Bacterial Adherence to Pharyngeal Cells: In Vitro Studies with Alpha-haemolytic Streptococci and Haemophilus influenzae

We examined the adherence to pharyngeal cells of alpha-haemolytic Streptococci (AHS) and Haemophilus influenzae , representing normal flora and otitis media (OM) pathogens, respectively. The bacteria were incubated with epithelial cells brushed from the tonsils, adenoid or tubal orifice of children and adults. Adherence varied among the clinical isolates of AHS and H. influenzae . AHS adhered better to epithelial cells from a child compared with those sampled from an adult. The bacteria adhered better to cells from the tubal orifice compared with those sampled from the adenoid. The selective attachment of AHS to certain cells but not to others could not be correlated to apoptotic/necrotic cells versus viable cells. Incubation of epithelial cells with an isolate of AHS with good inhibitory activity against OM pathogens showed almost no adherence of bacteria to the epithelial cells after 12 and 24 h of incubation. If, however, an isolate of AHS with weak inhibitory activity was incubated with the cells, the bacteria that were attached to the epithelial cells from the beginning showed overgrowth in the broth and increasing attachment to the cells after 12 and 24 h. Thus the inhibitory activity of AHS could also affect the adherence of potential pathogens to the mucosal surfaces. The adherence pattern may at least partially explain the difference in susceptibility to OM between children and adults.     

Outcome of Acute Otitis Media and its Relation to Clinical Features and Nasopharyngeal Colonization at the Time of Diagnosis

Children ( n = 115; age range 1-9 years) with a diagnosis of acute otitis media (AOM) were eligible for this study and were enrolled within 24 h of the onset of symptoms signs. A nasopharyngeal culture was obtained at the initial visit. Children were treated with a single oral antibiotic for 7 days. Changes in symptoms signs and tympanic membrane features assessed by a scoring system were monitored for 1 month and related to the nasopharyngeal pathogen recovered on Day 1. More than 80% of children had no symptoms signs by Day 3, and 60% of cases had normal tympanic membranes by Day 29. Children without any symptoms signs on Day 3 had significantly lower symptom sign scores on Day 1 ( p = 0.005). Seventy-nine percent of cases carried middle ear pathogens in the nasopharynx at diagnosis: Streptococcus pneumoniae (48%); Haemophilus influenzae (24%); and Moraxella catarrhalis (16%). Children with S. pneumoniae showed significantly higher tympanic membrane scores than children with no pathogen at Days 8, 15 and 29 ( p < 0.01 for each comparison). Multivariate regression analysis revealed that lower tympanic membrane score on Day 1, the absence of S. pneumoniae in the nasopharynx and treatment with amoxillin were independent factors for rapid normalization of the tympanic membrane. These data suggest that the clinical course of AOM may be predicted, in part, at the time of diagnosis by means of careful evaluation of symptoms signs and the tympanic membrane as well as knowledge of pathogens harbored in the nasopharynx.     

Early Structural Tympanic Membrane Reactions to Myringotomy: A Study in an Acute Otitis Media Model

Myringotomy (Myr) is one of the most frequently performed surgical procedures in children. However, events occurring in the early phases, i.e. a matter of hours, following Myr in the acute otitis media (AOM) model have not been described. The aim of the present study was to evaluate the early otomicroscopic and histopathologic reactions of the tympanic membrane (TM) after Myr during the course of AOM (AOM-Myr). The left tympanic bulla from 36 healthy Sprague-Dawley rats was inoculated with Streptococcus pneumoniae type 3. Forty-eight h later, at Day 0, 4 randomized animals were immediately sacrificed and the remaining animals were treated with bilateral Myr. Otomicroscopy and sacrifices were performed in series of 4 animals at 3, 6, 9, 12, 24 and 48 h, and 4 and 7 days. The AOM-Myr TMs were compared to non-infected Myr TMs (non-AOM-Myr). The TMs were then dissected free and routinely processed for light and electron microscopy. AOM developed in all inoculated ears at Day 0. In the pars tensa of the AOM-Myr TMs the reaction of the keratinocyte layer of the perforation border was already evident at 6 h. The lamina propria exhibited a strong inflammatory reaction, which became more organized from 12 h onwards. At Day 4 the perforations were closed in three-quarters of cases. At Day 7 all perforations were healed with a distorted scar. In the non-AOM Myr TMs a strong degranulation of mast cells and edema were found in the pars flaccida at 6 h. A keratin spur at the perforation border was not seen until 24 h. All perforations were patent on Day 7 and myringosclerotic deposits were abundant in these TMs. The infected TMs regenerated faster and closed their perforations at an earlier stage. These findings favor the hypothesis that there is a low risk of chronic perforations when myringotomizing AOM TMs.     

Maternal Pneumococcal Conjugate Immunization Protects Infant Chinchillas in the Pneumococcal Otitis Media Model

Infant immunization with pneumococcal polysaccharide-protein conjugate vaccines (PCVs) is unlikely to elicit protective serum antibody concentrations during the first 4-6 months of life, when recurrent pneumococcal otitis media (POM) often begins. We therefore investigated a maternal pneumococcal immunization strategy to prevent early infant POM. Pregnant chinchillas (dams) received injections of heptavalent PCV or saline. Post-partum maternal and infant (kits) blood samples were obtained, and kits were subsequently challenged by intranasal inoculation of a vaccine-type pneumococcal strain (19F). Anti-pneumococcal capsular polysaccharide IgG antibody (Ab) concentration was measured using an ELISA in maternal and kit serum samples. Immunized dams and their kits had significantly higher Ab titers than control dams and their kits. Antibody titer in kits declined with a half-life of 12 days. Maternal immunization significantly reduced both the incidence ( p = 0.05) and severity ( p < 0.01) of experimental POM in chinchilla kits, and was 82% effective at preventing mortality from invasive pneumococcal disease. Pre-challenge serum Ab concentration in kits was the single best predictor of POM severity ( r = -0.66). This experiment strongly supports the hypothesis that maternal immunization with PCV will reduce the burden of early infant POM and invasive pneumococcal disease.     

Endoscopic Adenoidectomy in Children With Otitis Media With Effusion and Mild Hearing Loss

ObjectivesSurgical management of children with chronic otitis media with effusion (OME) includes tympanostomy tube insertion or adenoidectomy, alone or with myringotomy and tube insertion. The aim of this study was to compare the effectiveness of transoral microdebrider endoscopic-assisted adenoidectomy (TOMEA) and traditional adenoidectomy in the management of children with mild hearing loss due to OME and chronic adenoiditis.MethodsThis prospective, double-blind and controlled study involved 120 consecutive patients aged 4–12 years, who were randomised 1:1 to undergo TOMEA or traditional adenoidectomy under general anesthesia. All the patients underwent a complete otolaryngological examination, including nasopharyngeal fibre endoscopy (NFE), pneumatic otoscopy, otomicroscopy, tympanometry and supraliminar tonal audiometry, upon enrolment, and three and nine months postoperatively.ResultsThere were no statistically significant differences in age or gender distribution between the TOMEA group (mean age, 4.9±1.1 years; 53.3% males) and the traditional adenoidectomy group (mean age, 5.3±0.9 years; 56.7% males). Both procedures led to a significant improvement in choanal patency (P<0.01) and all of the otological and audiological parameters (P<0.01) 3 and 9 months postoperatively, although postoperative NFE showed that the mean percentage of residual choanal obstruction was significantly less in the TOMEA group (P=0.02). There was no significant between-group difference in the percentage of children with tympanic membrane changes, but the postoperative prevalence of children with a type B tympanogram was significantly lower in the TOMEA group after 3 (15.0% vs. 31.7%, P=0.05) and 9 months (18.3% vs. 38.3%, P=0.02), as was the percentage of children with mild conductive hearing loss (3.3% vs. 23.3%, P<0.01; and 8.3% vs. 28.3%, P<0.01).ConclusionAlthough both TOMEA and traditional adenoidectomy are effective in treating children with mild hearing loss due to adenoidal hypertrophy and OME, the former achieves the greater reduction in residual adenoidal hypertrophy and better audiological outcomes.     

Myringotomy Delays the Tympanic Membrane Recovery in Acute Otitis Media: A Study in the Rat Model

Background/Hypothesis Acute otitis media is a major cause of visits to pediatric health care providers. Myringotomy in uncomplicated acute otitis media is debatable today. The study addressed this problem through the otomicroscopic and histopathological observations of the events occurring in the tympanic membrane during the first week after myringotomy. Study Design Randomized study in an experimental animal model. Methods Under anesthesia, the left middle ear of 36 Sprague‐Dawley rats was inoculated with Streptococcus pneumoniae type 3. Forty‐eight hours later, at day 0, four animals were immediately killed and the remaining animals were randomly assigned into a myringotomy group (n = 16, myringotomy on the left ear) and a non‐myringotomy group (n = 16, otomicroscopy without myringotomy). Otomicroscopy and killings were performed in series of four animals from each group at days 1, 2, 4, and 7 after myringotomy. Tympanic membranes were collected after fixation and processed for light and electron microscopy. Results All inoculated ears showed a manifest acute otitis media at day 0. An intense infiltration by inflammatory cells and edema distorted severely the tympanic membrane structure. These findings decreased the following days. However, inflammation as evaluated by the thickness and the cytoarchitecture of the tympanic membrane layers, recovered significantly faster in the membranes in the non‐myringotomy group. At day 7, all tympanic membranes in the myringotomy group were closed by a hypertrophic keratinizing epithelium and a remodeling connective tissue layer, whereas the animals in the non‐myringotomy group had a residual edema in the lamina propria. Conclusions The present infectious model induced an intense inflammatory reaction within the entire structure of the tympanic membrane. Myringotomy provoked a delayed recovery from the inflammatory process within the tympanic membrane. Therefore, if applicable to human conditions, the use of myringotomy in the management of acute otitis media should be restricted to selected cases of acute otitis media.     

The Evaluation of Oxidative Stress in the Serum and Tissue Specimens of Patients With Chronic Otitis Media

Objectives

To underline the effect of oxidative stress in chronic otitis media with and without cholesteatoma and to compare the oxidative stress values in the serum and tissue specimens in these two forms.

Methods

The study included a total of 75 individuals, 35 cases with chronic otitis media (COM; 16 females and 19 males) and a healthy control group of 40 cases (20 females and 20 males). The COM patient group was comprised of 18 patients with cholesteatoma and 17 patients without cholesteatoma. All patients underwent mastoidectomy. Serum specimens were taken prior to surgery and diseased tissue specimens from the ear were obtained during surgery from all patients. Only serum specimens were taken from the healthy control cases. The malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GHPx) were measured in the serum and tissue samples of the patient group and in the serum specimens of the control group.

Results

The age ranged from 14 to 48 years in the patient group (mean age, 20.4±12.2 years) and from 19 to 40 years in the control group (mean age, 26.4±4.64 years). When the serum values of all COM patients were compared with those of the control group, in the patient group MDA, which reflects lipid peroxidation, was found to be significantly higher (P<0.01) whereas the antioxidant enzymes SOD, CAT, and GHPx were found to be significantly lower (P<0.01). When the serum and tissue MDA, SOD, CAT, and GHPx values in patients with and without cholesteatoma were compared, no significant difference was found these parameters (P>0.01).

Conclusion

Although oxidative stress plays a role in the pathogenesis of COM with or without cholesteatoma, it may not reflect the severity of the disease. In patients with COM, the evaluation of only serum oxidative stress values without tissue evaluation may be sufficient for assessing oxidative stress.     

IL-5 and Eotaxin Levels in Middle Ear Effusion and Blood from Asthmatics with Otitis Media with Effusion

Objective--The purpose of this study was to evaluate eosinophil infiltration as well as IL-5 and eotaxin levels in middle ear effusion (MEE) and blood from otitis media with effusion (OME) patients with asthma and to compare the findings with those from OME patients without asthma (control group). Material and methods--Levels of IL-5 and eotaxin in MEE and blood were measured by means of enzyme-linked immunosorbent assay. Results--IL-5 levels in MEE were significantly higher than those in blood in both groups of patients and in OME patients with asthma than in the control group. In addition, in OME patients with asthma, there was a significant correlation between the percentage of eosinophils and IL-5 levels in MEE. Eotaxin levels in blood were significantly higher than those in MEE in both groups of patients and in OME patients with asthma than in the control group. In addition, in OME patients with asthma, the percentage of eosinophils and eotaxin levels in blood tended to correlate, but did not reach statistical significance. Conclusion--These data suggest that, in OME patients with asthma, eosinophilia in MEE depends more on IL-5 than on eotaxin, and that eotaxin may play an important role in the mobilization of eosinophils from the bone marrow into the blood.     

In VitroStreptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

Objectives

Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM.

Methods

In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1×10⁷ colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation.

Results

The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 µm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively.

Conclusion

The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.     

Changing Patterns of Bacterial Strains in Adults and Children With Otitis Media in Korean Tertiary Care Centers

Objectives

Otitis media (OM) is an infectious disease that affects all age brackets. Aural discharge is a typical symptom, occurring in all subtypes of OM. We have compared the identity and antibiotic sensitivity of bacteria isolated from aural discharges of adults and children with various types of OM, including acute OM (AOM), OM with effusion (OME), chronic OM (COM), and cholesteatomatous OM (CSOM).

Methods

The study involved 2,833 patients who visited five tertiary hospitals between January 2001 and December 2010 and were diagnosed with AOM, OME, COM, or CSOM. The patients were divided into a pediatric group and an adult group, and the distribution of cultured bacteria and their antibiotic sensitivity were compared in the two groups.

Results

Bacterial detection rates were higher in adults than in children with OME and COM (P=0.000 each). The majority of the bacteria cultured from patients with AOM and OME bacteria were methicillin-susceptible Staphylococcus aureus (MSSA) and Streptococcus pneumoniae. Bacteria cultured from children were more susceptible to antibiotics (P=0.002) and had higher antibiotic sensitivity (P=0.001) than were bacteria cultured from adults. The majority of bacteria culture from patients with COM and CSOM were MSSA and pathogenic Pseudomonas aeruginosa. The frequency of methicillin-resistant Staphylococcus aureus was significantly higher in adults than in children, and more strains of bacteria isolated from adults were sensitive to the antibiotics septrin, vancomycin, and teicoplanin.

Conclusion

Bacteria cultured from children were more susceptible to antibiotics and had higher antibiotic sensitivity than did bacteria cultured from adults.     

Evaluation of the Interleukin-1 Receptor Antagonist and Immunoregulatory Interleukin-10 in the Middle Ear in Chronic Otitis Media With Effusion in Children With and Without Atopy

ObjectivesThe role of pro-inflammatory cytokines in the course of chronic otitis media with effusion (COME) has been documented. However, there are fewer studies on the action of anti-inflammatory cytokines in the middle ear. We sought determine whether there is an association between COME and anti-inflammatory cytokines and whether there are any differences in the cytokine profile in COME children with and without atopy.MethodsEighty-four children were divided into 3 groups: 32 nonatopic children with COME (group NA), 31 atopic children with COME (group A), and 21 children without COME and without atopy (control group C). Specimens from the middle ear were collected and evaluated by enzyme-linked immunosorbent assay for the cytokines interleukin-1 receptor antagonist (IL-1Ra) and immunoregulatory IL-10.ResultsSignificantly higher IL-10 concentrations were found in both nonatopic and atopic children with COME compared to controls. No significant differences in IL-1Ra levels were found between atopic and nonatopic children with COME and the control group.ConclusionWe found no differences in the levels of IL-1Ra in atopic and nonatopic children with COME compared to controls. However, we found elevated IL-10 levels in the middle ear effusions from children with COME, with or without atopy. These elevated immunoregulatory cytokine levels suggest a role for new immunomodulatory treatments to prevent disease progression in COME, regardless of atopy.     

Long-term Changes of Hearing Thresholds and Eustachian Tube Function After Balloon Dilation of the Eustachian Tube in Patients With Chronic Otitis Media

ObjectivesThis study aimed to evaluate long-term changes after balloon dilation of the Eustachian tube (BDET) in chronic otitis media (COM) patients with Eustachian tube (ET) dysfunction that persisted after tympanomastoidectomy (TM).MethodsWe retrospectively reviewed the medical records of consecutive patients who were diagnosed with COM and ET dysfunction and underwent TM at our tertiary hospital from 2016 to 2017. The tympanic membrane status, the presence of a ventilation tube, ability to perform the Valsalva maneuver, and audiologic changes after dilation of the ET were analyzed.ResultsThis study included 20 patients (with 21 ears) who underwent TM but could not perform the Valsalva maneuver, showed a persistent air-bone gap, and eventually underwent BDET (male:female, 8:13; right:left, 11:10). Four ears showed perforation of the tympanic membrane after TM. Among the remaining 17 ears, 15 ears underwent ventilation tube insertion before BDET, while two ears underwent ventilation tube insertion and BDET simultaneously. Although none of the patients were capable of the Valsalva maneuver before BDET, 13 (62%) were able to perform the Valsalva maneuver successfully after BDET. When evaluating the tympanic membrane status at the latest follow-up, ventilation tubes were still present in eight ears. In the other 13 ears, intact tympanic membranes were present in nine out of 11 ears n the successful Valsalva group, whereas none of them were intact in the unsuccessful Valsalva group (P=0.014). The successful Valsalva group after BDET showed an improved air-bone gap of 8.9±12.4 dB, while the unsuccessful Valsalva group showed an aggravated air-bone gap of 3.8±11.8 dB at 1 year after BDET; this difference was statistically significant (P=0.031).ConclusionThe Valsalva maneuver could be performed successfully after BDET by 62% of patients with COM and ET dysfunction. BDET is helpful for successful hearing improvement and improved tympanic aeration in COM patients with ET dysfunction.     

Frequency of Alloicoccus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in children with otitis media with effusion (OME) in Iranian patients

Objective

To determine the presence of common bacterial agents of otitis media with effusion (OME), together with investigation these agent in the adenoid tissue and antimicrobial susceptibility pattern of isolated bacteria in Iranian children with OME.

Methods

Polymerase chain reaction (PCR) and bacterial culture methods were used for detection and isolation of Alloicoccus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in 63 middle ear fluid samples and 48 adenoid tissues from 48 OME patients. Fifteen patients were bilaterally affected. Antimicrobial susceptibility of all bacterial isolates were determined by disk agar diffusion (DAD) method.

Results

Bacteria were isolated from 47% (n = 30) of the middle ear fluid samples and 79% (n = 38) of the adenoid tissue specimens in OME patients. A. otitidis was the most common bacterial isolated from the middle ear fluid 23.8% by culture and 36.5% by PCR method. S. pneumoniae was the most prevalent pathogen (35.5% and 31.2% by culture and PCR) in the adenoid tissues. In 10 patients the same organisms were isolated from the middle ear fluid and adenoid tissue. Antimicrobial susceptibility pattern showed taht most isolates of bacteria were sensitive to ampicillin, Amoxicillin/Clavulanate and fluoroquinolones.

Conclusion

The present study, being the first report on the isolation of A. otitidis by culture method in Iran and Asian countries, shows that A. otitidis is the most frequently isolated bacterium in Iranian children having otitis media with effusion. In this study A. otitidis, S. pneumoniae, H. influenzae and M. catarrhalis are the major bacterial pathogens in patients with OME and we found that ampicillin and Amoxicillin/Clavulanate have the excellent activity against bacterial agents in Iranian children with OME.