DEVOS Ann 22/02/2005 1
RESISTANT BACTERIA RESISTANT BACTERIA
andand
ANTIBIOTICSANTIBIOTICS
Ann DEVOS 28-09-2004
Ann DEVOS 22-02-2005
INTRODUCTION
THE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNA MECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
DEVOS Ann 22/02/2005 2
Ann DEVOS 22-02-2005
Source: CDrom MicrobiologieNatuur & Techniek (Uitgeverij Veen magazines, Amsterdam)
BACTERIA BACTERIA
Ann DEVOS 22-02-2005
Visualizing small thingsVisualizing small things
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
PROCARYOTIC and EUCARYOTIC CELLSPROCARYOTIC and EUCARYOTIC CELLS
DEVOS Ann 22/02/2005 3
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
••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
Ann DEVOS 22-02-2005
RIBOSOMESRIBOSOMES
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
1940’s 1940’s MassMass penicillinpenicillin productionproduction
DEVOS Ann 22/02/2005 4
Ann DEVOS 22-02-2005
HISTORY OF ANTIBIOTIC RESISTANCEHISTORY OF ANTIBIOTIC RESISTANCE
198719721956Vancomycin
195619521948Tetracyclin
194719471944Streptomycin
194019401928Penicillin
Resistance identified
Used clinicallyDiscoveredAntibiotic
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
Resistance against Resistance against fluoroquinolonesfluoroquinolonesin in HawaianHawaian populationpopulation
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EXCHANGE DNA
MUTATIONTRANSFORMATIONCONJUGATION
PlasmidsTransposons or Transposable elements
TRANSDUCTIONMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
TransformationTransformation
Insertion
Crossingover
Ann DEVOS 22-02-2005
TransformationTransformation
DEVOS Ann 22/02/2005 6
Ann DEVOS 22-02-2005
TransformationTransformation
Ann DEVOS 22-02-2005
TransformationTransformation
Ann DEVOS 22-02-2005
TransformationTransformation
Ann DEVOS 22-02-2005
TransformationTransformation
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EXCHANGE DNA
MUTATIONTRANSFORMATIONCONJUGATION
PlasmidsTransposons or Transposable elements
TRANSDUCTIONMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
F- bacteria
ConjugationConjugationF plasmid
Mating Bridge
F+ bacteria
Fplasmid
Ann DEVOS 22-02-2005
ConjugationConjugation
F- bacteria
F plasmid
F+ bacteria
Fplasmid
Mating Bridge
Ann DEVOS 22-02-2005
ConjugationConjugation
F- bacteria
F plasmid
F+ bacteria
Fplasmid
Mating Bridge
Ann DEVOS 22-02-2005
ConjugationConjugation
F- bacteria
F plasmid
F+ bacteria
Fplasmid
Fplasmid
Mating Bridge
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
Transposable Genetic ElementsTransposable Genetic Elements
IS ISResistance Gene(s)
IS ISResistance Gene(s)
Carry genes except those involved in transposition
Structure : IS
IS IS
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EXCHANGE DNA
MUTATIONTRANSFORMATIONCONJUGATION
PlasmidsTransposons or Transposable elements
TRANSDUCTIONMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
BACTERIOPHAGEBACTERIOPHAGE
Ann DEVOS 22-02-2005
TransductionTransductionDestruction of the bacteria’s DNA
Lysis
Replication of the viral genome
Production of viral parts
Packaging
Infection
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC AbFUNGAL INFECTIONSCONCLUSIONS
DEVOS Ann 22/02/2005 9
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
ANTIBIOTIC RESISTANCE MECHANISMSANTIBIOTIC RESISTANCE MECHANISMS
• Bypass antibiotic-sensitive step• Altered antibiotic target/Overproduction of target
• Reduced antibiotic accumulation
•Impaired uptake
•Enhanced efflux
• Antibiotic inactivation
Ann DEVOS 22-02-2005
ANTIBIOTIC RESISTANCE MECHANISMSANTIBIOTIC RESISTANCE MECHANISMS
TrimethoprimSulfonamides
β-lactamsChloramphenicolErythromycin (AG)
TetracyclinsQuinolones
β-lactamsChloramphenicolErythromycin (AG)
StreptomycinQuinelonesRifampicin
β-lactamsChloramphenicolAminoglycosides
BypassReducedaccumulationAltered targetInactivation
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
SULFONAMIDESSULFONAMIDES
Chemical similarity between the sulfa drugs and PABA
DEVOS Ann 22/02/2005 10
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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)
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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)
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
The The bacterialbacterial RNA RNA polymerasepolymerasecomplexedcomplexed withwith rifampicinrifampicin
• Ligand: RFP
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab
IMPAIRED DNA SYNTHESISSulfonamidesQuinolones
IMPAIRED RNA SYNTHESISRifampicin
IMPAIRED PROTEIN SYNTHESISACTING ON THE CELL WALL
Beta-lactams (Penicillin, Ampicillin,..)ACTING ON THE CELL MEMBRANE
blabla
FUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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)
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
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?
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab
IMPAIRED DNA SYNTHESISSulfonamidesQuinolones
IMPAIRED RNA SYNTHESISRifampicin
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
Ann DEVOS 22-02-2005
BETA LACTAM ANTIBIOTICSBETA LACTAM ANTIBIOTICS
PenicillinsPenicillin GAmpicilin
CephalosporinsCephalexinCofotaximeCefuroxime
CephamycinsCefoxitin
CarbapenemsImipenem
MonobactamsAztreonam
OUTER
MEMBRANE
MUREIN
CYTOPLASMIC
MEMBRANE
Ann DEVOS 22-02-2005
StructureStructure of of PenicillinPenicillin
Penicillin V Ampicillin
DEVOS Ann 22/02/2005 15
Ann DEVOS 22-02-2005
SimilaritySimilarity betweenbetween the the Gram +Gram +veve cellcell wallwall and and PenicillinPenicillin
Image courtesy of the University of Texas-Houston Medical
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
RESISTANCE TO BETA LACTAMSRESISTANCE TO BETA LACTAMS
OUTER
MEMBRANE
Peptidoglycan
CYTOPLASMIC
MEMBRANE
BETA LACTAM
PBP
BETA-LACTAMASE
Enzymatic inactivation
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab
IMPAIRED DNA SYNTHESISSulfonamidesQuinolones
IMPAIRED RNA SYNTHESISRifampicin
IMPAIRED PROTEIN SYNTHESISTetracyclinesChloramphenicolErythromycinStreptomycinAminoglycosides (Kanamycin)
ACTING ON THE CELL WALLBeta-lactams (Penicillin, Ampicillin,..)
ACTING ON THE CELL MEMBRANEblabla
FUNGAL INFECTIONSCONCLUSIONS
DEVOS Ann 22/02/2005 16
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONANTIBIOTICSTHE BACTERIAL CELLHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab FUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
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.
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
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
Ann DEVOS 22-02-2005
INTRODUCTIONTHE BACTERIAL CELLANTIBIOTICSHOW BACTERIA EX-CHANGE DNAMECHANISMS OF Ab RESISTANCERESISTANCE TO SPECIFIC Ab FUNGAL INFECTIONSCONCLUSIONS
Ann DEVOS 22-02-2005
Ann DEVOS 22-02-2005
The wide spreading developement of antibiotic resistance by bacteria results in an increasing interest for alternativetherapies in the treatement of infectedwounds
CONCLUSIONCONCLUSION
Ann DEVOS 22-02-2005
CONCLUSIONCONCLUSION