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ANTONANTON VANVAN LEEUWENHOEKLEEUWENHOEK (1632(1632--1723)1723) –– AmateurAmateur

DiscoveredDiscovered microbesmicrobes inin thethe mouthmouth (~1680)(~1680)

LOUISLOUIS PASTEURPASTEUR (1822(1822--1895)1895) –– ChemistChemist DiscreditedDiscredited spontaneousspontaneous generationgeneration (~1860);(~1860); discovereddiscovered fermentation,fermentation, pioneeredpioneered purepure cultureculture techniquestechniques ROBERTROBERT KOCHKOCH (1843(1843––1910)1910) –– PhysicianPhysician EstablishedEstablished thethe germgerm theorytheory ofof diseasedisease (~1882);(~1882); discovereddiscovered thethe agentsagents ofof anthraxanthrax (1877),(1877), tuberculosistuberculosis (1882);(1882); andand choleracholera (1883)(1883) WILLOUGHBYWILLOUGHBY DAYTONDAYTON MILLERMILLER (1853(1853--1907)1907) –– DentistDentist FatherFather ofof oraloral microbiology;microbiology; associatedassociated bacterialbacterial productionproduction ofof acidacid withwith carriescarries (~1890)(~1890)

FirstFirst ImageImage ofof PlaquePlaque BacteriaBacteria

ImprovingImproving onon thethe recentrecent inventioninvention ofof thethe microscope,microscope, vanvan LeeuwenhoekLeeuwenhoek discovereddiscovered microbesmicrobes inin plaqueplaque andand recordedrecorded thethe diversitydiversity ofof thesethese organismsorganisms (1683)(1683) DottedDotted lineline betweenbetween CC andand DD indicatesindicates motilitymotility

organismsorganisms (1683)(1683) DottedDotted lineline betweenbetween CC andand DD indicatesindicates motilitymotility

VanVan Leeuwenhoek´sLeeuwenhoek´s famousfamous descriptiondescription ofof microbesmicrobes inin plaqueplaque (1683)(1683)

"In"In thethe morningmorning II usedused toto rubrub mymy teethteeth withwith

saltsalt andand rinserinse mymy mouthmouth withwith waterwater andand afterafter

eatingeating toto cleanclean mymy molarsmolars withwith aa toothpick toothpick

thenthen mostmost alwaysalways saw,saw, withwith greatgreat wonder,wonder, thatthat inin thethe saidsaid mattermatter therethere werewere manymany veryvery littlelittle

livingliving animalculesanimalcules,, veryvery prettilyprettily aa--moving.moving. TheThe biggestbiggest sortsort hadhad aa veryvery strongstrong andand swiftswift motion,motion, andand shotshot throughthrough thethe waterwater likelike aa pikepike doesdoes throughthrough thethe water;water; mostlymostly thesethese werewere ofof smallsmall numbersnumbers."."

II

ElectronElectron MicrographMicrograph ofof PlaquePlaque

Electron Electron Micrograph Micrograph of of Plaque Plaque
Electron Electron Micrograph Micrograph of of Plaque Plaque

PhysicalPhysical SizeSize

Physical Physical Size Size
Physical Physical Size Size

ProkaryotesProkaryotes VersusVersus EukaryotesEukaryotes

StructureStructure OrganellesOrganelles –– absentabsent NucleusNucleus -- absentabsent

CompositionComposition CytoplasmicCytoplasmic membranemembrane ComplexComplex phospholipids,phospholipids, sphingolipidssphingolipids –– absent/rareabsent/rare SterolsSterols -- absent,absent, exceptexcept forfor mycoplasmamycoplasma RibosomesRibosomes -- 70S70S ratherrather thanthan 80S80S

CellCell wallwall -- uniqueunique peptidoglycanpeptidoglycan ++ otherother uniqueunique elementselements GeneticGenetic OrganizationOrganization ChromosomesChromosomes –– oneone (usually)(usually) circularcircular DiploidyDiploidy –– absentabsent HistonesHistones andand nucleosomesnucleosomes -- absentabsent CouplingCoupling ofof TranscriptionTranscription andand TranslationTranslation –– presentpresent ColinearityColinearity ofof GeneGene ++ ProductProduct (mRNA(mRNA ++ Protein)Protein) -- presentpresent

GeneticGenetic SizeSize

ORGANISMORGANISM

DNDNAA (Haploid)(Haploid) (base(base pairspairs xx 1010 66 ))

GENESGENES

EukaryoteEukaryote

HumanHuman

2,8502,850

2020 -- 25,00025,000

EukaryoteEukaryote

SaccharomycesSaccharomyces cerevisiaecerevisiae

12.112.1

5,8855,885

GramGram--negativenegative

EscherichiaEscherichia colicoli

4.64.6

4,2884,288

BacillusBacillus

GramGram--negativenegative

HaemophilusHaemophilus influenzaeinfluenzae

1.81.8

1,7431,743

BacillusBacillus

CellCell wallwall--lackinglacking BacteriumBacterium

MycoplasmaMycoplasma genitaliumgenitalium

0.580.58

470470

VirusVirus

PoxvirusPoxvirus (vaccinia)(vaccinia)

0.190.19

250250

BacterialBacterial ShapeShape

Bacterial Bacterial Shape Shape

ArrangementArrangement ofof CocciCocci

Arrangement Arrangement of of Cocci Cocci
Arrangement Arrangement of of Cocci Cocci

CapsuleCapsule NegativeNegative StainStain

Capsule Capsule – – Negative Negative Stain Stain
Capsule Capsule – – Negative Negative Stain Stain

MultipleMultiple FlagellaFlagella SalmonellaSalmonella enteritidisenteritidis

Multiple Multiple Flagella Flagella – – Salmonella Salmonella enteritidis enteritidis
Multiple Multiple Flagella Flagella – – Salmonella Salmonella enteritidis enteritidis

FlagellarFlagellar RotorRotor -- GramGram--NegativeNegative CellCell

Flagellar Flagellar Rotor Rotor - - Gram Gram - - Negative Negative Cell Cell

FlagellarFlagellar RotationRotation andand MotilityMotility

CounterclockwiseCounterclockwise RotationRotation

BacteriaBacteria SwimSwim inin aa StraightStraight LineLine (A(A && C)C)

ClockwiseClockwise RotationRotation

BacteriaBacteria TumbleTumble inin aa RandomRandom FashionFashion (B(B && D)D)

ControlControl ofof thethe ExtentExtent ofof SwimmingSwimming andand TumblingTumbling EnablesEnables ChemotaxisChemotaxis

ofof thethe ExtentExtent ofof SwimmingSwimming andand TumblingTumbling EnablesEnables ChemotaxisChemotaxis

TheThe SexSex PilusPilus inin E.E. colicoli EnablesEnables aa ‘Male’‘Male’ CellCell toto AttachAttach toto aa ‘Female’‘Female’ CellCell

Enables a a ‘Male’ ‘Male’ Cell Cell to to Attach Attach to to a a ‘Female’

ConjugationConjugation

A)A) AttachmentAttachment toto FemaleFemale CellCell viavia SexSex PilusPilus

B)B) CellCell--CellCell AppositionApposition EnablingEnabling DNADNA TransferTransfer

Sex Sex Pilus Pilus B) B) Cell Cell - - Cell Cell Apposition Apposition Enabling Enabling

BacillusBacillus Species:Species: GramGram--PositivePositive

Bacillus Bacillus Species: Species: Gram Gram - - Positive Positive
Bacillus Bacillus Species: Species: Gram Gram - - Positive Positive

E.E. ColiColi :: GramGram--NegativeNegative

E. E. Coli Coli : : Gram Gram - - Negative Negative
E. E. Coli Coli : : Gram Gram - - Negative Negative

GramGram StainStain

Gram Gram Stain Stain
Gram Gram Stain Stain

GramGram--positivepositive

((StaphylococcusStaphylococcus aureusaureus))

GramGram--negativenegative ((EscherichiaEscherichia colicoli))

ComparisonComparison ofof G+G+ andand GG-- CellCell WallsWalls

Comparison Comparison of of G+ G+ and and G G - - Cell Cell Walls Walls

G+

Comparison Comparison of of G+ G+ and and G G - - Cell Cell Walls Walls

G-

ComparisonComparison ofof G+G+ andand GG-- CellCell WallsWalls

Comparison Comparison of of G+ G+ and and G G - - Cell Cell Walls Walls

G+:G+: THICKTHICK PEPTIDOGLYCANPEPTIDOGLYCAN ~~ 4040 LayersLayers (20(20--8080 nm)nm) contains:contains:

TEICHOICTEICHOIC ACIDACID LIPOTEICHOICLIPOTEICHOIC ACIDACID

GG--:: THINTHIN PEPTIDOGLYCANPEPTIDOGLYCAN 11 -- 22 LayersLayers (2(2--55 nm)nm) PERIPLASMICPERIPLASMIC SPACESPACE OUTEROUTER MEMBRANEMEMBRANE LipidLipid BilayerBilayer (10(10--3030 nm)nm) contains:contains:

LIPOPOLYSACCHARIDELIPOPOLYSACCHARIDE (LPS)(LPS) HYDROPHILICHYDROPHILIC PORESPORES (Porins)(Porins)

LIPOPOLYSACCHARIDE LIPOPOLYSACCHARIDE (LPS) (LPS) HYDROPHILIC HYDROPHILIC PORES PORES (Porins) (Porins)

GramGram--PositivePositive CellCell WallWall

Gram Gram - - Positive Positive Cell Cell Wall Wall

GramGram--NegativeNegative CellCell WallWall

Gram Gram - - Negative Negative Cell Cell Wall Wall
Gram Gram - - Negative Negative Cell Cell Wall Wall

ComparisonComparison ofof GG-- andand G+G+ PeptidoglycanPeptidoglycan NAMNAM--LL--AlanineAlanine--DD--isoGlutamateisoGlutamate

–– MuramylMuramyl dipeptidedipeptide oror MDPMDP –– HighlyHighly conservedconserved inin G+G+ andand GG-- peptidoglycanspeptidoglycans –– isoiso == peptidepeptide bondbond viavia γγ--carboxylcarboxyl groupgroup

ThirdThird aminoamino acidacid containscontains aa freefree ––NHNH22

–– TypicallyTypically LL--LysineLysine inin G+G+ –– TypicallyTypically DiaminopimelicDiaminopimelic acidacid (DAP)(DAP) inin GG--

CrosslinkCrosslink

–– TypicallyTypically directdirect inin GG-- –– TypicallyTypically aa shortshort peptidepeptide inin Gram+Gram+ –– PenicillinsPenicillins andand cephalosporinscephalosporins inhibitinhibit thethe crosslinkingcrosslinking stepstep inin bothboth G+G+ andand GG-- bacteriabacteria

ComparisonComparison ofof GG-- andand G+G+ PeptidoglycanPeptidoglycan

Comparison Comparison of of G G - - and and G+ G+ Peptidoglycan Peptidoglycan

DiaminoacidsDiaminoacids inin PeptidoglycanPeptidoglycan

33 rdrd PositionPosition ofof PentapeptidePentapeptide EnablesEnables CrosslinkingCrosslinking

3 r d r d Position Position of of Pentapeptide Pentapeptide – – Enables Enables Crosslinking

TeichoicTeichoic AcidsAcids

Teichoic Teichoic Acids Acids
Teichoic Teichoic Acids Acids

GramGram--PositivePositive CellCell WallWall

Gram Gram - - Positive Positive Cell Cell Wall Wall

LIPOPOLYSACCHARIDE (LPS) = ENDOTOXIN - Restricted to Gram- bacteria; major component of cell wall; anchored in the outer leaflet of the outer membrane by LIPID A.

in the outer leaflet of the outer membrane by LIPID A. O-ANTIGEN : 3 - 7

O-ANTIGEN: 3 - 7 sugar polysaccharide unit, repeated 50 –

100 times. Example:

A major antigenic determinant

E. coli serotype O157:H7

LIPID A: responsible for ENDOTOXIN activity

LPS (Endotoxin) initiates many systemic effects of Gram-negative infections

1. INFLAMMATION - LPS activates complement and has additional effects on innate immunity

2. FEVER (PYROGENICITY) - at moderate systemic LPS levels

3. LETHAL TOXICITY - at high systemic LPS levels – Hypotension, Shock, Death

E.E. ColiColi LipidLipid AA isis hexahexa--acyl’acyl’

E. E. Coli Coli Lipid Lipid A A is is ‘ ‘ hexa hexa - -

LipidLipid AA structuresstructures andand EndotoxinEndotoxin activityactivity varyvary betweenbetween bacterialbacterial speciesspecies

MaximalMaximal endotoxinendotoxin activityactivity occursoccurs withwith hexahexa--acylacyl LPSsLPSs’,’, whichwhich containcontain anan E.E. colicoli--likelike lipidlipid A:A: 66 fattyfatty acylacyl chainschains attachedattached toto diphosphoryldiphosphoryl diglucosaminediglucosamine TheThe number,number, lengthlength andand positionposition ofof thethe acylacyl chains,chains, andand thethe phosphorylationphosphorylation state,state, affectsaffects activityactivity TheThe periodontalperiodontal pathogenpathogen PorphyromonasPorphyromonas gingivalisgingivalis (Pg)(Pg) producesproduces tritri--,, tetratetra-- andand pentapenta-- acylatedacylated lipidlipid AA

(Pg) produces produces tri tri - - , , tetra tetra - - and and penta
(Pg) produces produces tri tri - - , , tetra tetra - - and and penta

––

thethe relativerelative amountamount ofof eacheach speciesspecies isis growthgrowth dependentdependent andand affectsaffects thethe inflammatoryinflammatory responseresponse toto thisthis organismorganism

ENDOTOXIN (LPS) is a heat stable toxin

Do not confuse with

EXOTOXINS, which are proteins and generally are heat labile

ENTEROTOXINS, which are exotoxins that cause an enteritis (diarrhea, vomiting)

GramGram--NegativeNegative CellCell WallWall

Gram Gram - - Negative Negative Cell Cell Wall Wall
Gram Gram - - Negative Negative Cell Cell Wall Wall

TheThe NN--TerminusTerminus ofof BacterialBacterial LipoproteinLipoprotein isis TriacylTriacyl-- oror DiacylDiacyl--CysteineCysteine ThisThis structurestructure isis absentabsent fromfrom EukaryotesEukaryotes

This This structure structure is is absent absent from from Eukaryotes Eukaryotes FA= FA= Fatty Fatty

FA=FA= FattyFatty AcidAcid

PEPTIDOGLYCANPEPTIDOGLYCAN--DEFICIENTDEFICIENT FORMSFORMS

MycoplasmaMycoplasma:: aa genusgenus withwith nono genesgenes forfor thethe cellcell wallwall

LL PhasePhase BacteriaBacteria:: naturallynaturally occurringoccurring wallwall--lessless variantsvariants ofof cellcell wallwall--containingcontaining species;species; cancan regainregain cellcell wall;wall; maymay contributecontribute toto somesome treatmenttreatment failurefailure

––

LL forfor ListerLister Institute,Institute, wherewhere discovereddiscovered

ACIDACID--FASTFAST BACTERIABACTERIA

StainedStained withwith aa dyedye (e.g.,(e.g., thethe redred dyedye carbolfuchsin)carbolfuchsin) thatthat resistsresists decolorizationdecolorization withwith acidacid--alcoholalcohol solutionssolutions

MembersMembers ofof thethe genusgenus MycobacteriumMycobacterium

(e.g.,(e.g., M.M. tuberculosistuberculosis;; M.M. lepraeleprae)) areare

stronglystrongly acidacid--fastfast –– AcidAcid--fastfast stainingstaining resultsresults fromfrom largelarge amountsamounts ofof MYCOLICMYCOLIC ACIDSACIDS andand mycolicmycolic acidacid--estersesters inin thethe cellcell wallwall

AcidAcid--FastFast CellCell WallWall

ThoughThough Gram+,Gram+, thethe acidacid--fastfast cellcell wallwall hashas anan outerouter membranemembrane asas wellwell asas porinsporins

–– likelike thethe GG-- cellcell wallwall

TheThe innerinner leafletleaflet ofof thethe outerouter membranemembrane containscontains mycolicmycolic acidsacids linkedlinked toto arabinogalactan,arabinogalactan, ((Arabinose)nArabinose)n-- (Galactose)n(Galactose)n,, whichwhich isis covalentlycovalently linkedlinked toto peptidoglycanpeptidoglycan TheThe outerouter leafletleaflet alsoalso containscontains mycolicmycolic acidacid structuresstructures

AA ModelModel ofof thethe MycobacterialMycobacterial CellCell WallWall

AA == Arabinose;Arabinose; GG == galactosegalactose

the the Mycobacterial Mycobacterial Cell Cell Wall Wall A A = = Arabinose; Arabinose; G G

MycolicMycolic AcidsAcids

HighHigh molecularmolecular weightweight (60(60--9090 carboncarbon atoms),atoms), branchedbranched--chain,chain, veryvery hydrophobic,hydrophobic, fattyfatty acidsacids thatthat areare responsibleresponsible forfor AcidAcid--FastFast StainingStaining,, VirulenceVirulence andand HighHigh--StabilityStability ofof MycobacteriaMycobacteria ShorterShorter homologshomologs occuroccur inin otherother generagenera

–– CorynobacteriumCorynobacterium:: corynomycoliccorynomycolic acidsacids (22(22--3636 carbons)carbons) –– NocardiaNocardia:: nocardomycolicnocardomycolic acidsacids (44(44--6060 carbons)carbons)

carbons)carbons) –– NocardiaNocardia :: nocardomycolicnocardomycolic acidsacids (44(44-- 6060 carbons)carbons)

AcidAcid--FastFast StainedStained LymphLymph NodeNode fromfrom anan AIDSAIDS Patient:Patient:

MycobacteriumMycobacterium AviumAvium--intracellulareintracellulare inin MacrophagesMacrophages

Patient: Mycobacterium Mycobacterium Avium Avium - - intracellulare intracellulare in in Macrophages Macrophages

EndosporesEndospores

CertainCertain bacteriabacteria cancan differentiatedifferentiate intointo aa dormant,dormant, heatheat--stablestable formform –– EndosporesEndospores

EndosporesEndospores areare aa meansmeans ofof survivalsurvival andand dissemination,dissemination, notnot reproductionreproduction

TheThe endosporeendospore formersformers ofof clinicalclinical interestinterest areare foundfound inin twotwo generagenera ofof G+G+ bacillibacilli

–– ClostridiumClostridium -- anaerobesanaerobes thatthat includeinclude C.C. botulinumbotulinum -- causescauses botulismbotulism C.C. tetanitetani –– causescauses tetanustetanus

–– BacillusBacillus –– aerobes/facultativeaerobes/facultative anaerobesanaerobes thatthat includeinclude B.B. anthracisanthracis –– causescauses anthraxanthrax

EndosporesEndospores ContinuedContinued

EndosporesEndospores areare likelike seedsseeds

–– nono metabolicmetabolic properties,properties, –– veryvery lowlow waterwater contentcontent -- thus,thus, veryvery refractilerefractile –– stablestable forfor longlong periodsperiods ofof timetime -- years,years, decadesdecades

EndosporesEndospores areare resistantresistant toto

–– heatheat –– desiccationdesiccation pHpH extremesextremes chemicalchemical agentsagents –– radiationradiation

––

––

TheseThese resistanceresistance traitstraits areare associatedassociated withwith

–– thethe lowlow waterwater contentcontent ofof thethe cocorere (which(which containscontains thethe DNA,DNA, ribosomes,ribosomes, etc.)etc.) –– SS--SS bondedbonded keratinkeratin--likelike proteinsproteins inin thethe sporespore coatcoat

SporulationSporulation

Sporulation Sporulation

SporesSpores AreAre RefractileRefractile

BacillusBacillus

ClostridiumClostridium

Spores Spores Are Are Refractile Refractile Bacillus Bacillus Clostridium Clostridium

ImportanceImportance ofof thethe CellCell EnvelopeEnvelope inin BacterialBacterial InfectionInfection

AntigenicAntigenic

AntiphagocyticAntiphagocytic

SourceSource ofof adhesins:adhesins: manymany bacteriabacteria

SiteSite ofof actionaction ofof cellcell wallwall antimicrobialantimicrobial agents:agents: e.g.,e.g., penicipenicillinsllins

ManyMany componentscomponents (e.g.,(e.g., LPS,LPS, polysaccharides,polysaccharides, teichoicteichoic acids)acids) activateactivate complementcomplement

ManyMany componentscomponents (e.g.,(e.g., LPS,LPS, lipoteichoiclipoteichoic acid,acid, lipoproteins,lipoproteins, peptidoglycan,peptidoglycan, flagellarflagellar protein)protein) stimulatestimulate additionaladditional elementselements ofof thethe innateinnate immuneimmune responseresponse suchsuch componentscomponents areare

PathogenPathogen--AssociatedAssociated MolecularMolecular PatternsPatterns (PAMPs)(PAMPs)