Objectives/Hypothesis Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations.



Study Design Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients.



Methods Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes.



Results Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes.



Conclusions Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.

Candidate's Thesis: Direct Evidence of Bacterial Biofilms in Otitis Media

Purpose : Bacterial deoxyribonucleic acid (DNA) has been previously detected by polymerase chain reactions (PCR) in a significant percentage of culturally-sterile pediatric middle-ear effusions. The current study was designed to determine whether this represents the existence of viable bacteria or the persistence of residual DNA in the middle-ear cleft. Materials and Methods : The middle-ear cavities of two sets of chinchillas were inoculated with either : 1) 100 colony-forming units (CFU) of live Haemophilus influenzae, 2.2 x 10 6 CFU of pasteurized Moraxella catarrhalis, and 1000 ng of DNA (>10 8 genomic equivalents) from Streptococcus pneumoniae ; or 2) 100 CFU of live S pneumoniae, 2.2 x 10 6 CFU of pasteurized M catarrhalis and 1000 ng of purified DNA from H influenzae. Animals were treated with ampicillin for 5 days beginning on day 3. A single-point longitudinal study design was used for sampling to eliminate the possibility of contamination. Results : No DNA was detectable from the heat-killed bacteria or the purified DNA after day 3. However, DNA from the live bacteria persisted through day 21, even though all specimens were culture-negative following the initiation of antimicrobial therapy. Conclusion : These findings indicate that purified DNA and DNA from intact but nonviable bacteria do not persist in the middle-ear cleft in the presence of an effusion, even following high copy inoculation. In contrast, antibiotic-treated bacteria persist in some viable state for weeks as evidenced by the differential ability of the PCR-based assay systems to detect the live bacteria, but not detect the heat-killed organisms.

PCR-based detection of bacterial DNA after antimicrobial treatment is indicative of persistent, viable bacteria in the chinchilla model of otitis media

BACKGROUND—Streptococcus pneumoniae is the leading cause of community acquired pneumonia; however, only a small proportion of cases can be detected by conventional methods. The ability of the polymerase chain reaction (PCR) test performed on whole blood samples to identify patients with pneumococcal pneumonia was investigated.
METHODS—One hundred and fourteen consecutive adult patients with community acquired pneumonia were evaluated by a wide battery of diagnostic tests in order to determine the aetiology. Blood samples from these patients and 50 controls were also tested by the nested PCR test to detect selected pneumolysin gene fragments of S pneumoniae.
RESULTS—The patients were divided into four groups: (1) 40 patients with pneumococcal pneumonia in 22 of whom (55%) the PCR was positive (eight of 11 with bacteraemia and 14 of 29 without); (2) 30 with pneumonia due to other pathogens in all of whom the PCR was negative; (3) 44 with pneumonia of unknown aetiology in 14 of whom (32%) PCR was positive, and (4) 50 controls in whom the PCR test was positive in two (4%). Thus, the sensitivity of the test was 55% and the specificity 100% (81% if positive PCR tests among undiagnosed patients are considered as false positive results).
CONCLUSION—PCR applied to whole blood samples appears to be a sensitive and very specific diagnostic test for identifying patients with pneumococcal pneumonia with a potential application in clinical practice.

https://thorax.bmj.com/content/thoraxjnl/55/2/133.full.pdf

Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study.

Like many pathogens inhabiting mucosal surfaces, nontypeable Haemophilus influenzae (NTHi) forms multicellular biofilm communities both in vitro and in various infection models. In the past 15 years much has been learned about determinants of biofilm formation by this organism and potential roles in bacterial virulence, especially in the context of chronic and recurrent infections. However, this concept has not been without some degree of controversy, and in the past some have expressed doubts about the relevance of NTHi biofilms to disease. In this review, I will summarize the present information on the composition and potential role(s) of NTHi biofilms in different clinical contexts, as well as highlight potential areas for future work.

/pdf/nontypeable-haemophilus-influenzae-biofilms-role-in-chronic-399q5l9yms.pdf

Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections.

Evaluation of the Polymerase Chain Reaction Method for Detection of Streptococcus pneumoniae DNA in Pleural Fluid Samples

Recent studies using the polymerase chain reaction (PCR) have identified bacterial and viral genomic sequences in culture-negative pediatric middle ear effusions. To evaluate this technique in adults, 19 effusions were analyzed to compare bacterial and viral culture and PCR detection of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and adenovirus. Effusions from 4 subjects positive for human immunodeficiency virus (HIV) were analyzed by PCR for HIV virus. Three of 19 effusions were culture-positive for bacteria, and 0 of 19 for viruses. Fifteen of 19 effusions were PCR-positive for bacterial genomic sequences, and 0 of 19 for adenovirus. Thirteen of 15 PCR-positive specimens demonstrated S pneumoniae, 5 of 15 H influenzae, and 0 of 13 M catarrhalis. All 4 effusions from HIV-positive subjects were PCR-positive for HIV. No effusion was culture-positive and PCR-negative. These results confirm that culture-negative middle ear effusions contain genomic sequences from bacterial pathogens. Finding of HIV RNA and DNA in effusion from HIV-positives suggests replicating virus in this fluid.

Analysis of adult otitis media: polymerase chain reaction versus culture for bacteria and viruses.

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Analysis of adult otitis media: polymerase chain reaction versus culture for bacteria and viruses.