Biofilms,%20Antibiotic%20Resistance%20and%20Implications%20for%20Medical%20Treatment

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Biofilms, Antibiotic Resistance and Implications
for Medical Treatment

• James M. Coticchia M.D.,F.A.C.S.
• Director of Pediatric Otolaryngology
• Associate Professor
• Vice Chairman
• Otolaryngology Head and Neck Surgery
• Wayne State University
• School of Medicine
• Giancarlo Zuliani MD
• Chief Resident
• Otolaryngology Head and Neck Surgery
• Wayne State University
• School of Medicine

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Biofilms

• Defined as an assemblage of microbial cells
  enclosed in a self-produced polymeric matrix that
  is irreversibly associated with an inert or
  living surface
• 65 of nosocomial infections whose treatment
  costs an estimated 1 billion dollars (CDC)

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Biofilm Formation

• Biofilms complex microbial lifestyle initiated by
  multiple genetic pathways
• Planktonic cells attach to a surface
• Cells then go on to form an attached monolayer

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Biofilm Formation

• Micro-colonies form
• Prolific EPS matrix with micro-organisms embedded
  in matrix forms
• Planktonic Shedding from the surface of biofilms

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Molecular Aspects of Biofilms

• Initial steps in the development of biofilms rely
  on altered gene expression
• A large number of genes are up-regulated or
  down-regulated as biofilm phenotypes develop
• Specific gene products are expressed to provide
  attachment
• Motility mechanisms are used to form
  multicellular aggregates
• Synthesis of extracellular matrix components EPS

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Molecular Aspects of Biofilms

• Multicellular biofilms communicate via quorum
  sensing, which may play important mechanism in
  antimicrobial resistance and dispersion of
  planktonic organisms

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Clinical Implications of Biofilms

• Bacteria in biofilms persist despite antibiotic
  concentration of 100 - 1000 x MLC
• Antimicrobial therapy can suppress planktonic
  organisms shed from biofilms and suppress
  clinical symptoms

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Clinical Implications of Biofilms

• Organisms embedded in biofilms resist
  antimicrobial therapy
• When antibiotic therapy ends, organisms in
  biofilm may reinfect the host in a recurrent and
  relapsing nature

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Clinical Implications of Biofilms

• Andrel Colleagues Antimicrobial Agents
  Chemotherapy 2000, 441818-24
• Demonstrated ß-lactamase negative Klebsiella
  pneumoniae, MIC 2mg/ml, survived as a biofilm in
  ampicillin concentration of 5000 mg/ml

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Clinical Implications of Biofilms

• Andrel Colleagues Antimicrobial Agents
  Chemotherapy 2000, 441818-24
• Dispersed planktonic organisms readily killed
• Suggests that standard resistance mechanisms such
  as efflux pumps may not play a central role in
  antibiotic resistance of biofilm organisms

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Biofilms and Antibiotic Resistance

• 10-1000 times more resistant than their
  planktonic counterparts
• Classic teaching resistance conferred via
  plasmids, transposons, and mutations
• Multicellular strategies

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Biofilms and Antibiotic Resistance

• Physical proximity of cells within a biofilm
  would be expected to favor conjugation over the
  same process in planktonic counterparts
• Ehlers and Bouwer demonstrated the conjugation
  rates between different species of Pseudomonas
  were significantly higher in biofilms than in
  their free-floating phenotype

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Putative mechanisms antimicrobial resistance
of bacterial biofilms

• Slow or incomplete penetration of antibiotics
  into the biofilm matrix
• Ampicillin readily penetrates ß-lactamase neg
  biofilms
• Ampicillin penetration retarded by wild strain
  ß-lactamase pos.
• Aminoglycoside antibiotics positive charge
  retarded by negative ions biofilm matrix

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Putative mechanisms antimicrobial resistance
of bacterial biofilms

• Altered chemical microenvironment within the
  biofilm
• pH gradients gt1 between fluid and solid phase
  inhibit some antibiotics
• Deeper layers of biofilm are anaerobic and
  decrease the efficacy of aminoglycoside
  antibiotics
• Depletion of nutritional substrate or elevation
  of waste products induces sessile growth phase
  that renders antibiotics less effective

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Putative mechanisms antimicrobial resistance
of bacterial biofilms

• Osmotic environment within biofilms may alter
  membrane permeability, alteration of porins and
  antibiotic penetration
• Subpopulation within biofilms form a unique
  phenotype similar to spore formation
• These phenotypes may be lt1 of population and
  develop even immature biofilms
• This phenotype is extremely resistant to both
  antimicrobial therapy and disinfectants

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Resistance Mechanisms

• Stewart et al. demonstrated the spatial
  physiologic heterogeneity within biofilms of
  Pseudomonas aeruginosa using visualization
  techniques that indicated protein synthesis,
  respiratory activity, and relative RNA content

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Resistance Mechanisms

• Quorum sensing
• lasI gene encodes protein for an acyl-homoserine
  lactone shown to be impotant for bacteria species
  (gm -) to monitor its own population density
• LasI mutants are arrested after micorcolony
  formation but before full maturation

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Resistance Mechanisms

• Antimicrobial diffusion may be affected by
  aggregates of micro-organisms
• Osmotic gradient may affect porins

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Resistance Mechanisms

• Quorum sensing influences small population of
  dormant micro-organisms
• Planktonic organisms revert to original
  sensitivity

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Host Immune Response Biofilms

• Bacteria within biofilms may elude normal host
  immune response
• Shiau Wu Microbiol Immunol, 42 33-40
• Demonstrated that the slime product of S.
  epidermidis affected phagocytosis by macrophages

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Host Immune Response Biofilms

• Ward Colleagues J. Med Microbiol, 36 406-413
• Demonstrated lack of phagocytosis of bacterial
  biofilm implanted device in immunized animals
• Meluleni Colleagues J. Immunol, 155 209-238
• Demonstrated opsonic antibody in Cystic Fibrosis
  patients to be ineffective in eliminating
  organisms within biofilms

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Host Immune Response Biofilms

• FISH imaging has also identified intracellular
  pod formation that may evade normal surveillance

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Therapeutic Options Biofilm Infections

• Mechanical Disruption
• Surgical debridement
• Device removal
• Ultrasonic treatment
• Increases efficacy gentamycin
• Chemical Disruption
• Saponification
• Enzyme degradation
• Alginate lyase

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Therapeutic Options Biofilm Infections

• Molecular Techniques
• Disruption of bacterial adherence
• Disruption of Quorum sensing pathway
• Inhibition of biofilm matrix synthesis
• Photodynamic therapy

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Therapeutic Options Biofilm Infections

• Antimicrobials
• Multidrug treatment regimens
• Clarithromycin decreases alginate and hexose
  biofilm matrix
• May have synergistic effect with other
  antibiotics like ofloxacin
• Multidrug regimens routinely used for treatment
  of H. pylori infection a biofilm disease

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Therapeutic Options Biofilm Infections

• Nanotechnology
• Succi Colleagues Chem Biology, 14 387-388
• Described development of viral nanoplatform
  (protein cage) delivery system Staphylococcus
  aureus biofilm bacterium
• Labeling
• Drug platform

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Thank-you / Grazie Mille

• Alessandro Fiocchi MD, Marcello Giovannini MD and
  the inviting committee
• James Coticchia MD, Aaron Duberstein MD, Michael
  Carlisle MD
• Division of Pediatric Otolaryngology, Department
  of Otolaryngology-Head and Neck Surgery, Wayne
  State University

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References

• Anderl JN. Role of antibiotic penetration
  limitation in Klebsiella pneumoniae biofilm
  resistance to ampicillin and ciprofloxacin.
  Antimicob Agents Chemother 2000 44 1818-1824.
• Cochran WL, McFeters GA, Stewart PS. Reduced
  susceptibility of thin Pseudomonas aeruginosa
  biofilms to hydrogen peroxide and monochloramine.
  J Appl Microbiol 2000 88 22-30.
• Ehlers LJ, Bouwer EJ. RP4 plasmid transfer among
  species of Pseudomonas in a biofilm reactor.
  Water Sci Technol 1999 7163-171.
• Leid JG, Willson CJ, Shirtliff ME, Hassett DJ,
  Parsek MR, Jeffers AK. The exopolysaccharide
  alginate protects Pseudomonas aeruginosa biofilm
  bacteria from IFN-gamma-mediated macrophage
  killing. J Immunol 2005 175(11) 7512-8.
• Mah T-F, OToole GA. Mechanisms of biofilm
  resistance to antimicrobial agents. Trends
  Microbiol 2001 9 34-9.
• Parsek MR, Greenberg EP. Acyl-homoserine lactone
  quorum sensing in gram-negative bacteria a
  signaling mechanism involved in associations with
  higher organisms. Proc Natl Acad Sci USA 2000
  97 8789-93.
• Stewart PS, Costerton JW. Antibiotic resistance
  of bacteria in biofilms. Lancet 2001 358 135-8.
• Xu KD, McFeters GA, Stewart PS. Biofilm
  resistance to antimicorbial agents. Microbiology
  2000 146 547-49.