Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

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 is an important cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD) Several lines of evidence suggest that the bacterium grows as a biofilm in the human respiratory tract Fifteen clinical isolates from middle ear fluid of children with otitis media and 15 isolates from sputum of adults with COPD were studied in an in vitro assay of biofilm formation Striking variability among isolates was observed in their ability to form biofilms Analysis of cell envelopes revealed minimal differences in banding patterns in polyacrylamide gels, alteration of expression of an epitope on lipooligosaccharide, and preservation of expression of selected epitopes on outer membrane proteins P2, P5 and P6 in biofilms compared to planktonically grown cells A pilus-deficient variant showed a marked impairment in biofilm formation compared to its isogenic parent Nontypeable H influenzae forms biofilms in vitro Clinical isolates show substantial variability in their ability to grow as biofilms Three major outer membrane proteins (P2, P5 and P6) are expressed during growth as a biofilm Expression of lipooligosaccharide is altered during growth as a biofilm compared to planktonic growth Pili are important in biofilm formation As the role of biofilms in human infection becomes better defined, characterization of biofilms may be important in understanding the pathogenesis of infection and immune response to nontypeable H influenzae in children with otitis media and adults with COPD

/pdf/biofilm-formation-by-nontypeable-haemophilus-influenzae-37imypbvkf.pdf

Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili.

The presence of endotoxin (detected by the Limulus amebocyte lysate assay) was compared to the presence of viable Haemophilus influenzae and Moraxella catarrhalis (detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.

Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family.

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Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family.