in the recently sequenced nontypeable H. influenzae strain R2846 that encodes predicted proteins exhibiting significant identity at the amino acid level to proteins involved in the utilization of the siderophore ferrichrome in other bacterial species. No siderophore biosynthesis genes were identified in the R2846 genome. Both comparative genomics and a PCR based analysis identified several additional H. influenzae strains possessing this operon. In growth curve assays strains containing the genes were able to utilize ferrichrome as an iron source. H. influenzae strains lacking the operon were unable to obtain iron from ferrichrome. An insertional mutation in one gene of the operon abrogated the ability of strains to utilize ferrichrome. In addition transcription of genes in the identified operon were repressible by high iron/heme levels in the growth media. CONCLUSIONS: We have identified an iron/heme-repressible siderophore utilization locus present in several nontypeable H. influenzae strains. The same strains do not possess genes encoding proteins associated with siderophore synthesis. The siderophore utilization locus may enable the utilization of siderophores produced by other microorganisms in the polymicrobial environmental niche of the human nasopharynx colonized by H. influenzae. This is the first report of siderophore utilization by H. influenzae.

Characterization of the Haemophilus influenzae tehB gene and its role in virulence.

The Haemophilus influenzae ORF designated HI1275 in the Rd KW20 genomic sequence encodes a putative S-adenosyl methyltransferase with significant similarity to tellurite-resistance determinants (tehB) in other species. While the H. influenzae tehB can complement an Escherichia coli tehB mutation, thus restoring tellurite resistance, its role in H. influenzae is unknown. In a previous study defining the iron and haem modulon of H. influenzae, we showed that transcription of this gene in H. influenzae Rd KW20 increases during growth in iron- and haem-restricted media. Since iron and haem uptake genes, and other known virulence factors, constitute the majority of the iron- and haem-regulated gene set, we postulated that tehB may play a role in nutrient acquisition and/or the virulence of H. influenzae. A tehB mutant was constructed in the H. influenzae type b strain 10810 and was evaluated for growth defects in various supplemented media, as well as for its ability to cause infection in rat models of infection. Deletion of tehB leads to an increase in sensitivity both to tellurite and to the oxidizing agents cumene hydroperoxide, tert-butyl hydroperoxide and hydrogen peroxide. The tehB mutant additionally showed a significantly reduced ability to utilize free haem as well as several haem-containing moieties including haem-human serum albumin, haemoglobin and haemoglobin-haptoglobin. Examination of the regulation kinetics indicated that transcription of tehB was independent of both tellurite exposure and oxidative stress. Paired comparisons of the tehB mutant and the wild-type H. influenzae strain 10810 showed that tehB is required for wild-type levels of infection in rat models of H. influenzae invasive disease. To our knowledge this is the first report of a role for tehB in virulence in any bacterial species. These data demonstrate that H. influenzae tehB plays a role in both resistance to oxidative damage and haem uptake/utilization, protects H. influenzae from tellurite exposure, and is important for virulence of this organism in an animal model of invasive disease.

The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization.

BACKGROUND: Haemophilus influenzae requires a porphyrin source for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. This porphyrin requirement may be satisfied by either heme alone, or protoporphyrin IX in the presence of an iron source. One protein involved in heme acquisition by H. influenzae is the periplasmic heme binding protein HbpA. HbpA exhibits significant homology to the dipeptide and heme binding protein DppA of Escherichia coli. DppA is a component of the DppABCDF peptide-heme permease of E. coli. H. influenzae homologs of dppBCDF are located in the genome at a point distant from hbpA. The object of this study was to investigate the potential role of the H. influenzae dppBCDF locus in heme utilization. FINDINGS: An insertional mutation in dppC was constructed and the impact of the mutation on the utilization of both free heme and various proteinaceous heme sources as well as utilization of protoporphyrin IX was determined in growth curve studies. The dppC insertion mutant strain was significantly impacted in utilization of all tested heme sources and protoporphyin IX. Complementation of the dppC mutation with an intact dppCBDF gene cluster in trans corrected the growth defects seen in the dppC mutant strain. CONCLUSION: The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae.

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant.

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin-haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.

The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon.

BACKGROUND: Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps in our knowledge remain. Unresolved issues include the identities of all genes exhibiting altered transcription in response to iron and heme availability, the fraction of such genes functioning in iron/heme acquisition, and the heterogeneity of this gene set among clinical isolates. Previously we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those observations by determining the iron/heme modulons of two clinical isolates, the type b isolate 10810 and the nontypeable isolate R2866. These data are used to begin to define the core iron/heme modulon of the species. RESULTS: Microarray studies were performed to compare gene expression on transition from iron/heme-restricted to iron/heme-replete conditions for each isolate. Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed: 233 were maximally transcribed under iron/heme-replete conditions and 130 under iron/heme-restricted conditions. Of the 1883 ORFs representing genes of strain 10810, 353 were significantly differentially transcribed: 150 were preferentially transcribed under iron/heme-replete conditions and 203 under iron/heme-restricted conditions. Comparison of the data sets indicated that 163 genes exhibited similar regulation in both isolates and that 74 of these exhibited similar patterns of regulation in Rd KW20. These comprise the putative core iron/heme modulon. CONCLUSION: This study provides evidence for a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and/or heme in the growth environment. Elucidation of this modulon provides a means to identify genes with unrecognized roles in iron/heme acquisition or homeostasis, unanticipated responsiveness to environmental levels of the micronutrients or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

Catalase as a source of both X- and V-factor for Haemophilus influenzae.

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis.

Lipoprotein e (P4) of Haemophilus influenzae is a phosphomonoesterase, encoded by the hel gene, that has been implicated in the acquisition of heme by this fastidious organism. However, lipoprotein e (P4) is also involved in the utilization of NAD and NMN. Some reports have concluded that the reported heme-related growth defect actually reflects a growth defect for NAD. In the current study, hel insertion mutants were constructed and a role for e (P4) in heme acquisition was demonstrated independent of its role in NAD or NMN acquisition. In addition, a rat model of infection demonstrated a role for e (P4) in the pathogenesis of invasive disease.