biofilms , antibiotic resistance and implications for medical treatment
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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 - PowerPoint PPT PresentationTRANSCRIPT
Biofilms, Antibiotic Biofilms, Antibiotic Resistance and Implications Resistance and Implications
for Medical Treatmentfor Medical Treatment
James M. Coticchia M.D.,F.A.C.S.James M. Coticchia M.D.,F.A.C.S.Director of Pediatric OtolaryngologyDirector of Pediatric Otolaryngology
Associate ProfessorAssociate ProfessorVice ChairmanVice Chairman
Otolaryngology Head and Neck SurgeryOtolaryngology Head and Neck SurgeryWayne State UniversityWayne State University
School of MedicineSchool of Medicine
Giancarlo Zuliani MDGiancarlo Zuliani MDChief ResidentChief Resident
Otolaryngology Head and Neck SurgeryOtolaryngology Head and Neck SurgeryWayne State UniversityWayne State University
School of MedicineSchool of Medicine
BiofilmsBiofilmsDefined as an Defined as an assemblage of microbial assemblage of microbial cells enclosed in a self-cells enclosed in a self-produced polymeric produced polymeric matrix that is irreversibly matrix that is irreversibly associated with an inert associated with an inert or living surfaceor living surface65% of nosocomial 65% of nosocomial infections whose infections whose treatment costs an treatment costs an estimated 1 billion estimated 1 billion dollars (CDC)dollars (CDC)
Biofilm FormationBiofilm FormationBiofilms complex microbial lifestyle initiated by multiple genetic pathwaysBiofilms complex microbial lifestyle initiated by multiple genetic pathways
Planktonic cells attach to a surfacePlanktonic cells attach to a surface
Cells then go on to form an attached monolayerCells then go on to form an attached monolayer
Biofilm FormationBiofilm Formation
Micro-colonies formMicro-colonies formProlific EPS matrix with micro-organisms Prolific EPS matrix with micro-organisms embedded in matrix formsembedded in matrix formsPlanktonic Shedding from the surface of biofilmsPlanktonic Shedding from the surface of biofilms
Molecular Aspects of BiofilmsMolecular Aspects of Biofilms
Initial steps in the development of biofilms Initial steps in the development of biofilms rely on altered gene expressionrely on altered gene expression
A large number of genes are up-regulated or A large number of genes are up-regulated or down-regulated as biofilm phenotypes developdown-regulated as biofilm phenotypes develop
Specific gene products are expressed to provide Specific gene products are expressed to provide attachmentattachment
Motility mechanisms are used to form multicellular Motility mechanisms are used to form multicellular aggregatesaggregates
Synthesis of extracellular matrix components: EPSSynthesis of extracellular matrix components: EPS
Molecular Aspects of BiofilmsMolecular Aspects of Biofilms
Multicellular biofilms communicate via quorum Multicellular biofilms communicate via quorum sensing, which may play important mechanism in sensing, which may play important mechanism in antimicrobial resistance and dispersion of antimicrobial resistance and dispersion of planktonic organismsplanktonic organisms
Clinical Implications of Clinical Implications of BiofilmsBiofilms
Bacteria in biofilms persist despite Bacteria in biofilms persist despite antibiotic concentration of 100 - 1000 x antibiotic concentration of 100 - 1000 x MLCMLC
Antimicrobial therapy can suppress Antimicrobial therapy can suppress planktonic organisms shed from biofilms planktonic organisms shed from biofilms and suppress clinical symptomsand suppress clinical symptoms
Clinical Implications of Clinical Implications of BiofilmsBiofilms
Organisms embedded in biofilms resist Organisms embedded in biofilms resist antimicrobial therapyantimicrobial therapy
When antibiotic therapy ends, organisms When antibiotic therapy ends, organisms in biofilm may reinfect the host in a in biofilm may reinfect the host in a recurrent and relapsing naturerecurrent and relapsing nature
Clinical Implications of Clinical Implications of BiofilmsBiofilms
Andrel & ColleaguesAndrel & ColleaguesAntimicrobial Agents Chemotherapy Antimicrobial Agents Chemotherapy 2000, 44:1818-242000, 44:1818-24
– Demonstrated Demonstrated ββ-lactamase -lactamase negative negative Klebsiella pneumoniae,Klebsiella pneumoniae, MIC 2mg/ml, MIC 2mg/ml, survived as a biofilm in ampicillin survived as a biofilm in ampicillin concentration of 5000 mg/mlconcentration of 5000 mg/ml
Clinical Implications of Clinical Implications of BiofilmsBiofilms
Andrel & ColleaguesAndrel & ColleaguesAntimicrobial Agents Chemotherapy Antimicrobial Agents Chemotherapy 2000, 44:1818-242000, 44:1818-24
– Dispersed planktonic organisms readily killedDispersed planktonic organisms readily killed
– Suggests that standard resistance Suggests that standard resistance mechanisms such as efflux pumps may not mechanisms such as efflux pumps may not play a central role in antibiotic resistance of play a central role in antibiotic resistance of biofilm organismsbiofilm organisms
Biofilms and Antibiotic Biofilms and Antibiotic ResistanceResistance
10-1000 times 10-1000 times more resistant than more resistant than their planktonic their planktonic counterpartscounterpartsClassic teaching: Classic teaching: resistance resistance conferred via conferred via plasmids, plasmids, transposons, and transposons, and mutationsmutationsMulticellular Multicellular strategiesstrategies
Biofilms and Antibiotic Biofilms and Antibiotic ResistanceResistance
Physical proximity of cells within a biofilm Physical proximity of cells within a biofilm would be expected to favor conjugation would be expected to favor conjugation over the same process in planktonic over the same process in planktonic counterpartscounterpartsEhlers and Bouwer demonstrated the Ehlers and Bouwer demonstrated the conjugation rates between different conjugation rates between different species of Pseudomonas were species of Pseudomonas were significantly higher in biofilms than in their significantly higher in biofilms than in their free-floating phenotypefree-floating phenotype
Putative mechanisms : antimicrobial Putative mechanisms : antimicrobial resistance of bacterial biofilmsresistance of bacterial biofilms
Slow or incomplete penetration of Slow or incomplete penetration of antibiotics into the biofilm matrixantibiotics into the biofilm matrix
Ampicillin readily penetrates Ampicillin readily penetrates ββ-lactamase -lactamase neg neg biofilmsbiofilms
Ampicillin penetration retarded by wild strain Ampicillin penetration retarded by wild strain ββ--lactamase lactamase pos.pos.
Aminoglycoside antibiotics : positive charge Aminoglycoside antibiotics : positive charge retarded by negative ions biofilm matrixretarded by negative ions biofilm matrix
Putative mechanisms : antimicrobial Putative mechanisms : antimicrobial resistance of bacterial biofilmsresistance of bacterial biofilms
Altered chemical microenvironment within Altered chemical microenvironment within the biofilmthe biofilm
pH gradients >1 between fluid and solid phase pH gradients >1 between fluid and solid phase inhibit some antibioticsinhibit some antibiotics
Deeper layers of biofilm are anaerobic and Deeper layers of biofilm are anaerobic and decrease the efficacy of aminoglycoside antibioticsdecrease the efficacy of aminoglycoside antibiotics
Depletion of nutritional substrate or elevation of Depletion of nutritional substrate or elevation of waste products induces sessile growth phase that waste products induces sessile growth phase that renders antibiotics less effectiverenders antibiotics less effective
Putative mechanisms : antimicrobial Putative mechanisms : antimicrobial resistance of bacterial biofilmsresistance of bacterial biofilms
Osmotic environment within biofilms may alter Osmotic environment within biofilms may alter membrane permeability, alteration of porins and membrane permeability, alteration of porins and antibiotic penetrationantibiotic penetration
Subpopulation within biofilms form a unique Subpopulation within biofilms form a unique phenotype similar to spore formationphenotype similar to spore formation
These phenotypes may be <1% of population and These phenotypes may be <1% of population and develop even immature biofilmsdevelop even immature biofilms
This phenotype is extremely resistant to both This phenotype is extremely resistant to both antimicrobial therapy and disinfectantsantimicrobial therapy and disinfectants
Resistance MechanismsResistance Mechanisms
Stewart et al. Stewart et al. demonstrated the spatial demonstrated the spatial physiologic heterogeneity physiologic heterogeneity within biofilms of within biofilms of Pseudomonas Pseudomonas aeruginosa aeruginosa using using visualization techniques visualization techniques that indicated protein that indicated protein synthesis, respiratory synthesis, respiratory activity, and relative RNA activity, and relative RNA contentcontent
Resistance MechanismsResistance Mechanisms
Quorum sensingQuorum sensing– lasIlasI gene encodes gene encodes
protein for an acyl-protein for an acyl-homoserine lactone homoserine lactone shown to be impotant shown to be impotant for bacteria species for bacteria species (gm -) to monitor its (gm -) to monitor its own population densityown population density
– LasI mutants are LasI mutants are arrested after arrested after micorcolony formation micorcolony formation but before full but before full maturationmaturation
Resistance MechanismsResistance Mechanisms
Antimicrobial diffusion may be affected Antimicrobial diffusion may be affected by aggregates of micro-organismsby aggregates of micro-organisms
Osmotic gradient may affect porinsOsmotic gradient may affect porins
Resistance MechanismsResistance Mechanisms
Quorum sensing influences small Quorum sensing influences small population of dormant micro-organismspopulation of dormant micro-organisms
Planktonic organisms revert to original Planktonic organisms revert to original sensitivitysensitivity
Host Immune Response & Host Immune Response & BiofilmsBiofilms
Bacteria within biofilms may elude normal Bacteria within biofilms may elude normal host immune responsehost immune response
Shiau & Wu; Shiau & Wu; Microbiol & ImmunolMicrobiol & Immunol, 42: , 42: 33-4033-40
Demonstrated that the slime product of Demonstrated that the slime product of S. S. epidermidisepidermidis affected phagocytosis by affected phagocytosis by macrophagesmacrophages
Host Immune Response & Host Immune Response & BiofilmsBiofilms
Ward & Colleagues; Ward & Colleagues; J. Med MicrobiolJ. Med Microbiol, 36: , 36: 406-413406-413
Demonstrated lack of phagocytosis of bacterial Demonstrated lack of phagocytosis of bacterial biofilm implanted device in immunized animalsbiofilm implanted device in immunized animals
Meluleni & Colleagues; Meluleni & Colleagues; J. ImmunolJ. Immunol, 155: , 155: 209-238209-238
Demonstrated opsonic antibody in Cystic Fibrosis Demonstrated opsonic antibody in Cystic Fibrosis patients to be ineffective in eliminating organisms patients to be ineffective in eliminating organisms within biofilmswithin biofilms
Host Immune Response & Host Immune Response & BiofilmsBiofilms
FISH imaging has also identified FISH imaging has also identified intracellular pod formation that may evade intracellular pod formation that may evade normal surveillancenormal surveillance
Therapeutic Options : Biofilm Therapeutic Options : Biofilm InfectionsInfections
Mechanical DisruptionMechanical DisruptionSurgical debridementSurgical debridement
Device removalDevice removal
Ultrasonic treatmentUltrasonic treatment– Increases efficacy gentamycinIncreases efficacy gentamycin
Chemical DisruptionChemical DisruptionSaponificationSaponification
Enzyme degradationEnzyme degradation– Alginate lyaseAlginate lyase
Therapeutic Options : Biofilm Therapeutic Options : Biofilm InfectionsInfections
Molecular TechniquesMolecular TechniquesDisruption of bacterial adherenceDisruption of bacterial adherence
Disruption of Quorum sensing pathwayDisruption of Quorum sensing pathway
Inhibition of biofilm matrix synthesisInhibition of biofilm matrix synthesis
Photodynamic therapyPhotodynamic therapy
Therapeutic Options : Biofilm Therapeutic Options : Biofilm InfectionsInfections
AntimicrobialsAntimicrobialsMultidrug treatment regimensMultidrug treatment regimens
Clarithromycin decreases alginate and hexose Clarithromycin decreases alginate and hexose biofilm matrixbiofilm matrix
May have synergistic effect with other antibiotics May have synergistic effect with other antibiotics like ofloxacinlike ofloxacin
Multidrug regimens routinely used for treatment of Multidrug regimens routinely used for treatment of H. pyloriH. pylori infection : a biofilm disease infection : a biofilm disease
Therapeutic Options : Biofilm Therapeutic Options : Biofilm InfectionsInfections
NanotechnologyNanotechnologySucci & Colleagues; Chem & Biology, 14: 387-388Succi & Colleagues; Chem & Biology, 14: 387-388
Described development of viral nanoplatform Described development of viral nanoplatform (protein cage) delivery system : (protein cage) delivery system : Staphylococcus Staphylococcus aureusaureus biofilm bacterium biofilm bacterium
LabelingLabeling
Drug platformDrug platform
Thank-you / Grazie MilleThank-you / Grazie Mille
Alessandro Fiocchi MD, Marcello Giovannini Alessandro Fiocchi MD, Marcello Giovannini MD and the inviting committee MD and the inviting committee James Coticchia MD, Aaron Duberstein MD, James Coticchia MD, Aaron Duberstein MD, Michael Carlisle MDMichael Carlisle MDDivision of Pediatric Otolaryngology, Division of Pediatric Otolaryngology, Department of Otolaryngology-Head and Neck Department of Otolaryngology-Head and Neck Surgery, Wayne State UniversitySurgery, Wayne State University
ReferencesReferencesAnderl JN. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm Anderl JN. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicob Agents Chemother 2000; 44: 1818-resistance to ampicillin and ciprofloxacin. Antimicob Agents Chemother 2000; 44: 1818-1824.1824.
Cochran WL, McFeters GA, Stewart PS. Reduced susceptibility of thin Pseudomonas Cochran WL, McFeters GA, Stewart PS. Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol 2000; 88: aeruginosa biofilms to hydrogen peroxide and monochloramine. J Appl Microbiol 2000; 88: 22-30.22-30.
Ehlers LJ, Bouwer EJ. RP4 plasmid transfer among species of Pseudomonas in a biofilm Ehlers LJ, Bouwer EJ. RP4 plasmid transfer among species of Pseudomonas in a biofilm reactor. Water Sci Technol 1999; 7:163-171.reactor. Water Sci Technol 1999; 7:163-171.
Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK. The Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol 2005; 175(11): 7512-8.gamma-mediated macrophage killing. J Immunol 2005; 175(11): 7512-8.
Mah T-F, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Mah T-F, O’Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9: 34-9.Microbiol 2001; 9: 34-9.
Parsek MR, Greenberg EP. Acyl-homoserine lactone quorum sensing in gram-negative Parsek MR, Greenberg EP. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl Acad Sci USA 2000; 97: 8789-93.Acad Sci USA 2000; 97: 8789-93.
Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: 135-8.135-8.
Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicorbial agents. Microbiology Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicorbial agents. Microbiology 2000; 146: 547-49.2000; 146: 547-49.