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

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.

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)60245-7/pdf

The polymicrobial nature of biofilm infection

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

Utility of the polymerase chain reaction for detecting s. pneumoniae in blood

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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

Utility of the polymerase chain reaction for detecting s. pneumoniae in blood