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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
Biofilms
Defined as an
assemblage of
microbial cells
enclosed in a selfproduced 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)
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
Biofilm Formation
Micro-colonies form
Prolific EPS matrix with micro-organisms
embedded in matrix forms
Planktonic Shedding from the surface of biofilms
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
Molecular Aspects of Biofilms
Multicellular biofilms communicate via quorum
sensing, which may play important mechanism in
antimicrobial resistance and dispersion of
planktonic organisms
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
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
Clinical Implications of
Biofilms
Andrel & Colleagues
Antimicrobial Agents Chemotherapy
2000, 44:1818-24
– Demonstrated β-lactamase negative
Klebsiella pneumoniae, MIC 2mg/ml, survived
as a biofilm in ampicillin concentration of 5000
mg/ml
Clinical Implications of
Biofilms
Andrel & Colleagues
Antimicrobial Agents Chemotherapy
2000, 44:1818-24
– 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
Biofilms and Antibiotic
Resistance
10-1000 times
more resistant than
their planktonic
counterparts
Classic teaching:
resistance
conferred via
plasmids,
transposons, and
mutations
Multicellular
strategies
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
Putative mechanisms : antimicrobial
resistance of bacterial biofilms
Slow or incomplete penetration of
antibiotics into the biofilm matrix
Ampicillin readily penetrates β-lactamase neg
biofilms
Ampicillin penetration retarded by wild strain βlactamase pos.
Aminoglycoside antibiotics : positive charge
retarded by negative ions biofilm matrix
Putative mechanisms : antimicrobial
resistance of bacterial biofilms
Altered chemical microenvironment within
the biofilm
pH gradients >1 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
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 <1% of population and
develop even immature biofilms
This phenotype is extremely resistant to both
antimicrobial therapy and disinfectants
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
Resistance Mechanisms
Quorum sensing
– lasI gene encodes
protein for an acylhomoserine 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
Resistance Mechanisms
Antimicrobial diffusion may be affected
by aggregates of micro-organisms
Osmotic gradient may affect porins
Resistance Mechanisms
Quorum sensing influences small
population of dormant micro-organisms
Planktonic organisms revert to original
sensitivity
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
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
Host Immune Response & Biofilms
FISH imaging has also identified
intracellular pod formation that may evade
normal surveillance
Therapeutic Options : Biofilm
Infections
Mechanical Disruption
Surgical debridement
Device removal
Ultrasonic treatment
– Increases efficacy gentamycin
Chemical Disruption
Saponification
Enzyme degradation
– Alginate lyase
Therapeutic Options : Biofilm
Infections
Molecular Techniques
Disruption of bacterial adherence
Disruption of Quorum sensing pathway
Inhibition of biofilm matrix synthesis
Photodynamic therapy
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
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
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
References
Anderl JN. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm
resistance to ampicillin and ciprofloxacin. Antimicob Agents Chemother 2000; 44: 18181824.
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; 7:163-171.
Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK. The
exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFNgamma-mediated macrophage killing. J Immunol 2005; 175(11): 7512-8.
Mah T-F, O’Toole 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.