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Relative or complete lack of effect of antimicrobial against a previously susceptible
microbe
Increase in MIC
Figure 20.20
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
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
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 resistance
G-neg bacteria are resistant to vancomycin (large
molecule)
Tetracyclines are hydrophobic, G-neg bacilli are
resistant
Acquired resistance
Mutations (PBP)
Disseminated by plasmids and transposons
Spontaneous mutations
Types of resistance
Mechanisms of antibiotic resistance
1. Production of enzymes
destroying 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)
Bacteriophagevirus, infecting bacteria (virus of bacteria)
Integronslice(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)
antibiotics
ß-lactamases
Main mechanism of resistance in ß-lactam
antibiotics
Penicillin-resistant S.aureus
Ampicillin-resistant E.coli
Production of enzymes modifying antibiotics
Aminoglycosides, chloramphenicol
(1) Mechanisms of resistance
Resistance mechanisms: inactivating enzymes (2)
Degrading enzymes will bind to the
antibiotic and essentially degrade it
or 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,
AmpC
ESBL - 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
Examples
TEM-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
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
Altered permeability
› Altered efflux
tetracycline
Microbe Library
American Society for
Microbiology
www.microbelibrary.org
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 (macrolideresistant
S.pneumoniae)
Mechanisms of resistance
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 enterococci
70% 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 drug
resistant microorganisms
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 ab
binds to PBP
2. Inhibition of
peptidoglycan
synthesis
3. Cell death
Structure of peptidoglycan
ß-lactams inhibit synthesis of crosslinks
Penicillins
Cephalosporins
Initially isolated form
the mould Cephalosporium
Compared with penicillins:
More resistant to ß-
lactamase hydrolysis
Wider antibacterial spectrum
Improved PK-properties
Resistance to ß-lactam
antibiotics
Resistance to ß-lactam antibiotics
Production of ß-lactamases
Penicillin-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
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
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 peptidoglycanprecursors
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-positive
G-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
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 action
AG 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 identified
Genes encoding resistance located in plasmids
Gene 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 synthetic
Narrow 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 producing
acetyltransferase 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 action
Blocking 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 Blocking
Translation 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
Resistance
Intrinsic 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
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. Sequencing
Universal 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 genesresponsible for drug resistance development
follwed by hybridization with oligonucleotideprobes immobilized on membranes;
Both commercial kits and in-house macro-arrays have been reported to demonstratehigh sensitivity and specificity
Molecular tests for the detection of resistance to RIF and INH
GenoType® MTBDRplus test procedure
Reaction zones of GenoType® MTBDRplus (examples)
Exposure to sub-optimal levels of antimicrobial
Exposure to microbes carrying resistance
genes
Prescription not taken correctly
Antibiotics for viral infections
Antibiotics sold without medical supervision
Spread of resistant microbes in hospitals due to lack of hygiene
Lack of quality control in manufacture or outdated antimicrobial
Inadequate surveillance or defective susceptibility assays
Poverty or war
Use of 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
Infections resistant to available antibiotics
Increased cost of treatment
Methicillin-Resistant Staphylococcus aureus
Most frequent nosocomial (hospital-acquired) pathogen
Usually resistant to several other antibiotics
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)
Antisense agents
› Complementary DNA or peptide nucleic acids that binds to a pathogen's virulence
gene(s) and prevents transcription
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