INTRODUCTION RESISTANT BACTERIA THE BACTERIAL CELLusers.skynet.be/fa055557/...Resistente_Bacterien.pdf · Gram +ve and Gram -ve bacteria Electron Micrograph of a Gram-Negative Cell

DEVOS Ann 22/02/2005 1

RESISTANT BACTERIA RESISTANT BACTERIA

andand

ANTIBIOTICSANTIBIOTICS

Ann DEVOS 28-09-2004


INTRODUCTION

THE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS


INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS


BACTERIA and HUMANSBACTERIA and HUMANS• Before birth

• Normally no contact with bacteria

After birthBacteria as commensals from persons and environment

All parts of the body which are in contact with the environment

- are permanently colonized by bacteria

- not harmful under normal conditions

On incidental penetration of bacteria in tissues,

the body reacts by a combined action of

- blood clotting

- circulating antibodies

- fagocytosis


BACTERIA and HUMANSBACTERIA and HUMANS• Opportunistic pathogens

• Cannot infect under normal conditions (no penetration into tissues, cannot

fix to the host cells, cannot multiply, cannot produce toxins…

e.g. E. coli )

• Infect when

• The host resistance mechanism has been weakened by medication, malnutrition ..

• Local disruption of the epithelial barriers of the skin and epithelial mucus

secreting tissues = wounds

• Pathogens are harmful• Production of poisonous products

• Destruction of host cells

• Excessive growth and depletion of energy reserves of host


INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNA MECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS

DEVOS Ann 22/02/2005 2


Source: CDrom MicrobiologieNatuur & Techniek (Uitgeverij Veen magazines, Amsterdam)

BACTERIA BACTERIA


Visualizing small thingsVisualizing small things


BACTERIABACTERIA

MycobacteriumMycobacterium tuberculosistuberculosisare are becomingbecoming increasinglyincreasingly resistantresistant toto

antibioticsantibiotics.. ..

Salmonella Salmonella enteritidisenteritidis10.000x, 10.000x, entericenteric bacteriumbacteriumrelatedrelated toto E. E. colicoli

EndosporesEndospores of of ClostridiumClostridium and Bacillus speciesand Bacillus species

DesulfovibrioDesulfovibrio 15.000x, 15.000x, withwith single single polarpolarflagellumiflagellumi

ShigellaShigella is a is a nonnon--motilemotileentericenteric bacteriumbacterium

E. coli


CharacterizationCharacterization of Bacteriaof Bacteria

From: http://

By shapeBacillus anthracis Bordetella holmessii Clostridium tetanii Enterococcus faecalis

Esherichia coli en E.coli hemorragic type Haemophilus influenza Listeria monocytogenes

Neisseria menengitidis Spirullina spp. Staphylococcus aureus Streptococcus faecalis

Strept.pneumoniae Salmomella typhii Salmonella enteritidis Vibrio cholerae


Source: figure CDrom Microbiologie, Natuur & Techniek (Uitgeverij Veen magazines, A’dam)Images:http://commons.wikimedia.org/wiki/Image:Staphylococcus_aureus_Gram.jpg

microscopic image of Escherichia coli (ATCC 11775). Gram staining, magnification:1,000.

microscopic image of Staphylococcus aureus (ATCC 25923). Gram staining, magnification:1,000.

CharacterizationCharacterization of Bacteriaof BacteriaBy Gram-staining (Hans Christian Gram)based on a chemical reaction with the cell wall of bacteria


PROCARYOTIC and EUCARYOTIC CELLSPROCARYOTIC and EUCARYOTIC CELLS

DEVOS Ann 22/02/2005 3


Gram +Gram +veve and Gram and Gram --veve bacteriabacteria

Electron Micrograph of a Gram-Negative Cell Wall

Structure of a Gram-Negative Cell Wall

Electron Micrograph of a Gram-Positive Cell Wall

Structure of a Gram-Positive Cell Wall


••Cover of Cover of Computer Graphics and ApplicationsComputer Graphics and Applications, July, July--August, August, 20012001

TranscriptionTranscription and and TranslationTranslationof the of the insulininsulin proteinprotein

1: The DNA coded information (blue helix) in the cell nucleus is copied, or transcribed, to an RNA mirror image (red strand)

2: The ribosome translates the linear pattern described in the RNA to construct a protein strand. This translation is based on the genetic code. Transfer RNAs(cross-like shapes) ferry amino acids to the growing protein chain based on the 3-codon RNA sequence

3: Finally, the insulin proetin strand folds itself into its active form- and to the end result of the transcription-translation process, LIFE itself


RIBOSOMESRIBOSOMES




History ofHistory of AntibioticsAntibiotics

Time line of events

1900 2000

1928, Penicillin discovered

1932, Sulfonamides

discovered

1940’s: Penicillin becomes commercially

available and Cephalosporins are

synthesized

1952, Erythromycin

discovered

1956, Vancomycin introduced

1962, Quinolones discovered

1980’s, Fluorinated Quinolones available

Linezolid becomes available


1940’s 1940’s MassMass penicillinpenicillin productionproduction

DEVOS Ann 22/02/2005 4


HISTORY OF ANTIBIOTIC RESISTANCEHISTORY OF ANTIBIOTIC RESISTANCE

198719721956Vancomycin

195619521948Tetracyclin

194719471944Streptomycin

194019401928Penicillin

Resistance identified

Used clinicallyDiscoveredAntibiotic


INCREASE IN ANTIBIOTIC RESISTANCEINCREASE IN ANTIBIOTIC RESISTANCE

>7019966,01985Multidrug (2)S. typhymurium

12,019961,01994ciprofloxacin

6,519942,91989ciprofloxacin

5,719942,71989ceftazidimeKlebsiella

10,919953,31989erythromycin

2,919950,31989penicillinS. pneumoniae

10,819950,41988VancomycinEnterococci

13,719962,21990MethicillinS. Aureus (MRSA)

1,219950,61981Multidrug (1)M. tuberculosis

%Year%YearResistanceBacterium

1 INH, rifampicin, ethambutol2 Ampicillin, chloramphenicol, sulfonamides, tetracycline


Penicillin Resistance in the USPenicillin Resistance in the US

PenI(MIC 0.12-1.0 µg/mL)

PenR(MIC ≥2.0 µg/mL)

% PenR or PenI0

1996-971994-951992-931990-911988-89

198719861985198419831982198119801979

10 20 30 40

1997-981999-002000-01

Doern GV. Am J Med. 1995;99(Suppl 6):3S-7S.Thornsberry C, et al. Diagn Microbiol Infect Dis. 1997;29(4):249-257.PROTEKT 2000; PROTEKT US 2001.Thornsberry C, et al. J Antimicrob Chemother. 1999;44(6):749-759.


Resistance against Resistance against fluoroquinolonesfluoroquinolonesin in HawaianHawaian populationpopulation


ANTIBIOTICS: General ConceptsANTIBIOTICS: General ConceptsSelectivity/Toxicity

Toxicity levels for bacteria ≠ from that of host High doses w/o toxic effectsNo antibiotic is completely safe!

Antibiotics must survive their route of administrationBroad and narrow spectrum drugsCategories of Ab:

Bactericidal: usually Ab of choiceBacteriostatic: duration of treatment sufficient for host defences

CombinationsSynergismAntagonismIndifference


ANTIBIOTICS: General Concepts ANTIBIOTICS: General Concepts

Antibiotic susceptibility testing (in vitro)

Minimum inhibitory concentration (MIC)= Lowest concentration that results in inhibition of visible growth

Minimum bactericidal concentration (MBC)= Lowest concentration that kills 99.9% of the original inoculum

DEVOS Ann 22/02/2005 5




ANTIBIOTIC RESISTANCEANTIBIOTIC RESISTANCE•INTRINSIC RESISTANCE

•Inherent features of bacterial species which prevent antibiotic action

•Usually expressed by chromosomal genes

•ACQUIRED RESISTANCE

•MUTATIONS in chromosomal genes

The spontaneous mutational frequency is 10-7

Drug-resistant tuberculosis arises this way

•TRANSFORMATION

•CONJUGATION

•TRANSDUCTION


INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EXCHANGE DNA

MUTATIONTRANSFORMATIONCONJUGATION

PlasmidsTransposons or Transposable elements

TRANSDUCTIONMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS


TransformationTransformation

Insertion

Crossingover



DEVOS Ann 22/02/2005 6















CONJUGATIONCONJUGATION

Is the transfer of one or more genesfrom one bacterium to another in a single process that can take as little as an hour to complete.

Also been called bacterial sex or horizontal gene transfer.

Allows bacteria to become resistant toantibiotics by acquiring DNA from a bacterium that already has acquiredresistance to the antibiotic.

Bacteria are capable of transferring genes to or acquiring genesfrom bacteria of a different species or genus.

Bacteria, E. coli as an example:transfer a portion of their chromosome to a recipient with which they are in direct contact.

DEVOS Ann 22/02/2005 7


PLASMIDSPLASMIDS

Plasmids

•Extrachromosomal genetic elements that replicate independently

•Plasmids are mobile by conjunction

•Frequently carry Ab resistance genes

•Up to 7 different resistance genes on one plasmid

•In the absence of Ab, the plasmid is often lost from the majority of cells

•Cost of carrying a resistance gene

•Exposure of the Ab results in all cells having the plasmid (s)

•Sensitive cells are killed and plasmids are mobilized


F- bacteria

ConjugationConjugationF plasmid

Mating Bridge

F+ bacteria

Fplasmid


ConjugationConjugation

F- bacteria

F plasmid

F+ bacteria

Fplasmid

Mating Bridge



F- bacteria

F plasmid

F+ bacteria

Fplasmid

Mating Bridge



F- bacteria

F plasmid

F+ bacteria

Fplasmid

Fplasmid

Mating Bridge


Range of resistance exchangeRange of resistance exchange

Gram +veBacteria

YEAST Non Conjugative

Gram –ve bacteria

Streptomycetes

Amabile-C

uevas&

Chicurel, Bacterialplasm

idsand gene flux. C

ell70: 189-199 (1992)

Conjugative gram –ve bacteria

Mycobacteria

DEVOS Ann 22/02/2005 8


TRANSPOSABLE GENETIC ELEMENTSTRANSPOSABLE GENETIC ELEMENTS

•Mobile DNA sequences which can move from one location on

the DNA molecule onto another molecule

•Not capable of self-replication (no replicon)

•Transposition is mediated by site-specific recombination, mediated by a transposase

•Classified as TransposoN Tn551, Tn4291Tn551, Tn4291 etc.

•Central region of transposon often carries Ab resistance gene(s)

Results in a spread of antibiotic resistance


Transposable Genetic ElementsTransposable Genetic Elements

IS ISResistance Gene(s)

IS ISResistance Gene(s)

Carry genes except those involved in transposition

Structure : IS

IS IS







BACTERIOPHAGEBACTERIOPHAGE


TransductionTransductionDestruction of the bacteria’s DNA

Lysis

Replication of the viral genome

Production of viral parts

Packaging

Infection



DEVOS Ann 22/02/2005 9


GENETIC DETERMINANTSGENETIC DETERMINANTS• Intrinsic resistance

•Resistance determined by chromosomal genes

•Eg Beta-lactamases of gram-ve bacteria inactivate beta-lactam antibiotics

• Acquired resistance

•Resistance determined by chromosomal genes

•Eg Nalidixic acid resistance in E.coli is caused by the mutation at the N-terminus of the GyrA protein which reduces nalidixic acid binding


ACQUIRED DETERMINANTSACQUIRED DETERMINANTS

Resistance determined by plasmidsExtrachromosomal genetic elements that replicateindependently of the chromosome

Resistance determined by transposonsMobile genetic elements capable of transferring (transposing) themselves from one DNA molecule toanother


ANTIBIOTIC RESISTANCE MECHANISMSANTIBIOTIC RESISTANCE MECHANISMS

• Bypass antibiotic-sensitive step• Altered antibiotic target/Overproduction of target

• Reduced antibiotic accumulation

•Impaired uptake

•Enhanced efflux

• Antibiotic inactivation


ANTIBIOTIC RESISTANCE MECHANISMSANTIBIOTIC RESISTANCE MECHANISMS

TrimethoprimSulfonamides

β-lactamsChloramphenicolErythromycin (AG)

TetracyclinsQuinolones

β-lactamsChloramphenicolErythromycin (AG)

StreptomycinQuinelonesRifampicin

β-lactamsChloramphenicolAminoglycosides

BypassReducedaccumulationAltered targetInactivation


INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab

IMPAIRED DNA SYNTHESISSulfonamidesQuinolones

IMPAIRED RNA SYNTHESISRifampicin (α-amanitin, actinomycin)

IMPAIRED PROTEIN SYNTHESISTetracyclinesChloramphenicolMacrolides (Erythromycin, Clathromycin)Aminoglycosides (Streptomycin, Gentamycin, Tobramycin, Kanamycin)

ACTING ON THE CELL WALLBeta-lactams (Penicillin, Ampicillin,..)

ACTING ON THE CELL MEMBRANEblabla

FUNGAL INFECTIONSCONCLUSIONS


SULFONAMIDESSULFONAMIDES

Chemical similarity between the sulfa drugs and PABA

DEVOS Ann 22/02/2005 10


SULFONAMIDESSULFONAMIDES: : MechanismMechanism of of actionaction

Recent studies indicate that substituents on the N(1) nitrogen may play the role of competing for a site on the dihydropteroate synthetase (DHPS) enzyme surface reserved for the glutamate residue in p-aminobenzoic acid-glutamate through one of the following two ways:a) Direct competition in the linking of PABA-glutamate with the pteridine derivative.b) Indirect interference with the coupling of glutamate to dihydropteroic acid.

FA is synthesized in two steps in bacteria


RESISTANCE TO SULPHONAMIDESRESISTANCE TO SULPHONAMIDESChromosomal-encoded

Hyperproduction of PABAMutation of dihydropteroatesynthetase (DHPS) lowers affinityfor sulphonamides

Plasmid-encoded

Duplication of DHPS enzymeAt least 2 types (I and II) of the DHPS enzymes have been foundwhich are only 50% homologous in sequenceBind sulphonamides 10.000-fold less efficiently

PteridineDHPS +PABA

+SA Dihydroptereic acid

Dihydrofolic acid

Tetrahydrofolic acid

DNA synthesis (Pu)


QuinolonesQuinolonesQuinolone antibacterial drugs:

nalidixic acidNorfloxacinOfloxacinciprofloxacin

Broad-spectrum agentsRapidly kill bacteriaAct by inhibiting the activity of the bacterial DNA gyrase,preventing the normal functioning of DNA.

Bacterial DNA exists in a supercoiled formThe enzyme DNA gyrase, a topoisomerase, is responsible for introducing negative supercoils into the structure.

Humans do possess DNA gyrase but it is structurally distinct from the bacterialenzyme and remains unaffected by the activity of quinolones.


RESISTANCE TO QUINOLONESRESISTANCE TO QUINOLONES

Only chromosomal mutations

gyrA mutations confer nalixidicacid resistance only

N-terminal point mutation in DNA gyrase which reduceaffinity of binding of quinolones

gyrB mutations confer resistance tonalidixic acid and to ciprofloxacin

Amino acid substitutions whichreduce affinity of binding

Supercoiled DNADNA gyrase

Double Strand breakage

DNA unwinding

DNA replication

Quinolones causedouble strand breaks




IMPAIRED RNA SYNTHESISRifampicin (α-amanitin, actinomycin)






RIFAMPICINRIFAMPICINThe bacterial DNA-dependent RNA polymerase is inhibited

Used as an antimycobacterialBactericidal

Little effect on eukaryotic cells.

It is active against the mitochondrial(and chloroplast) RNA polymerasebut its penetration into mitochondriais so poor that it displays very littleactivity in intact eukaryotic cells.

used in treating tuberculosis and against meningococcal meningitis.

DEVOS Ann 22/02/2005 11


Eukaryotic and Prokaryotic RNA PolymeraseEukaryotic and Prokaryotic RNA Polymerase

• 3 classes of RNA polymerase,I,II,and III, binds to promoters toInitiate transcription• Nuclear RNAP is complex enzyme- containing 2 LSU+ several SSU, 5 are common

• Prokaryotic RNAP consist four Core subunits (2α; β; β’) and one regulatory subunit (σ)


The The bacterialbacterial RNA RNA polymerasepolymerasecomplexedcomplexed withwith DNADNA

The bacterial RNA polymerase bound to a short piece of DNA containing a promoter sequence.

The closed complex - because the DNA is not melted.

The open complex - where the RNA polymerase actually melts DNA to expose one of the double-helical strands to the core enzyme which will use it as the template to synthesize RNA.


The The bacterialbacterial RNA RNA polymerasepolymerasecomplexedcomplexed withwith rifampicinrifampicin

• Ligand: RFP


RESISTANCE TO RIFAMPICINRESISTANCE TO RIFAMPICIN

Only chromosomal mutations

Altered DNA-dependent RNA polymerase

Beta sub-unit of the RNA polymerase does no longer bind rifampicin

DNA

RNA polymerase

RNA

Rifampicin binds to the beta-sub unit of the RNA polymerase and inhibitstranscription




IMPAIRED RNA SYNTHESISRifampicin

IMPAIRED PROTEIN SYNTHESISACTING ON THE CELL WALL

Beta-lactams (Penicillin, Ampicillin,..)ACTING ON THE CELL MEMBRANE

blabla



Protein Synthesis InhibitorsProtein Synthesis InhibitorsBactericidal

AminoglycosidesStreptomycin, Kanamycin, NeomycinGentamicin, Tobramycin, Amikacin, Netilmicin

Oxazolidone (Linezolid)

Bacteriostatic

ChloramphenicolTetracyclines

Doxycycline, Minocycline

MacrolidesErythromycin, Azithromycin, ClarithromycinClindamycin

StreptograminsQuinupristin/Dalfopristin (Synercid)

DEVOS Ann 22/02/2005 12


InhibitionInhibition of of proteinprotein synthesissynthesis byby AbAb

http://www.elmhurst.edu/~chm/vchembook/654antibiotic.html Ann DEVOS 22-02-2005

TETRACYCLINSTETRACYCLINSTetracyclins include: Aureomycin

Terramycin,Panmycin

Isolated from Streptomyces spp.BacteriostaticHave the broadest spectrum of antimicrobial activity.

Inhibit the codon-anticodon interaction.

Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism.


TETRACYCLINS TETRACYCLINS blocksblocks translationtranslationduringduring bacterialbacterial proteinprotein synthesissynthesis

The tetracyclins (Tc, doxycycline, demeclocycline, minocycline, etc. ) block bacterial translation by binding reversible to the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAscannot align properly with the codons of the mRNA

http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/tetres.html


MECHANISM OF TETRACYCLINE MECHANISM OF TETRACYCLINE RESISTANCERESISTANCE

Plasmid/transposon-encoded

Membrane proteins are encoded bythe R-genes

Mechanism involves energy-dependent efflux

Decreased accumulation of the antibiotic

Tc Tc RR

Tc in Tc in Tc outTc out


Chloramphenicol member: Chloromycetin

Broad spectrum antibioticwith similar to the tetracylines. Bacteriostatic

At present, it is the only antibiotic prepared synthetically.

Reserved for treatment of serious infectionsbecause it is potentially highly toxic to bone marrow cells.

It inhibits protein synthesis by attaching to the ribosomeInterferes with the formation of peptide bonds between amino acids. It behaves as an antimetabolite for the essential amino acid phenylalanine at ribosomal binding sites

CHLORAMPHENICOLCHLORAMPHENICOL


RESISTANCE TO CHLORAMPHENICOLRESISTANCE TO CHLORAMPHENICOL

Plasmid/transposon-encoded

Enzymatic inactivationPlasmid/transposon-encodedchloramphenicol acetyltransferase (cat)

Impaired uptakePlasmid-encoded cml-geneencodes a protein which reducesuptake of the antibiotic

Ribosome of procaryote

ChloramphenicolCat

Chloramphenicolmonoacetate-

INACTIVE

Protein synthesis

Cat leads to the acetylation of chloramphenicol that preventsbinding to the ribosome

DEVOS Ann 22/02/2005 13


MACROLIDESMACROLIDESMacrolides are Erythromycin,

Clarithromycin,Azithromycin

Discovered in 1952Metabolic products of a strain of Actinomyces (Soil bacteria, Streptomyces erythreus), originallyobtained from a soil sample.

Erythromycin is an orally effectiveantibiotic with bacteriostatic activity

Used in penicillin-allergic patientsagainst Staphylococcus, Streptococcus, Pneumococcus

Erythromycin


MACROLIDES MACROLIDES blockingblocking translationtranslationduringduring bacterialbacterial proteinprotein synthesissynthesis

Mode of action of Macrolides:

http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/macresr_anim.html

The macrolides (erythromycin, azitthromycin, clarithromycin, dirithromycin, troleandomycin, etc.)

- bind reversibly to the 50S subunit. - They appear to inhibit elongation of the

protein by preventing the enzymepeptidyltransferase from forming peptide bonds between the amino acids(macresp).

- They may also prevent the transfer of the peptidyl tRNA from the A-site to the P-site as show here (macresr)


ERYTHROMYCIN : ERYTHROMYCIN : mechanismmechanism of of actionaction

Erythromycin (and other macrolides) inhibit protein synthesis

bind to the 23S rRNA molecule of the bacterial ribosome

The association between erythromycin and the ribosome is reversible.

Gram +ve bacteria accumulate about 100 times more erythromycin than do gram -ve micro-organisms.

Certain resistant microorganisms with mutational changes in components of the 30S subunit fail to bind the drug.


ErythromycinErythromycin--ResistanceResistance MethylasesMethylases::Different classes of Different classes of genesgenes involvedinvolved

ChromosomeBacillus sphericusermG

pBF4Bacteroides fragilesermF

ChromosomeArthrobacter sp.ermA’

ChromosomeStreptomyces erytheusermE

ChromosomeBacillus licheniformisermD

pE194Staphylococcus aureusermC

pAM77Streptococcus sanguisermAMTn554Staphylococcus aureusermA

Ann DEVOS 22-02-2005 Ann DEVOS 22-02-2005

AMINOGLYCOSIDESAMINOGLYCOSIDES• Aminoglycoside family includes streptomycin

gentamicinneomycintobramycinkanamycin ….

• Clinically important group of antibiotics• Broad-spectrum of activity• Are bactericidal in action.

• Have a variety of effects within the bacterial cell• Principally they inhibit protein synthesis by binding to the 30S ribosomal subunit

to prevent the formation of an initiation complex with messenger RNA.

• They also cause misreading of the messenger RNA message, leading to the production of nonsense peptides.

• Aminoglycosides increase membrane leakage at high doses.

• Antibiotics such as gentamicin and kanamycin exist as mixtures of severalclosely related structural compounds.

DEVOS Ann 22/02/2005 14


STREPTOMYCINSTREPTOMYCIN

Effective against gram-ve bacteria

Also used in the treatmentof tuberculosis.

Binds to the 30S ribosomeChanges its shapeInhibits protein synthesis by causing a misreading of messenger RNA information.


AgriculturalAgricultural useuse of of streptomycinstreptomycinleadsleads toto resistanceresistance

In regions of dense apple and pear production, streptomycin is applied by air-blast spray equipment to hundreds of hectares of nearly contiguous orchards. While growers strive to minimize drift by spraying during calm weather, non-target organisms on plants, in the soil, and in water are exposed to low doses of streptomycin. Low doses of antibiotics applied to large areas over long periods of time contribute to the build-up of resistance in clinical bacteria. Might a similar scenario be played out in apple and pear orchards?


RESISTANCE TO AMINOGLYCOSIDESRESISTANCE TO AMINOGLYCOSIDES

Plasmid/transposon-encodedenzymes

Effect antibiotic uptakeEnzymatic modifications of the antibioticThree classes of enzyme

Acetyltransferases (AAC)Adenyltransferases (AAD)Phosphotransferases (APH)

Enzymes divided into sub-typeson the basis of the sites theymodify in the antibiotics (> 30 in total)

Ribosome of procaryote

Kanamycin

ModifiedKanamycin

Protein synthesis

Inhibitors of protein synthesis










BETA LACTAM ANTIBIOTICSBETA LACTAM ANTIBIOTICS

PenicillinsPenicillin GAmpicilin

CephalosporinsCephalexinCofotaximeCefuroxime

CephamycinsCefoxitin

CarbapenemsImipenem

MonobactamsAztreonam

OUTER

MEMBRANE

MUREIN

CYTOPLASMIC

MEMBRANE


StructureStructure of of PenicillinPenicillin

Penicillin V Ampicillin

DEVOS Ann 22/02/2005 15


SimilaritySimilarity betweenbetween the the Gram +Gram +veve cellcell wallwall and and PenicillinPenicillin

Image courtesy of the University of Texas-Houston Medical


ββ--LACTAMSLACTAMS

• Are bactericidal

• interfere with cell wall synthesis by binding to penicillin-binding proteins (PBPs) which are located in bacterial cell walls

• inhibition of PBPs leads to inhibition of peptidoglycan synthesis

Penicillin works outside the cellmimics the D-ala-L-ala tail of the peptidoglycanblocks transpeptidase


RESISTANCE TO RESISTANCE TO ββ--LACTAMSLACTAMSProduction of β-lactamase enzymes

Most important and common mechanism of resistance in clinical isolates

Hydrolyses the β-lactam ring causinginactivation

Alteration in PBPs leading todecreased binding affinity

major cause of resistance in Staphylococcusaureus (MRSA) and Streptococcus species

Alteration of outer membraneleading to decreased penetration

a major cause of resistance in Psuedemonasaeruginosa and other pathogenic gram –vespecies


RESISTANCE TO BETA LACTAMSRESISTANCE TO BETA LACTAMS

OUTER

MEMBRANE

Peptidoglycan

CYTOPLASMIC

MEMBRANE

BETA LACTAM

PBP

BETA-LACTAMASE

Enzymatic inactivation


VRSA?VRSA?

1992: laboratory transmission of resistance from VRE to S. aureasshowed VRSA is possible

Late 1990’s: First reports of vancomycin insensitive Staph

Proved to be bugs with vastly thickened cell wall

Thick cell wall of VRSAreduces virulence

These isolates retainedother antibiotic sensitivities





IMPAIRED PROTEIN SYNTHESISTetracyclinesChloramphenicolErythromycinStreptomycinAminoglycosides (Kanamycin)




DEVOS Ann 22/02/2005 16


CellCell membranemembrane

Antibiotics including the polymixins and gramicidin act by interfering with the functioning of the bacterial cellmembrane by increasing its permeability.

Gramicidin is one of a family of cyclic decapeptidesactive against Gram +ve bacteria.

Polymixins have a smaller peptide ring attached to a peptide chain ending with a branched fatty acid.

They act specifically against Gram -ve bacteria, althoughchemically modified derivatives do have a broaderspectrum of activity.

These antibiotics are toxic to humans and are now rarelyused in clinical practice.

Polymixins

gramicidin


Gating (opening & closing) of a gramicidin channel is thought to involve reversible dimerization.

An open channel forms when two gramicidin molecules join end to end to span the membrane.

This model is consistent with the finding that, at high concentrations of gramicidin, the overall transport rate depends on [gramidicin]2.

GatingGating of of gramicidingramicidin channelschannels


INTRODUCTIONANTIBIOTICSTHE BACTERIAL CELLHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab FUNGAL INFECTIONSCONCLUSIONS


FUNGAL INFECTIONSFUNGAL INFECTIONS

Caused by eukaryotic organisms

For that reason they generally present more difficult therapeutic problems than do bacterial infections.

There are relatively few agents that can beused to treat fungal infections.


YEAST and FUNGAL INFECTIONSYEAST and FUNGAL INFECTIONSThe fungal cell wall may be considered to be a prime target for selectively toxic antifungalagentsIt consists of a chitin structure, absent fromhuman cells.

No clinically available inhibitor of chitin synthesisanalogous to the β-lactams exists at present.Much effort is being directed towards developingsuch agents.

Other targets are currently being exploited. Ann DEVOS 22-02-2005

PolyenePolyene AB: AB: NystatinNystatin• Polyene antibiotics bind to sterols (ergosterols) within the fungal

membrane, disrupting its integrity.

• This makes the membrane leaky, leading to a loss of small molecules from the fungal cell.

• Polyene antibiotics include nystatin, used topically for candidainfections and amphotericin B.

DEVOS Ann 22/02/2005 17


CellularCellular targets of targets of antifungalantifungal agentsagents

Calcium?OomycotaStrep. griseusStreptomycin

Protein synthesisSomeStrep. kasugaensisKasugamycin

Protein synthesisSomeStrep. griseochromogenesBlasticidin-S

MorphogenSomeStrep. hygroscopicusValidamycin A

Chitin synthesisMany (not Oomycota)Strep. cacaoiPolyoxins

Cell membrane Many (not Oomycota)Streptomyces spp.Polyene macrolides

Fungal tubulinsMany (not Oomycota)Penicillium griseofulvumGriseofulvin

Site/mode of actionFungi affectedProduced byAntibiotic

Antifungal antibiotics used for control of plant or human mycoses

http://helios.bto.ed.ac.uk/bto/chap17.htmAnn DEVOS 22-02-2005

Candida species have adapted several mechanisms of fluconazole resistance:The most important of these consisting of

-an alteration in the target enzyme 14 alpha-demethylase (change in binding site or overexpression of the enzyme)

-from enhanced drug efflux caused by plasma membrane transporters. The net effect of the efflux pump is to decrease the intracellular concentration of fluconazoleand thus reduce the concentration of drug that reaches the active site at the enzyme 14 alpha-demethylase.

MechanismsMechanisms of of resistanceresistancewithwith FluconazoleFluconazole--ResistantResistant CandidiasisCandidiasis

http://depts.washington.edu/hivaids/oral/case4/discussion.html


INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab FUNGAL INFECTIONSCONCLUSIONS



The wide spreading developement of antibiotic resistance by bacteria results in an increasing interest for alternativetherapies in the treatement of infectedwounds

CONCLUSIONCONCLUSION


CONCLUSIONCONCLUSION

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INTRODUCTION RESISTANT BACTERIA THE BACTERIAL CELLusers.skynet.be/fa055557/...Resistente_Bacterien.pdf · Gram +ve and Gram -ve bacteria Electron Micrograph of a Gram-Negative Cell