Biofilms in chronic suppurative otitis media and cholesteatoma

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BIOFILMS IN CHRONIC SUPPURATIVE OTITIS MEDIA AND CHOLESTEATOMA: SCANNING ELECTRON MICROSCOPY FINDINGS. SAUNDERS J , MURRAY M , ALLEMAN A . Section of Otolaryngology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA Camino Ear,Nose and Throat Clinic, Sn Jose,CA,USA Dpet. Of Radiology,University ofOkhlahoma Health Science Center ,Okhlahoma City,OK, USA

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Transcript of Biofilms in chronic suppurative otitis media and cholesteatoma

Page 1: Biofilms in chronic suppurative otitis media and cholesteatoma

BIOFILMS IN CHRONIC SUPPURATIVE OTITIS MEDIA AND CHOLESTEATOMA: SCANNING

ELECTRON MICROSCOPY FINDINGS.SAUNDERS J, MURRAY M, ALLEMAN A.

Section of Otolaryngology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA

Camino Ear,Nose and Throat Clinic, Sn Jose,CA,USA Dpet. Of Radiology,University ofOkhlahoma Health

Science Center ,Okhlahoma City,OK, USA

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introduction

Biofilms play a role in the pathogenesis of a variety of otorhinolaryngologic diseases.

OBJECTIVE: to detect evidence of biofolms in human chronic ear infections with scanning electron microscopy(SEM)

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Biofilm definition

Biofilms are complex bacterial communities that adhere to the surface of implanted biomaterial or mucosa .

Embedded in a slim-like extracellular matrix composed of proteins, polysaccharides, and nucleic acids known as extracellular polymeric substances (EPS)

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WHERE DO THE BIOFILMS GROW

Wherever we find a combination of moisture, nutrients and a surface, we are likely to find biofilm.

Biofilm forms when bacteria adhere to surfaces in moist environments by excreting a slimy, glue-like substance. 

all kinds of surfaces

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Formation of biofilmFive stages of biofilm development: (1) Initial attachment to the mucosa electrostatically(planktonic form), (2) Irreversible attachment by chemical agents, (3) Maturation I:aggregation & formation of EPS, (4) Maturation II:formation of biofilm structure and (5) Dispersion. Important for biofilm survival & biofilm spreading. Each stage of development in the diagram is paired with a photomicrograph of a developing P. aeruginosabiofilm.

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Role of biofilms in otolaryngology

The role of biofilms in the persistence of chronic, mucosal-based ENT-related infections was first recognized in otitis media. 

chronic bacterial infections are biofilm related is fundamental to developing rationale strategies for treatment and prevention.

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Characteristics of biofilm

1. Biofilms are complex, dynamic community structures

dynamic and responsive to their environment. 

biofilms can also migrate across surfaces over a period of time in a variety of ways, as illustrated in image.

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2. Genetic expression is different in biofilm bacteria when compared toplanktonic bacteria

 we see proteins from the outer membranes of planktonic (outlined in blue, Lanes 1-4 and 6) and biofilm (outlined in red, Lane 5) bacteria, of a single strain ofPseudomonas aeruginosa. The bands of proteins are strikingly different.

SDS PAGE preparation of the outer membrane proteins (OMPs) ofPseudomonas aeruginosa cells in planktonic and biofilm states

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3. Biofilm cells can coordinate behavior via intercellular "communication" using biochemical signaling molecules

communicate using chemical signals.   chemical signals, produced by cells and

passed through their outer membranes It involves complex species of bacteria

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Bacterial populations will activate some genes only when they are able to sense, via cell signaling.

Ex:some bacterial pathogens will not produce toxins until they sense that an adequate population has been established to survive host defenses.

population recognition has been termed "quorum sensing.“

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MECHANISM OF PROTECTION FROM ANTIMICROBIALS

A. Free-floating cells utilize nutrients, but do not have sufficient metabolic activity to deplete substrates from the neighborhood of the cells. 

In contrast, the collective metabolic activity of groups of cells in the biofilm leads to substrate concentration gradients and localized chemical microenvironments.Reduced metabolic activity may result in less susceptibility to antimicrobials.

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B. Free-floating cells carry the genetic code for numerous protective stress responses. Planktonic cells, however, are readily overwhelmed by a strong antimicrobial challenge. These cells die before stress responses can be activated. 

In contrast, stress responses are effectively implemented in some of the cells in a biofilm at the expense of other cells which are sacrificed.

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C. Free-floating cells neutralize the antimicrobial agent. The capacity of a lone cell, however, is insufficient to draw down the antimicrobial concentration in the neighborhood of the cell.

In contrast, the collective neutralizing power of groups of cells leads to slow or incomplete penetration of the antimicrobial in the biofilm.

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D. Free-floating cells spawn protected persister cells. But under permissive growth conditions in a planktonic culture, persisters rapidly revert to a susceptible state.

In contrast, persister cells accumulate in biofilms because they revert less readily and are physically retained by the biofilm matrix.

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Current methods in biofilm studies

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Scanning electron microscopy

SEM is a well-established basic method provides information about the morphology of

biofilm, presence of EPS and the nature of corrosion products (crystalline or amorphous).

Many of the conclusions about biofilm development, composition, distribution, and relationship to substratum have been derived from scanning electron microscopy .

SEM is a powerful technique for revealing the fine structure of living systems and has been applied to biofilms.

able to measure and quantify data in three dimensions

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Scanning electron microscopy

A, Control at ×5500 magnification. This specimen was obtained at time 0 in an animal that was not inoculated with Haemophilus influenzae; B, H influenzae microcolonies on middle-ear mucosa 24 hours after inoculation at ×5000; C, mature H influenzae biofilm on middle-ear mucosa 5 days after inoculation at ×5000; D, mature biofilm 10 days after inoculation at ×2000.

Chinchilla inoculated with H.influenza after 72 hrs treadted with ampicillin and sterilized effusion. And biofilms are found in all effusions.

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Confocal laser scanning microscopy

Elucidate structure-function relationship in biofilms

Red dye shows dye bacteria with live host nuclei.

Green dye shows live bacteriaUnfixed specimens were obtained under general anesthesia, placed in buffer,

and shipped via overnight courier from the Center for Genomic Sciences to the Center for Bioengineering for imaging. Specimens were stained using the Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, Ore), which uses the 2 dyes, SYTO 9 (green) and propidium iodide (red). Green indicates uncompromised bacterial cell membranes (ie, live cells); red indicates dead cells and is also taken up by the host cell nuclei. Panel A shows an early-stage biofilm, panels B and C show mature biofilms with characteristic tower structure (arrowheads); and panel D shows a biofilm at 21 days after inoculation

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Atomic force microscopy

This atomic force microscope (AFM) image of the surface structure of a hydrated biofilm reveals microcolonies of bacteria

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FLOURESCENCE INSITU HYBRIDIZATION

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 A sample was considered to have a biofilm if 3 criteria were met:

(1) presence of bacterial-sized and -shaped objects;

(2) presence of an amorphous material, consistent with glycocalyx around the bacteria;

(3) surface binding.

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Specific ENT Diseases

AOM- infection by planktonic bacteria -susceptable to antibiotics. CSOM- with persistant efussion

commonly form biofilm. It usually begins with AOM. culture is –ve but still bacterial based

infection. PCR detects DNA of bacteria. Refractory to treatment.

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Cholesteatoma: form biofilm of pseudomonal firm attachment on microscopy.

Infections of implanted devices which form biofilms – PEtubes

-Cochlear implants, artificial ossicles

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Chronic rhinosinusitis(CRS) Adenoid tissues present with biofilm if pt

had CRS Crypts of tonsills are possitive with

biofilms for both infected ,hypertrophic tonsils.

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OTHER THAN ENT

Ctheters infection Dental plaque Gingivitis Contact lenses Joint prosthesis Heart valves IUCD

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 Effective prevention and management strategies include interruption of quorum sensing, inhibition of related genes, disruption of the protective extrapolymer matrix, macrolides (clarithromycin and erythromycin), and mechanical debridement of the biofilm-bearing tissues.