Chinchilla Otitis Media Research Articles

Increased Virulence of an Encapsulated Streptococcus pneumoniae Upon Expression of Pneumococcal Surface Protein K

Current Streptococcus pneumoniae vaccines selectively target capsular polysaccharide of specific serotypes, leading to an increase in nonencapsulated S. pneumoniae (NESp). Cocolonization by encapsulated pneumococci and NESp increases the opportunity for intraspecies genetic exchange. Acquisition of NESp genes by encapsulated pneumococci could alter virulence and help vaccine-targeted serotypes persist in the host. Adhesion and invasion assays were performed using immortalized human pharyngeal or lung epithelial cells. In vivo models assessing murine nasopharyngeal colonization and pneumonia, as well as chinchilla otitis media (OM), were also used. Pneumococcal surface protein K (PspK) expression increased encapsulated pneumococcal adhesion and invasion of lung cells and enhanced virulence during pneumonia and OM. Additionally, PspK increased nasopharyngeal colonization, persistence in the lungs, and persistence in the middle ear when expressed in a capsule deletion mutant. Competition experiments demonstrated encapsulated pneumococci expressing PspK also had a selective advantage in both the lungs and nasopharynx. PspK increases pneumococcal virulence during pneumonia and OM. PspK also partially compensates for loss of virulence in the absence of capsule. Additionally, PspK provides a selective advantage in a competitive environment. Therefore, acquisition of PspK increases encapsulated virulence in a condition-dependent manner. Together, these studies demonstrate risks associated with pneumococcal intraspecies genetic exchange.

Treatment of otitis media by transtympanic delivery of antibiotics.

Otitis media is the most common reason U.S. children receive antibiotics. The requisite 7- to 10-day course of oral antibiotics can be challenging to deliver in children, entails potential systemic toxicity, and encourages selection of antimicrobial-resistant bacteria. We developed a drug delivery system that, when applied once to the tympanic membrane through the external auditory canal, delivers an entire course of antimicrobial therapy to the middle ear. A pentablock copolymer poloxamer 407-polybutylphosphoester (P407-PBP) was designed to flow easily during application and then to form a mechanically strong hydrogel on the tympanic membrane. U.S. Food and Drug Administration-approved chemical permeation enhancers within the hydrogel assisted flux of the antibiotic ciprofloxacin across the membrane. This drug delivery system completely eradicated otitis media from nontypable Haemophilus influenzae (NTHi) in 10 of 10 chinchillas, whereas only 62.5% of animals receiving 1% ciprofloxacin alone had cleared the infection by day 7. The hydrogel system was biocompatible in the ear, and ciprofloxacin was undetectable systemically (in blood), confirming local drug delivery and activity. This fast-gelling hydrogel could improve compliance, minimize side effects, and prevent systemic distribution of antibiotics in one of the most common pediatric illnesses, possibly minimizing the development of antibiotic resistance.

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.

Virulence phenotypes of low-passage clinical isolates of nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media.

BackgroundThe nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease.ResultsThe local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA – PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME.ConclusionCollectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

A Mutation in the sap Operon Attenuates Survival of Nontypeable Haemophilus influenzae in a Chinchilla Model of Otitis Media

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla beta-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.

Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin–haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media

Since Haemophilus influenzae lacks enzymes necessary for synthesis of the porphyrin ring, it has an absolute growth requirement for a porphyrin source. This requirement can be satisfied in vitro by hemoglobin and hemoglobin complexed to haptoglobin. The products of the hgp genes mediate the utilization of heme from hemoglobin–haptoglobin. These genes are also involved in the use of heme from hemoglobin, although additional gene products independently mediate the acquisition of heme from this substrate. Different strains of H. influenzae possess one to four hgp genes. A nontypeable H. influenzae mutant lacking all the hgp genes was constructed and compared to the wild-type strain in a chinchilla ( Chinchilla lanigera) model of otitis media. Compared to the wild-type strain, the hgp-deficient mutant exhibited a significantly delayed onset of detectable middle ear infection and significantly reduced duration of infection as assessed by both video otoscopy and tympanometry and as evidenced by viable bacterial counts in middle ear effusions. In addition, the maximum bacterial load in the middle ears of chinchillas infected with the mutant strain was significantly reduced when compared to the parent. These data indicate that the hemoglobin/hemoglobin–haptoglobin binding proteins are required for bacterial proliferation during H. influenzae-induced otitis media in chinchillas.

Progress in the prevention of otitis media through immunization.

To review the progress that has been made in developing effective vaccines against the major bacterial pathogens responsible for acute otitis media. Review of the literature with the aid of the MEDLINE database using the search terms otitis media and otitis media and vaccine. Data were collected from clinical trials and laboratory studies. The heptavalent pneumococcal conjugated vaccine, Prevnar, reduced the incidence of acute otitis media from all causes by 7% in one study and by 6% in another study. For culture-positive pneumococcal otitis media, the point estimate of efficacy was 66.7% in one study, and the reduction in incidence was 34% in another study. A Phase I clinical trial has been completed successfully for a conjugated vaccine against nontypeable Haemophilus influenzae (NTHi), which has high immunogenicity for mice and rabbits, induces complement-mediated bactericidal activity against NTHi in rabbits, and is protective against NTHi otitis media in chinchillas. A conjugated vaccine against Moraxella catarrhalis elicits strong immune responses in mice and rabbits and induces complement-mediated bactericidal activity in rabbits. The prevention of otitis media is likely to require multivalent pneumococcal, NTHi, and M. catarrhalis vaccines, and these vaccines likely can be developed within a decade.

Passive transfer of antiserum specific for immunogens derived from a nontypeable Haemophilus influenzae adhesin and lipoprotein D prevents otitis media after heterologous challenge.

We recently determined that passive transfer of serum directed against a synthetic peptide called LB1 or a recombinant fusion protein immunogen [LPD-LB1(f)(2,1,3)] could prevent otitis media after challenge with a homologous nontypeable Haemophilus influenzae (NTHI) isolate. NTHI residing in the nasopharynx was rapidly cleared from this site, thus preventing it from ascending the eustachian tube and inducing otitis media in chinchillas compromised by an ongoing viral upper respiratory tract infection. While LB1 is based solely on one NTHI adhesin, the latter immunogen, LPD-LB1(f)(2,1,3), was designed to incorporate two NTHI antigens shown to play a role in the pathogenesis of otitis media; lipoprotein D (LPD) and the P5-homologous fimbrin adhesin. The design of LPD-LB1(f)(2,1,3) also accommodated for the recently demonstrated existence of three major groupings, based on amino acid sequence diversity, in the third surface-exposed region of P5-fimbrin. LPD-LB1(f)(2,1,3) was thus designed to potentially confer broader protection against challenge by diverse strains of NTHI. Chinchillas were passively immunized here with serum specific for either LB1 or for LPD-LB1(f)(2,1,3) prior to challenge with a member of all three groups of NTHI relative to diversity in region 3. The transferred serum pools were also analyzed for titer, specificity, and several functional activities. We found that both serum pools had equivalent ability to mediate C'-dependent killing and to inhibit adherence of NTHI strains to human oropharyngeal cells. When passively transferred, both serum pools significantly inhibited the signs and incidence of otitis media (P </= 0.01) induced by any of the three challenge isolates. Despite providing protection against disease, the ability of these antisera to induce total eradication of NTHI from the nasopharynx was not equivalent among NTHI groups. These data thus suggested that while early, complete eradication of NTHI from the nasopharynx was highly protective, reduction of the bacterial load to below a critical threshold level appeared to be similarly effective.

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.

Detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins confers protection against otitis media in chinchillas.

Detoxified-lipooligosaccharide (dLOS)-protein conjugates from nontypeable Haemophilus influenzae (NTHi) elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (X.-X. Gu, C.-M. Tsai, T. Ueyama, S. J. Barenkamp, J. B. Robbins, and D. J. Lim, Infect. Immun. 64:4047-4053, 1996). In this study, we evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-conjugated tetanus toxoid, dLOS-conjugated high-molecular-weight proteins from NTHi, or saline (control) in Freund's adjuvant and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS antibody, and 49% (19 of 39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all 19 controls and 56% (22 of 39) of the vaccinated animals during a period of 21 days (P < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (P < 0.01). The incidences of infection in the unchallenged ear or inner ear were 26 or 28% in the vaccine groups and 53 or 58% in the controls (P < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicate that active immunization with LOS-based conjugates reduces the incidence of NTHi-induced otitis media.

Inhibition of proteases in Pseudomonas otitis media in chinchillas

The treatment of chronic suppurative otitis media caused by Pseudomonas aeruginosa remains a challenging problem. The virulence of Pseudomonas is related to its secretion of two matrix metalloproteinases, alkaline protease and elastase. This experiment examines the effects of a synthetic inhibitor of matrix metalloproteinases GM 6001, or N-[2(R)-2-(hydroxyamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide), in a chinchilla Pseudomonas otitis media model. Thirty chinchillas underwent bilateral subtotal tympanic membrane perforations. Twenty-four chinchillas underwent bilateral middle ear inoculation with P. aeruginosa. Chinchillas were divided into four groups of six animals after the establishment of otitis media. Animals in one group were controls; the other three groups received either gentamicin, GM 6001, or gentamicin plus GM 6001 into the external auditory canal three times daily for 4 weeks. Clearance of Pseudomonas infection occurred in three ears of three animals, all in gentamicin groups, with or without GM 6001. Otorrhea ( p = 0.0014) and external canal erythema ( p = 0.025) were mild in the two gentamicin groups and moderate in the GM 6001 group when compared with bacterial controls. Animals in the GM 6001 group had the highest survival rate, less severe facial paralysis, and less vestibular toxicity than the gentamicin, gentamicin plus GM 6001, or control groups, although these differences were not statistically significant. This pilot study showed encouraging results for a role of ototopical protease inhibitors in the treatment of Pseudomonas otitis media. (Otolaryngol Head Neck Surg 1996;115:342-51.)

Effect of Prostaglandin, Leukotriene, and Arachidonic Acid on Experimental Otitis Media with Effusion in Chinchillas

Although previous studies have shown that prostaglandins (PGs), leukotrienes (LTs), and other arachidonic acid (AA) metabolites play an important role in the pathogenesis of otitis media with effusion (OME), the exact role of each AA metabolite in OME is still unknown. The purpose of this study was to examine the effect of several individual AA metabolites alone or in combination and AA itself on experimental otitis media in chinchillas. Normal chinchillas were inoculated daily with normal saline, PGE2, LTC4, LTC4 + PGE2, and AA through the superior bullae over 7 days. Animals were followed by otoscopy and tympanometry, samples of middle ear effusion were collected for biochemical assay, and temporal bones were processed for histopathology. The highest number of ears that developed OME was in the group inoculated with PGE2 + LTC4. The degree of inflammatory change was more pronounced in groups injected with LTC4 than any other group. The findings of this study suggest that eicosanoids PGE2, LTC4, and AA alone or in combination inoculated into the middle ear can induce OME.

Proteases and Protease Inhibitors in Immune Complex Otitis Media in Chinchillas

Using an immune complex-induced (IC) otitis media model in chinchillas sensitized with human serum albumin (HSA), we measured the levels of protease activity, protease inhibitors, and HSA in the middle ear fluid (MEF). The effect of a corticosteroid agent (triamcinolone) on the degree of IC otitis media also was studied. The levels of protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin in the MEF were significantly higher than in nonsensitized animals. Fibrinolytic (plasminogen activator [PA]) activity was detected in the MEF; however, only a small level of activity of nonspecific proteases was detected because of the significant level of antitryptic activity in the MEF. The levels of protease inhibitors and PA activity were significantly reduced by the steroid treatment. It is concluded that protease inhibitors play an important role in the protection of the middle ear mucosa and that corticosteroid treatment can reduce the severity of IC otitis media.

Antiinflammatory Effects of a Corticosteroid and Protease Inhibitor Agents on Antigen-Induced Otitis Media in Chinchillas

Antiinflammatory Effects of a Corticosteroid and Protease Inhibitor Agents on Antigen-Induced Otitis Media in Chinchillas

Recurrence of antigen-inducedotitis media by thiol compound

Reduced glutathione (GSH), a potential factor in the chronicity of human otitis media, was tested to determine its potency in the recurrence of antigen-induced otitis media in chinchillas sensitized with human serum albumin (HSA). GSH instillation into the healing middle ear bulla reproduced a level of otitis media similar to the original antigen-induced otitis media with cellular involvement occurring earlier, according to cytologic analyses. This study indicates that GSH is probably a factor in recurrent antigen-induced otitis media, controlling the balance between thiol proteases and thiol protease inhibitors during middle ear inflammation.

Antiinflammatory effects of a corticosteroid and protease inhibitor agents on antigen-induced otitis media in chinchillas

Using an antigen-induced otitis media (OM) model in chinchillas sensitized with human serum albumin (HSA), we studied the antiinflammatory effect of a corticosteroid (triamcinolone) and a protease (kallikrein) inhibitor (aprotinin) by conducting both biochemical and cytologic analyses of middle ear fluid (MEF). The levels of HSA, alpha 1-antitrypsin, alpha 2-macroglobulin, and total protein concentration were measured in the MEF to evaluate the degree of OM. Both vascular leakage and leukocyte infiltration were significantly reduced by corticosteroid and inhibitor treatments. HSA levels in the MEF were markedly reduced after HSA challenge, and both treatments reduced the drop in HSA levels. In conclusion, the kallikrein-kinin system is related to the early stage of antigen-induced OM. Both corticosteroid and inhibitor treatments effectively reduce the degree of antigen-induced OM in chinchillas, suggesting that both may be useful local therapeutic agents in the treatment of human OM.

Prevention of pneumococcal otitis media in chinchillas with human bacterial polysaccharide immune globulin

Clinical and experimental observations suggest that immune globulin may prevent otitis media (OM) in children. We performed experiments in chinchillas to test the hypothesis that human bacterial polysaccharide immune globulin (BPIG) might prevent OM caused by Streptococcus pneumoniae. Animals were given BPIG or saline intraperitoneally on day 0. On day 3 the epitympanic bulla was inoculated with S. pneumoniae type 7F. All 12 saline-treated and none of 12 BPIG-treated animals developed pneumococcal OM by day 7 (P less than 0.0001). Bacteremia developed in 6 of 12 saline- and 0 of 12 BPIG-treated animals (P = 0.007). Death with pneumococcal OM occurred within 28 days in 5 of 12 saline- and 0 of 12 BPIG-injected animals (P = 0.02). A chinchilla-specific immunoassay was used to show that surviving saline-injected animals developed serum anticapsular antibody; BPIG-treated animals had no detectable response. At levels of anticapsular immunoglobulin G similar to those of human adults, BPIG given systemically prevented pneumococcal OM and disseminated infection in chinchillas. BPIG may be of value in preventing human bacterial infection and may also inhibit development of antibody if it affects local infection or colonization. Specific immunoglobulin G antibody may provide an important antibacterial defense of mucosal surfaces of the respiratory tract.

The Contribution of Pneumococcal Cell Wall to the Pathogenesis of Experimental Otitis Media

We studied the contribution of pneumococcal cell wall to the pathogenesis of otitis media in chinchillas after middle ear inoculation of killed, encapsulated type 7F Streptococcus pneumoniae; killed, unencapsulated R6 S. pneumoniae; and isolated R6 pneumococcal cell wall. Ears inoculated with encapsulated and unencapsulated pneumococci had significantly higher concentrations of polymorphonuclear and mononuclear leukocytes and lysozyme in middle ear fluid and developed more epithelial metaplasia and granulation tissue than did saline-inoculated ears. The mean concentration of lysozyme in middle ear fluid was higher in ears inoculated with killed, unencapsulated than encapsulated pneumococci. The middle ear mucoperiosteum of ears inoculated with pneumococcal cell wall showed significantly more polymorphonuclear leukocytes, epithelial metaplasia, subepithelial congestion, and granulation tissue than did control ears. Because nonviable, unencapsulated pneumococci and pneumococcal cell wall caused middle ear inflammation in the chinchilla model of otitis media, it is possible that cell envelope and cell wall components released during bacterial lysis may contribute to chronic otitis media with effusion in humans.

Histopathology of Animal Models of Otitis Media in Chinchillas

Histopathology of Animal Models of Otitis Media in Chinchillas

Modification of otitis media in chinchillas rechallenged with nontypable Haemophilus influenzae and serological response to outer membrane antigens.

Otitis media was produced in chinchillas by right-sided intrabullar inoculation with nontypable Haemophilus influenzae, and susceptibility to reinfection was investigated. After resolution of initial right-sided infection, animals underwent ipsilateral or contralateral intrabullar rechallenge with the same strain. After ipsilateral rechallenge right ears were completely protected against reinfection; previously uninfected left ears were similarly protected on contralateral rechallenge. Previously infected ears remained fully susceptible to infection with a heterologous strain of nontypable H. influenzae. Using an enzyme-linked immunosorbent assay, we measured the serological response to outer membrane protein and lipopolysaccharide antigens during initial infection. A greater than or equal to 10-fold rise in titer of antibody to homologous outer membrane proteins was observed in all 11 animals tested. Most animals exhibited a minimal serological response to lipopolysaccharide. Thus experimental otitis media due to nontypable H. influenzae induces strain-specific protective immunity and a concomitant serological response to outer membrane proteins.