Modification of otitis media following vaccination with the capsular polysaccharide of Streptococcus pneumoniae in chinchillas.

An animal model was used to determine whether vaccination with the capsular polysaccharide of Streptococcus pneumoniae could alter the development of experimental otitis media induced by the same type of S. pneumoniae as the vaccine. Following vaccination with the capsular polysaccharide of S. pneumoniae type 7, 36(63%) of 57 chinchillas seroconverted with at least a 100% increase in concentration of antibody in serum which remained elevated for at least six weeks. The middle ears of 23 chinchillas that were vaccinated and seroconverted, 13 that were vaccinated and did not seroconvert, and 47 that were not vaccinated were inoculated with S. pneumoniae type 7. Vaccinated animals that seroconverted developed otitis media as readily as nonseroconverting and unvaccinated animals but had fewer pneumococci in their middle ears, a lower incidence of bacteremia, and lower mortality rates during the first week after inoculation of bacteria. Animals that did not seroconvert showed no evidence of modification of their middle ear infections. These results indicate that type 7 pneumococcal capsular polysaccharide vaccine is antigenic for the chinchilla and modifies experimental otitis media due to type 7 S. pneumoniae.     

Experimental otitis media in chinchillas following nasal colonization with type 7F Streptococcus pneumoniae: prevention after vaccination with pneumococcal capsular polysaccharide.

Chinchillas were colonized intranasally with type 7F Streptococcus pneumoniae, and pneumococcal otitis media developed in greater than 50% of the animals during the first week after negative middle ear pressure (-25 mm Hg) was briefly applied. Twenty-three chinchillas were vaccinated subcutaneously with the capsular polysaccharde of type 7F S. pneumoniae to determine whether vaccination could prevent the development of experimental otitis media. Following vaccination, 14 animals seroconverted with at least a twofold rise in serum antibody concentration; nine animals that were vaccinated did not seroconvert. All of 23 vaccinated animals and 42 of 42 unvaccinated control animals became colonized after intranasal inoculation with pneumococci. Only one (7%) of the vaccinated seroconverting animals developed pneumococcal otitis media, whereas 26 (62%) of the control animals developed middle ear infection with type 7F pneumococci. Four (44%) of nine vaccinated nonseroconverting animals developed pneumococcal otitis media. Protection was associated with high levels of serum antibody prior to intranasal inoculation. Higher antibody levels were found in sterile middle ear effusions than in S. pneumoniae-infected effusions. Vaccination with type 7F pneumococcal capsular polysaccharide significantly reduced the incidence of pneumococcal otitis media following intranasal inoculation of type 7F S. pneumoniae in chinchillas.     

The relative role of bacterial cell wall and capsule in the induction of inflammation in pneumococcal meningitis

The relative contribution of bacterial components to the induction of inflammation during Streptococcus pneumoniae meningitis is unknown. Several strains of pneumococci with differences in cell surface characteristics (capsule or cell wall) were compared for the effect on the inflammatory response evoked during infection of the cerebrospinal fluid (CSF) in vivo. The presence of bacterial capsular polysaccharide was not necessary for bacterial growth in CSF in vivo but correlated with greater CSF bacterial density. CSF inflammatory changes began to appear when the bacterial concentration exceeded 10(5) cfu/ml, regardless of the pneumococcal strain. CSF inflammatory changes could be invoked by cisternal instillation of 10(5)-10(6) cell equivalents of whole, heat-killed unencapsulated strains or their isolated cell walls but not by similar concentrations of heat-killed encapsulated strains or isolated capsular polysaccharide. Hypoglycorrhachia was observed only during inflammation caused by live bacteria. The inflammatory response characteristic of naturally acquired pneumococcal meningitis can be reproduced by challenge with both encapsulated and uncapsulated bacteria. The bacterial cell wall appears to be the most potent pneumococcal surface component in inducing CSF inflammation.     

Pneumococcal carriage and otitis media induce salivary antibodies to pneumococcal surface adhesin a, pneumolysin, and pneumococcal surface protein a in children

Local antibodies probably contribute to defense against Streptococcus pneumoniae. This study examined whether pneumococcal carriage and acute otitis media (AOM) induce mucosal antibodies to potential vaccine candidates pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), and pneumococcal surface protein A (PspA). IgA to all 3 proteins was detected by EIA in saliva of 329 children at ages 6, 12, 18, and 24 months and of 17 adults. A higher proportion of IgA-positive samples and higher antibody concentrations were seen in children with pneumococci-positive cultures of nasopharyngeal samples or middle ear fluid than in children with all cultures negative for pneumococci. The strong correlation between IgA and the presence of the secretory component suggests that the IgA was secretory. The findings indicate that pneumococcal carriage and AOM induce local production of anti-PsaA, anti-Ply, and anti-PspA antibodies early in life.     

Mechanism of killing of pneumococci by lysozyme.

Lysozyme is abundant in respiratory secretions and may play a role in lung host defenses. Mechanisms by which lysozyme killed Streptococcus pneumoniae, an important respiratory pathogen, were studied. Lysozyme caused optical clearing of pneumococcal suspensions and released fragments containing [3H]choline from their cell walls. Electron micrographs revealed wide-spread cell wall destruction and bacteriolysis. Breakdown of the cell wall appeared to be mediated mostly by the major pneumococcal autolysin, N-acetylmuramoyl-L-alanine amidase, because it was blocked by phosphorylcholine, a specific inhibitor of amidase, or by substitution of ethanolamine for choline in the cell wall. Blockade of amidase did not greatly increase survival of lysozyme-treated pneumococci on blood agar. Pneumococci in which amidase was blocked appeared intact immediately after treatment with lysozyme, but when they were reincubated at 37 degrees C in fresh culture medium they swelled and lysed. Thus, widespread triggering of the major pneumococcal autolysin is not essential for the bactericidal effect of lysozyme.     

Lysozyme activity and immunoglobulins in middle ear effusion fluid in acute purulent otitis media and in otitis media with effusion.

147 samples of punctured middle ear effusion fluid from cases of otitis media with effusion and 150 samples from patients with acute purulent otitis media were tested for lysozyme activity. In otitis media with effusion the concentration was 182.0 U/ml, in acute otitis 433.8 U/ml. The lysozyme concentration in otitis media with effusion depended upon the nature of the effusion. Serous fluid showed an activity of 124.8 U/ml and mucoid 311.6 U/ml, respectively. In culture-positive cases of acute otitis media the lysozyme level was 423.4 U/ml. Culture-negative cases showed about the same concentration, 438.3 U/ml. The possible role of lysozyme in defence systems of the middle ear is discussed.     

Experimental otitis media due to Streptococcus pneumoniae: immunopathogenic response in the chinchilla.

Acute otitis media was produced in 110 chinchillas by inoculation of type 23 Streptococcus pneumoniae directly into the middle ear cavity by tympanotomy. During the first three days after inoculation, inflammatory cells were seen in the mucoperiosteum of the middle ear. After four to seven days, there was purulent exudation in the middle ear cavity, and 40% of the animals had pneumococcal meningitis and/or bacteremia. The middle ears were sterile in five of 28 animals sacrificed during the second week and in six of seven animals sacrificed at six weeks, although subepithelial changes persisted in the mucoperiosteum. Levels of antibody to S. pneumoniae in serum were measured by radioimmunoassay; mean values were 6.1 ng of pneumococcal antibody nitrogen/ml in 28 uninfected control animals and 16.5 ng of antibody nitrogen/ml in 29 animals sacrificed two weeks after inoculation (P less than 0.025). Opsonic activity of serum against S. pneumoniae was evaluated in infected and uninfected chinchillas. The opsonic titer was significantly higher in infected animals sacrificed at six weeks than in uninfected controls. Although pneumococcal polysaccharide antigen was found by counterimmunoelectrophoresis in 25 of 30 middle ear effusions, it could not be detected in the serum from infected animals. Methods for infection and sacrifice of chinchillas yielded reproducible results. This model should permit evaluation of the pathologic response to other serotypes of S. pneumoniae and possibly to prophylactic and therapeutic regimes.     

Experimental viral infections of the temporal bone. I. Experimental otitis media due to vaccinia virus in hamsters.

Clinical and pathologic studies of patients with smallpox suggest that conductive hearing loss is a frequent complication of smallpox and that it is due to otitis media. A possible experimental model for this hearing loss has been developed by study of the pathogenesis of acute vaccinia virus infections of hamster middle ears. Infant hamsters developed severe necrotic otitis media, and vaccinia viral antigen was demonstrated by fluorescent antibody staining in large confluent areas of epithelial cells lining the middle ear. Juvenile hamsters developed a patchy, less severe infection of the middle ear, most of which present along the tympanic membrane and middle ear ossicles, Adult hamsters did not develop middle ear infection. These findings support the argument that variola (smallpox) virus can cause otitis media in humans and that viral infection of the middle ear may be the cause of hearing loss.     

Improved host defense against pneumococcal pneumonia in platelet-activating factor receptor-deficient mice

Platelet-activating factor (PAF) is a phospholipid with proinflammatory properties that binds to a specific receptor (PAF receptor [PAFR]) that is expressed on many different cell types. PAFR is able to bind phosphorylcholine, which is present in both PAF and the pneumococcal cell wall. Activation of respiratory epithelial cells in vitro results in up-regulation of PAFR, which, in turn, facilitates invasion of Streptococcus pneumoniae. To determine the role of PAFR in host defense against pneumococcal pneumonia, PAFR-deficient (PAFR(-/-)) and wild-type (wt) mice were inoculated intranasally with S. pneumoniae. PAFR(-/-) mice were relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection (all P<.05, vs. wt mice). PAFR(-/-) mice also had less pulmonary inflammation. These data provide evidence that PAFR is used by S. pneumoniae to induce lethal pneumonia.     

Antibiotic treatment in acute Otitis Media promotes superinfection with resistant Streptococcus pneumoniae carried before initiation of treatment

Antibiotic-resistant pneumococci are difficult to eradicate from middle ear fluid (MEF) and the nasopharynx (NP). Bacteriologic eradication from the NP and MEF during acute otitis media (AOM) by 3 common antibiotic drugs was prospectively evaluated. In 19 (16%) of 119 MEF culture-positive patients, an organism susceptible to the treatment drug (Haemophilus influenzae, Streptococcus pneumoniae, or both) was isolated from the initial MEF, whereas resistant S. pneumoniae was present in the NP; in 9 (47%) patients, the initial resistant NP organism (identified by serotyping, resistance to the administered drug, and pulsed-field gel electrophoresis) replaced the susceptible MEF organism within only a few days after initiation of treatment. In regions where resistant pneumococci are prevalent, antibiotics may not only fail to eradicate the organisms, but they may often induce MEF superinfection with resistant pneumococci initially carried in the NP. This is an important mechanism by which, in recently treated patients, AOM infections often become refractory to treatment.     

Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children

A trial with a 7-valent pneumococcal-conjugate vaccine in children with recurrent acute otitis media showed a shift in pneumococcal colonisation towards non-vaccine serotypes and an increase in Staphylococcus aureus-related acute otitis media after vaccination. We investigated prevalence and determinants of nasopharyngeal carriage of Streptococcus pneumoniae and S aureus in 3198 healthy children aged 1-19 years. Nasopharyngeal carriage of S pneumoniae was detected in 598 (19%) children, and was affected by age (peak incidence at 3 years) and day-care attendance (odds ratio [OR] 2.14, 95% CI 1.44-3.18). S aureus carriage was affected by age (peak incidence at 10 years) and male sex (OR 1.46, 1.25-1.70). Serotyping showed 42% vaccine type pneumococci. We noted a negative correlation for co-colonisation of S aureus and vaccine-type pneumococci (OR 0.68, 0.48-0.94), but not for S aureus and non-vaccine serotypes. These findings suggest a natural competition between colonisation with vaccine-type pneumococci and S aureus, which might explain the increase in S aureus-related otitis media after vaccination.     

Pneumococcal carriage and otitis media induce salivary antibodies to pneumococcal capsular polysaccharides in children

Mucosal immunity likely plays an important role in the defense against Streptococcus pneumoniae. This study examined whether pneumococcal carriage and acute otitis media induce mucosal antibodies to pneumococcal capsular polysaccharides (Pnc-PSs) of types 1, 6B, 11A, 14, 19F, and 23F. Immunoglobulin (Ig) A, IgG, and secretory (S) Ig anti-Pnc-PS antibodies were measured by enzyme immunoassay in the saliva of children at ages 6, 12, 18, and 24 months and were analyzed in relation to the previous pneumococcal findings. A larger proportion of IgA-positive samples and higher concentrations of type-specific IgA antibodies were detected in samples of children with pneumococci of the given types cultured before sampling from nasopharyngeal samples or middle-ear fluid, compared with children who had cultures negative for pneumococci of the indicated types or of all types. The IgA and S-Ig concentrations correlated strongly, which suggests that the anti-Pnc-PS IgA was secretory. Salivary anti-Pnc-PS IgG was detected only rarely.     

The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio.

Streptococcus pneumoniae, type 23F, resistant to penicillin (MIC, 2 micrograms/mL) and multiple other antimicrobic agents, was isolated from middle ear fluid of a child with otitis media attending a day care center in Ohio. To determine the extent of spread of this strain, nasopharyngeal culture surveys were done, and 52 carriers were identified among 250 children attending the index day care center. No carriers were found among 121 children at two other day care centers in the same urban area. Use of prophylactic doses of antibiotics (P < .001) and frequent use of antibiotics (P < 0.001) were risk factors for nasopharyngeal carriage. Carriers were more likely to have had frequent otitis media episodes (P < .02) and otitis media not responsive to antimicrobial therapy (P < .001). Strategies to limit the spread of highly resistant pneumococcal strains should include encouraging judicious use of antimicrobic agents and reevaluating indications for prophylactic use of antimicrobic agents.     

A pneumococcal pilus influences virulence and host inflammatory responses

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.     

The contribution of specific pneumococcal serogroups to different disease manifestations: implications for conjugate vaccine formulation and use, part II.

To assess whether certain serogroups of Streptococcus pneumoniae are preferentially associated with specific disease manifestations, we analyzed all recent pneumococcal disease studies and assessed the relative frequency of isolation of each serogroup by clinical site (as a proxy for different disease states). In all age groups, serogroups 1 and 14 were more often isolated from blood, and serogroups 6, 10, and 23 were more often isolated from cerebrospinal fluid (CSF); in young children, serogroups 3, 19, and 23 were more often isolated from middle ear fluid (MEF). Serogroups represented in conjugate vaccines were isolated slightly less frequently from CSF than from blood or MEF. Nonetheless, serogroups in the 9-valent conjugate vaccine formulation still comprised approximately 75% of pneumococcal isolates from the CSF of young children in Europe and in the United States and Canada. These analyses indicate that pneumococcal conjugate vaccines could potentially prevent a substantial proportion of episodes of bacteremic disease, pneumonia, meningitis, and otitis media, especially in young children.     

Effective treatment of experimental acute otitis media by application of volatile fluids into the ear canal

Essential oils are volatile and can have good antimicrobial activity. We compared the effects of oil of basil (Ocimum basilicum) and essential oil components (thymol, carvacrol, and salicylaldehyde) to those of a placebo when placed in the ear canal of rats with experimental acute otitis media caused by pneumococci or Haemophilus influenzae. Progress was monitored by otomicroscopic examination and middle ear cultures. The treatment with oil of basil or essential oil components cured or healed 56%-81% of rats infected with H. influenzae and 6%-75% of rats infected with pneumococci, compared with 5.6%-6% of rats in the placebo group. Essential oils or their components placed in the ear canal can provide effective treatment of acute otitis media.     

Demonstration of Chlamydia pneumoniae in the adenoid from children with and without secretory otitis media using immunohistochemistry and PCR

Chlamydia pneumoniae has been found in patients with middle ear inflammation. The adenoid, which has a central role in the development of secretory otitis media (SOM), may act as a reservoir for bacteria causing ear infection. Adenoid tissue was examined for the presence of C. pneumoniae. Twenty children undergoing adenoidectomy because of hyperplastic adenoids, 10 with SOM and 10 without SOM, were examined with nasopharyngeal swabs for routine bacteriological culture, serology for C. pneumoniae and throat swabs for C. pneumoniae PCR. The removed tissues were analyzed for C. pneumoniae using immunohistochemical (IHC) analysis and PCR. In the group of children with SOM samples were also taken from the middle ear fluid for routine bacteriological culture and PCR for C. pneumoniae. C. pneumoniae was found in the adenoid by PCR in 3 cases from each group and from all 20 children by IHC. Four children in each group had increased levels of specific antibodies to C. pneumoniae. Two children with SOM had high antibody titers and a positive PCR from a throat swab. Two children were PCR-positive for C. pneumoniae in fluid from the middle ear. The significance of these findings is not yet clear.     

Role of Streptococcus pneumoniae and Haemophilus influenzae in the development of acute otitis media and otitis media with effusion in a gerbil model

The efficacy of amoxicillin/clavulanate and cefuroxime was determined in a gerbil model of otitis media with a mixed Streptococcus pneumoniae plus Haemophilus influenzae middle ear (ME) infection. Results were compared with those obtained in a previous single H. influenzae model. All untreated animals inoculated with the mixed inoculum developed acute otitis media (AOM), whereas 86.7% of those inoculated with H. influenzae developed otitis media with effusion (OME). Antibiotics eradicated H. influenzae from the ME more efficiently in AOM than in OME, and this difference was highly significant (P80% of animals developed culture-negative OME.     

Interaction between Streptococcus pneumoniae and Branhamella catarrhalis obtained from double-colonized, healthy nasopharynx and double-infected, diseased middle ear cavity

Streptococcus pneumoniae and Branhamella catarrhalis were obtained from the double-colonized nasopharynx of 3 healthy carriers and from the double-infected middle ear cavity of 3 patients suffering from acute otitis media. The bacterial strains were isolated and injected both separately and together into brain-heart infusion broth. Separately injected, both S. pneumoniae and B. catarrhalis survived in the broth for at least 48 h. When injected together, B. catarrhalis was completely suppressed after 16-24 h, whereas S. pneumoniae survived for at least 48 h. Thus S. pneumoniae had a remarkable inhibitory growth effect on B. catarrhalis, which can explain why double-infected middle ear cavities so seldom are found during acute otitis media.     

Innate Immunity and the Role of Defensins in Otitis Media

Otitis media is the most common pediatric disease in developed countries and a significant cause of morbidity and hearing loss in developing countries. The innate immune system is essential to protecting the middle ear from infection. Defensins, broad-spectrum cationic antimicrobial peptides, have been implicated in prevention of and the early response to acute otitis media; however, the mechanisms by which defensins and other antimicrobial molecules mediate this protection have not been completely elucidated. In both animal otitis media models and human middle ear epithelial cell culture models, β-defensins are highly induced and effectively kill the common pathogens associated with otitis media. We review the importance of innate immunity in protecting the middle ear and recent advances in understanding the roles of defensins and other antimicrobial molecules in the prevention and treatment of otitis media. The extremely high prevalence of otitis media, in spite of sophisticated innate and adaptive immune systems, is a vexing problem for clinicians and scientists. We therefore also review mechanisms by which bacteria evade innate immune defenses.