ContextChronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection.ObjectiveTo test the hypothesis that chronic OM in humans is biofilm-related.Design, Setting, and PatientsMiddle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice.Main Outcome MeasuresConfocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively.ResultsOf the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation.ConclusionDirect detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

https://jamanetwork.com/journals/jama/articlepdf/211068/JPC60005.pdf

Direct Detection of Bacterial Biofilms on the Middle-Ear Mucosa of Children With Chronic Otitis Media

Moraxella catarrhalis (formerly known as Branhamella catarrhalis) has
 emerged as a significant bacterial pathogen of humans over the past two
 decades. During this period, microbiological and molecular diagnostic
 techniques have been developed and improved for M. catarrhalis, allowing
 the adequate determination and taxonomic positioning of this pathogen.
 Over the same period, studies have revealed its involvement in respiratory
 (e.g., sinusitis, otitis media, bronchitis, and pneumonia) and ocular
 infections in children and in laryngitis, bronchitis, and pneumonia in
 adults. The development of (molecular) epidemiological tools has enabled
 the national and international distribution of M. catarrhalis strains to
 be established, and has allowed the monitoring of nosocomial infections
 and the dynamics of carriage. Indeed, such monitoring has revealed an
 increasing number of B-lactamase-positive M. catarrhalis isolates (now
 well above 90%), underscoring the pathogenic potential of this organism.
 Although a number of putative M. catarrhalis virulence factors have been
 identified and described in detail, their relationship to actual bacterial
 adhesion, invasion, complement resistance, etc. (and ultimately their role
 in infection and immunity), has been established in a only few cases. In
 the past 10 years, various animal models for the study of M. catarrhalis
 pathogenicity have been described, although not all of these models are
 equally suitable for the study of human infection. Techniques involving
 the molecular manipulation of M. catarrhalis genes and antigens are also
 advancing our knowledge of the host response to and pathogenesis of this
 bacterial species in humans, as well as providing insights into possible
 vaccine candidates. This review aims to outline our current knowledge of
 M. catarrhalis, an organism that has evolved from an emerging to a
 well-established human pathogen.

/pdf/moraxella-catarrhalis-from-emerging-to-established-pathogen-nvju43cf6i.pdf

Moraxella catarrhalis: from Emerging to Established Pathogen

Biofilms associated with the human body, particularly in typically sterile locations, are difficult to diagnose and treat effectively because of their recalcitrance to conventional antibiotic therapy and host immune responses. The study of biofilms in medicine today requires a translational approach, with examination of clinically relevant biofilms in the context of specific anatomic sites, host tissues, and diseases, focusing on what can be done to mitigate their pathologic consequences. This review, which grew out of a discussion session on clinical biofilms at the 5th ASM Biofilm Conference in Cancun, Mexico, is designed to give an overview of biofilm-associated infections (BAI) and to propose a platform for further discussion that includes clinicians, medical microbiologists, and biofilm researchers who are stakeholders in advancing the scientific pursuit of better diagnosis and treatment of BAI to mitigate their human and healthcare costs. It also highlights the need for better diagnostic markers, which exploit the difference between planktonic and biofilm cells.

Towards diagnostic guidelines for biofilm-associated infections

Nontypeable Haemophilus influenzae colonizes the respiratory tract of adults with chronic obstructive pulmonary disease (COPD) and causes intermittent exacerbations Isolates of H influenzae collected monthly in a prospective study were subjected to molecular typing During a 7-year study spanning 345 patient-months of observation, 122 episodes of negative cultures lasting 1 month or more, and that were preceded and followed by isolation of an apparently identical strain of H influenzae, were found Seventeen such episodes of negative cultures, lasting 6 months or more and spanning 203 patient-months, were studied in detail to test the hypothesis that these periods of negative cultures represented continuous colonization by the same strain of H influenzae Molecular typing by three independent methods established that the strains preceding and following the episodes of negative cultures were indeed identical Strain-specific H influenzae DNA was detected in some of the sputum samples that had yielded negative cultures These results indicate that some patients with COPD are persistently colonized with H influenzae and that sputum cultures underestimate the frequency of colonization of the respiratory tract by H influenzae in COPD This observation has a significant impact on understanding bacterial colonization in COPD

Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease.

Context.—Otitis media with effusion (OME) can lead to significant hearing loss
in children. Although previous studies have shown that bacterial DNA is present
in a significant percentage of effusions sterile by culture, whether the DNA
represents viable organisms or "fossilized remains" is unknown.Objective.—To determine if bacterial messenger RNA (mRNA), as detected by a reverse
transcriptase–polymerase chain reaction (RT-PCR)–based assay,
is present in chronic pediatric middle ear effusions that contain bacterial
DNA but are sterile by standard cultural methods. Bacterial mRNAs have a half-life
measured in seconds to minutes; therefore, detection of bacteria-specific
mRNAs would be evidence that metabolically active organisms are present.Design.—Blinded comparative study.Patients.—A total of 93 effusions from pediatric outpatients seen for myringotomy
and tube placement for chronic (>3 months) OME (median age of children, 17
months).Setting.—Tertiary care pediatric hospital.Main Outcome Measures.—Percentage of positive test results for RT-PCR–based assays compared
with culture for Haemophilus influenzae and concordance
between RT-PCR and PCR-based findings for bacterial nucleic acids.Results.—Eleven (11.8%) of the 93 specimens tested positive by culture, PCR,
and RT-PCR for H influenzae. A total of 29 specimens
(31.2%) were positive by PCR but negative by culture for H influenzae. All 29 specimens were positive by RT-PCR for H influenzae–specific mRNA.Conclusions.—The RT-PCR–based assay system can detect the presence of bacterial
mRNA in a significant percentage of culturally sterile middle ear effusions,
establishing the presence of viable, metabolically active, intact organisms
in some culture-negative OME.

https://jamanetwork.com/journals/jama/articlepdf/187171/JCI71023.pdf

Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.

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

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day I from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.

Comparative Evaluation of Culture and PCR for the Detection and Determination of Persistence of Bacterial Strains and DNAS in the Chinchilla Laniger Model of Otitis Media

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

Comparative Evaluation of Culture and PCR for the Detection and Determination of Persistence of Bacterial Strains and DNAS in the Chinchilla Laniger Model of Otitis Media

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