Post on 08-May-2015
Antimicrobial agents andantimicrobial resistance
BLS 206 Lecture
Hoza, A . S
Antimicrobial Resistance
Relative or complete lack of effect of antimicrobial against a previously susceptible microbe
Increase in MIC
Figure 20.20
Antibiotic Resistance
Horizontal Gene Transfer
A = Transformation; B = Conjugation; C = Transduction
• Enzymatic destruction of drug
• Prevention of penetration of drug
• Alteration of drug's target site
• Rapid ejection of the drug
Mechanisms of Antibiotic Resistance
Antimicrobial Drug ResistancePrinciples and Definitions
Clinical resistance vs actual resistance Resistance can arise by mutation or by gene transfer (e.g.
acquisition of a plasmid) Resistance provides a selective advantage Resistance can result from single or multiple steps
Cross resistance vs multiple resistance› Cross resistance -- Single mechanism-- closely related
antibiotics› Multiple resistance -- Multiple mechanisms -- unrelated
antibiotics
Antibiotic Selection for Resistant Bacteria
Resistant organism MICs of organism are higher than achieved drug
concentrations in tissues
Intermediately resistant the antibiotic may still be effective but higher doses
should be used
Highly resistant the antibiotic tissue concentrations are likely not to
exceed MICs of the microorganisms
Terminologies
Intrinsic or natural resistanceG-neg bacteria are resistant to vancomycin (large molecule)
Tetracyclines are hydrophobic, G-neg bacilli are resistant
Acquired resistanceMutations (PBP)
Disseminated by plasmids and transposons
Spontaneous mutations
Types of resistance
Mechanisms of antibiotic resistance
1. Production of enzymesdestroying and modifying AB ß-lactamases AG modifying enzymes
2. Decrease of cell membrane permeability
3. Active efflux of AB from cell
4. Modification of AB target sites
Genetics and spread of drug resistance
Viridans Streptococci
S.pneumoniae
S.Epidermidis S.aureus
E.faecium S.aureus
Transposon . genes moving from one point to another (jumping genes)
Bacteriophage virus, infecting bacteria (virus of bacteria)
Integron slice(s) of DNA, cassette of gene that may be entered into other cell
Plasmidcircular double stranded DNA molecule, located separately of the chromosomal RNA
Production of enzymes inactivating (destroying) antibioticsß-lactamases
Main mechanism of resistance in ß-lactam antibiotics
Penicillin-resistant S.aureus
Ampicillin-resistant E.coli
Production of enzymes modifying antibioticsAminoglycosides, chloramphenicol
(1) Mechanisms of resistance
Resistance mechanisms: inactivating enzymes (2)
Degrading enzymes will bind to the antibiotic and essentially degrade itor make the antibiotic inactive
Blocking enzymes attach side chains to the antibiotic that inhibit its function.E.g. ß-lactamases
PBP & ß-lactamase
Serine proteases (PBP) a metalloenzymes (Zn-binding thiole group as coenzyme)200 different enzymes e.g. penicillinases, cephalosporinases, ESBL, AmpCESBL - extended spectrum ß-lactamases (broad spectrum of activity);encoded in plasmids, can be transferred from organism to organism
Production of ß-lactamases: mechanism of action
ExamplesTEM-1 is a widespread ß- lactamase of Enterobacteriaciae that attacks Penicillin G and narrow spectrum cephalosporins
>50% AmpR E.coli isolates are caused by TEM-1
Antimicrobial Drug ResistanceMechanisms
Altered permeability› Altered influx
Gram negative bacteria
Antibiotics are removed via active efflux pump
Universal efflux pump
specific efflux pump
quinolones, tetracyclines, chloramphenicol
Efflux Mechanisms of resistance
Resistance mechanisms: efflux pump
The efflux pump is a membrane bound protein that "pumps" the antibiotic out of the bacterial cell
Microbe Library
American Society for Microbiology
www.microbelibrary.org
Antimicrobial Drug ResistanceMechanisms
Altered permeability› Altered efflux
tetracycline
Microbe Library
American Society for Microbiology
www.microbelibrary.org
Antimicrobial Drug ResistanceMechanisms
Inactivation› ß-lactamase
› Chloramphenicol acetyl transferase
Microbe Library
American Society for Microbiology
www.microbelibrary.org
Modification of target sites
altered PBP (PRSP)
new PBP (MRSE, MRSA)
Modification in ribosomes (macrolideresistantS.pneumoniae)
Mechanisms of resistance
Antimicrobial Drug ResistanceMechanisms
Altered target site› Penicillin binding
proteins (penicillins)
› RNA polymerase (rifampin)
› 30S ribosome (streptomycin)
Microbe Library
American Society for Microbiology
www.microbelibrary.org
Modification of AB target sites:disruption in protein synthesis
VRE . vancomycin-resistant enterococci70% of E. faecium strains in USA
GISA . glycopeptide intermediately susceptible S.aureus
VISA . vancomycin intermediately susceptible S.aureus
VRSA & VRSE . vancomycin-resistant S.aureus and S.epidermidis (MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA)
ESBL producing K.pneumoniae . Extended spectrum ß-lactamase producing K. pneumoniae
PRSP penicillin-resistant S. pneumoniae
Important terms among drugresistant microorganisms
Interference with cell wallsynthesis
ß-lactam antibiotics:
penicillins
cephalosporines
carbapenems
Alexander Fleming
P. chrysogenum(original strain of Fleming)
destroy Staphylococcus aureus 1928
ß-lactam structure is presented in red and blue
Side chain is presented in black
Penicillins
Carbapenems
Cephalosporins
Mechanism of action of ß-lactam antibiotics
1ß-lactam abbinds to PBP
2. Inhibition ofpeptidoglycansynthesis
3. Cell death
Structure of peptidoglycan
ß-lactams inhibit synthesis of crosslinks
Penicillins
Cephalosporins
Initially isolated formthe mould Cephalosporium
Compared with penicillins:More resistant to ß- lactamase hydrolysis
Wider antibacterial spectrum Improved PK-properties
Resistance to ß-lactamantibiotics
Resistance to ß-lactam antibiotics
Production of ß-lactamasesPenicillin-resistant S.aureus (>95%) - Synthetic
Penicillins
ESBL K.pneumoniae - IV generation cephalosporins, carbapenems
Ampicillin-resistant E.coli – cephalosporins
Changes in the structure of PBP
(altered PBP) Penicillin-resistant S.pneumoniae - larger doses of penicillin
New PBP - MRSA, MRSE . vancomycin
Disruption of bacterial cell wall
Glycopeptides
vancomycin
teicoplanin
Vancomycin: mechanism of action
Mechanism - vancomycin inhibits cross linkage between peptidoglycan layers
Vancomycin can bind only to D-Ala-D-Ala and not to D-Ala-D-lac
Originally obtained form Streptomyces orientalis
Active only against G+ bacteria (large molecule unable to penetrate outer membrane of G+ bacteria)
Used for treatment of oxacillin resistant G+ infections
Glycopeptide resistance Intrinsic resistance (pentapetide end with D-Ala-D-Lac)
Leuconostoc, Lactobacillus, Pediococcus
Or with D-Ala-D-Ser Enetrococcus gallinarum, Enetercoccus caselliflavus
Acquired resistance A thickening of the PG layer, and Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-
lactate Gene (vanA, B, C, D, G, E) is carried on plasmids & may be
transferred from organism to organism Importance
VRE - vancomycin resistant E. faecium, E.faecalis VISA - vancomycin intermediately resistant S.aureus GISA - glycopeptide intermediately resistant S.aureus VRSA - vancomycin resistant S.aureus (MIC> 32 µg/ml; 1st
clinical case reported 2002 in US)
Mechanism of Resistance to Vancomycin
Polypeptides Bacitracin (cyclic peptides) is isolated form
Bacillus licheniformis Topically applied agent against G+ bacteria
Interferes with the dephoshorylation and recycling of the lipid carrier responsible for moving peptidoglycan precursors
Polymyxin (cyclic polypeptides) derived from Bacillus polymyxa Interact with the lipopolysaccharides and phospholipids in
the outer membrane and thus increase cell permeability
Mostly active against G- bacilli (G+ bacilli do not have outer membrane)
Activity of antibiotics to bacterial cell wall
G-positiveG-negative
polypeptides ß-lactamsglycopeptides
Inhibition of protein synthesis
Aminoglycosides
Tetracyclines
Oxazolidones
Chloramphenicol
Macrolides
Clindamycin
Streptogramins
Protein synthesis
Substance binding to 30S subunit
Antibiotics that act at the level of protein synthesis initiation
Antibiotics that act at the level of the elongation phase of protein synthesis
Aminoglycosides Consists of aminosugars that are
linked through glycosidic rings
Origin Streptomyces - streptomycin, neomycin, kanamycin, tobramycin
Micromonospora - gentamicin, Sisomicin
Synthetic derivates Amikacin = kanamycin Netilmycin = sisomycin
Mainly active against G-negative bacteria
Gentamycin
Aminoglycoside: mode of actionAG pass through cell wall, cytoplasmic membrane to cytoplasma (mainly of Gbacteria, no penetration through cytoplasmic membrane of strepto- and entrococci)
Bind irreversible to the 30S subunit of bacterial ribosomes and block the attachment of the 50S subunit to the initiation complex
As a result production of aberrant proteins and misreading of RNA occurs
Aminoglycoside: mode of action
1. Passage through cytoplasmic membrane of G- bacteria (no penetration through cytoplasmic membrane of strepto- and enterococci)
2. Binding to 30S subunit
3. Misreading the codon along mRNA
4. Inhibition of protein synthesis
Enzymatic modification (common) of the drug High level resistance>50 enzymes identifiedGenes encoding resistance located in plasmidsGene transfer occurs across species
Reduced uptake or decreased permeability of bacterial cell wall
Resistance in anaerobes (transport through cytoplasmic membrane depends on anaerobic respiration)
Altered ribosome binding sites (rare) Microbes bind to multiple sitesLow level resistance
Aminoglycoside resistance
TetracyclinesOrigin
Tetracyclin, oxytetracyclin isolated from Streptomyces
Minocyclin, doxycyclin are synthetic
Broad spectrum bacteriostatic antibiotics
Antibacterial spectrum similar to macrolides (incl. Clamydia, Mycoplasma, Rickettsia)
Resistance (widespread)
Energy dependent efflux pump (most common)
Alteration of ribosomal target (ribosome protection)
Enzymatic change
The tetracyclines block bacterial translation by binding reversibly to the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA
Tetracyclines
Newest class of antibiotics; completely syntheticNarrow spectrum of activity (G+ bacteria, includingVRE, MRSA)
G-neg bacteria resistant due to efflux pump
Mode of action: unique mechanism among antibiotics; interferes with the initiation complex at the 50S ribosome subunit (V domain of 23S rRNA)
Resistance confers to mutation at 23S rRNA
Resistance is rare; cross-resistance unlikely because 23S rRNA is encoded by several genes
Oxazolidones: linezolid
Oxazolidones: mode of action
Inhibit the formation of an initiation complex by binding to the 50S ribosomal subunit (domain V of the 23S rRNA), disrupting the preliminary phases of protein synthesis
Binds irreversible to peptidyl transferase component of 50S ribosome and blocks peptide elongation, thus interferes with protein synthesis
Bacteriostatic antibiotic with broad spectrum of antibacterial activity
Interferes with the protein synthesis of bone marrow cells causing aplastic anaemia
Limited clinical use in Western world due to side Effect
Resistance is associated with producingacetyltransferase which catalyses acetylation of 3-hydroxy group of chloramphenicol
Chloramphenicol
Macrolides (1)Erythromycin was derived from Streptomyces erythreus
The basic structure is a lactone ring
14-membered lactone ring . erthromycin, clarithromycin, roxithromycin, telithromyin (ketolide)
15-membered lactone ring . Azithromycin
16-membered lactone ring . spiramycin, josamycin
Acitivity .broad spectrum G+ bacteria and some G- bacteria including
Chlamydia, Mycoplasma, Legionella, Rickettsia, Neisseria
Azithromycin, Clarithromycin active against some mycobacteria
Macrolides: mode of actionBlocking Translation during Bacterial Protein
Synthesis
The macrolides bind reversibly to the 50S subunit.
They can inhibit elongation of the protein by the peptidyltransferase, the enzyme that forms peptide bonds between the amino acids.
erythromycin
Mode of Action of Macrolides in BlockingTranslation during Bacterial Protein
Synthesis
The macrolides bind reversibly to the 50S subunit.
They can inhibit elongation of the protein by blocking the translocation of the ribosome to the next codon on mRNA
Macrolide resistance
ResistanceIntrinsic resistance- hydrophobic macrolides have low permeability through outer membrane (G- bacilli)
Acquired resistance
Ribosomal modification
Efflux pump
Enzyme inactivation
Clindamycin, lincomycin
Family of lincosamide antibiotics originally isolated from Streptomyces lincolnensis
Mode of action: bind 50S ribosome subunit and blocks protein elongation
Resistance is related to 23S ribosomal RNA Methylation
Active against staphylococci and G-ve anaerobic bacilli.
No activity against aerobic
Antimicrobial Drug ResistanceMechanisms
Replacement of a sensitive pathway› Acquisition of a
resistant enzyme (sulfonamides, trimethoprim)
Molecular Drug Susceptibility Testing
• Genotypic methods: the drug target and nature of the gene mutation are known
• Usually molecular amplification of target DNA or RNA followed by some means of detecting mutation in the product.
Molecular methods of drug susceptibility testing
1. SequencingUniversal and reliable methodExpensive, time-consuming and not suitable for everyday routine testingApplied as reference method to verify results of other tests.
2. PCR-based methods
PCR-Single Strand Conformation Polymorphism (PCR-SSCP)
Mutations cause alterations in conformation of single-strand DNA fragments and it is registered in non-denaturizing PAGE
Other molecular methods of drug susceptibility testing:
Molecular beacons
Real-Time fluorescent PCR combines amplification and detection: minimises amplicon contamination
PCR+hybridization
Based on amplification of fragments of genes responsible for drug resistance development follwed by hybridization with oligonucleotide probes immobilized on membranes;
Both commercial kits and in-house macro-arrays have been reported to demonstrate high sensitivity and specificity
Molecular tests for the detection of resistance to RIF and INH
GenoType® MTBDRplus test procedure
Reaction zones of GenoType® MTBDRplus (examples)
What Factors Promote Antimicrobial Resistance?
Exposure to sub-optimal levels of antimicrobial
Exposure to microbes carrying resistance genes
Inappropriate Antimicrobial Use
Prescription not taken correctly
Antibiotics for viral infections
Antibiotics sold without medical supervision
Spread of resistant microbes in hospitals due to lack of hygiene
Inappropriate Antimicrobial Use
Lack of quality control in manufacture or outdated antimicrobial
Inadequate surveillance or defective susceptibility assays
Poverty or war
Use of antibiotics in foods
Antibiotics in Foods Antibiotics are used in animal feeds and
sprayed on plants to prevent infection and promote growth
Multi drug-resistant Salmonella typhi has been found in 4 states in 18 people who ate beef fed antibiotics
Consequences of Antimicrobial Resistance
Infections resistant to available antibiotics
Increased cost of treatment
MRSA “mer-sah”
Methicillin-Resistant Staphylococcus aureus
Most frequent nosocomial (hospital-acquired) pathogen
Usually resistant to several other antibiotics
Proposals to Combat Antimicrobial Resistance
Speed development of new antibiotics
Track resistance data nationwide
Restrict antimicrobial use
Direct observed dosing (TB)
Use more narrow spectrum antibiotics Use antimicrobial cocktails
Ecology of Antimicrobial Resistance
Antimicrobial peptides› Broad spectrum antibiotics from plants
and animals Squalamine (sharks)
Protegrin (pigs)
Magainin (frogs)
The Future of Chemotherapeutic Agents
Antisense agents› Complementary DNA or peptide nucleic
acids that binds to a pathogen's virulence gene(s) and prevents transcription
The Future of Chemotherapeutic Agents
Doctors are men who prescribe medicines of which they know little: to cure diseases of which they know less: in human being of who they know nothing’’
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