biofilms, antibiotic resistance and implications for medical treatment james m. coticchia...

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


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


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


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


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


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


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


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


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


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


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


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


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


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.

biofilms, antibiotic resistance and implications for medical treatment james m. coticchia...

Documents

surface of biofilms

development of biofilms

eps slide

mgml slide

dollars cdc slide

relapsing nature slide

freefloating phenotype

attached monolayer slide