Download Mechanisms of drug resistance

MECHANISMS OF
DRUG RESISTANCE
Dr.Ihsan Edan Alsaimary
Dept.Microbiology , College Of
Medicine , University Of Basrah
INTRODUCTION
• Chemotherapy is the primary means of
treating bacterial infections.
• Successful chemotherapy depends in a
large part on the ability to exploit
metabolic differences between the
pathogen and the host.
• A problem confronting chemotherapy is
the ability of the pathogen to mutate and
become drug resistance.
Why Is Antibiotic Resistance A
Public Health Issue?
• No new classes of antibiotics have been developed to combat
infectious diseases since 1970. Research and development of
antibiotics takes 10 to 20 years (WHO 2000).
• For example, Shigella dysenteriae, which claimed the lives of
more than 2.2 million people in 1998, has become resistant to
every available drug except ciprofloxacin within the past ten
years.
• Even non-pathogenic resistant bacteria can pass resistance
genes to pathogenic bacteria by processes such as conjugation.
Antibiotics in Animal Feed
• Promotes growth
• Decreases amount of feed
needed
• Prevents infectious diseases
• Facilitates confinement housing
• Lowers costs
ANTIMICROBIAL RESISTANCE:
The role of animal feed antibiotic additives
• 48% of all antibiotics by weight is added to animal feeds to
promote growth. Results in low, subtherapeutic levels
which are thought to promote resistance.
• Farm families who own chickens feed tetracycline have an
increased incidence of tetracycline resistant fecal flora
• Chickens at Spanish supermarkets have >90% of cultured
campylobacter resistant to quinolones
• 39% of enterococci in the fecal flora of pigs from the
Netherlands is resistant to vancomycin vs 0% in Sweden.
(Sweden bans antibiotic additives in animal feed)
What Are Sub-therapeutic
Antibiotics?
• In modern large-scale agriculture, chickens, pigs, and other
livestock are often given sub-therapeutic doses of antibiotics
as a prophylactic and as a growth promoter.
• When antibiotics are administered at sub-therapeutic doses,
the hardiest bacteria will survive treatment and reproduce to
create an increasingly resistant population of bacteria.
What is MRSA?
• Staphylococcus aureus is an opportunistic bacterium that lives on
the skin and in the nasal passages of people and animals.
• S. aureus can become resistant to methicillin by acquiring the
mecA gene. MecA positive strains are the main cause of
nosocomial infections worldwide.
• There is concern that feeding antibiotics to animals subtherapeutically can promote the development and growth of
Methicillin-Resistant Staphylococcus aureus (MRSA).
• According to recent publications, pets and farm animals and their
caretakers can act as reservoirs of MRSA (Corrente).
The magic bullet
• Antibiotics revolutionised medicine
• The first antibiotic, penicillin, was discovered
by Alexander Fleming in 1929
• It was later isolated by Florey and Chain
• It was not extensively used until the 2nd World
War when it was used to treat war wounds
• After 2nd World War many more antibiotics
were developed
• Today about 150 types are used
• Most are inhibitors of the protein synthesis,
blocking the 70S ribosome, which is
characteristic of prokaryotes
Resistance
• It took less than 20 years for, bacteria
to show signs of resistance
• Staphylococcus aureus, which causes
blood poisoning and pneumonia,
started to show resistance in the 1950s
• Today there are different strains of S.
aureus resistant to every form of
antibiotic in use
© 2008 Paul Billiet ODWS
Multiple resistance
• It seems that some resistance was already
naturally present in bacterial populations
• The presence of antibiotics in their
environment in higher concentrations
increased the pressure by natural
selection
• Resistant bacteria that survived, rapidly
multiplied
• They passed their resistant genes on to
other bacteria (both disease causing
pathogens and non-pathogens)
© 2008 Paul Billiet ODWS
Transposons & Integrons
• Resistance genes are often associated
with transposons, genes that easily move
from one bacterium to another
• Many bacteria also possess integrons,
pieces of DNA that accumulate new genes
• Gradually a strain of a bacterium can build
up a whole range of resistance genes
• This is multiple resistance
• These may then be passed on in a group
to other strains or other species
© 2008 Paul Billiet ODWS
Antibiotics promote resistance
• If a patient taking a course of antibiotic treatment
does not complete it
• Or forgets to take the doses regularly,
• Then resistant strains get a chance to build up
• The antibiotics also kill innocent bystanders
bacteria which are non-pathogens
• This reduces the competition for the resistant
pathogens
• The use of antibiotics also promotes antibiotic
resistance in non-pathogens too
• These non-pathogens may later pass their
resistance genes on to pathogens
Resistance gets around
• When antibiotics are used on a person,
the numbers of antibiotic resistant
bacteria increase in other members of
the family
• In places where antibiotics are used
extensively
e.g. hospitals and farms
antibiotic resistant strains increase in
numbers
Antibiotic use and abuse
• Viral infections are not stopped by
antibiotics
• Yet doctors still prescribe (or are
coerced into prescribing) antibiotics to
treat them
Drug Action
• Drugs act by specifically interfering with
cellular or biochemical processes, often
called 'targets‘
– The classic example of a drug target is an
enzyme (inhibition)
• Drugs to be effective need to exhibit a
selective toxicity for the pathogen as
compared to host. Many factors contributing
to this selective toxicity:
–
–
–
–
unique target in bacteria
discrimination between host and bacteria targets
greater drug accumulation by bacteria
drug activation by bacteria
Potential mechanisms involved in
drug resistance
• Conversion of the drug to an inactive
form by an enzyme.
• Modification of a drug sensitive site.
• Increased efflux or decreased influx
• Alternative pathway to bypass inhibited
reaction.
• Increase in the amount of an enzyme
substrate (ie to compete with the drug).
• Failure to activate the drug.
Potential mechanisms involved
in drug resistance
• These modifications can arise in a
population of bacteria by a number of
mechanisms.
– Physiological adaptations
– Differential selection of resistant
individuals from a mixed population of
susceptible and resistant individuals.
– Spontaneous mutations followed by
selection.
– Changes in gene expression. (gene
amplification)
MICROBIAL GENETICS - PLASMIDS / OTHER
MOBILE GENETIC ELEMENTS
PLASMIDS
Characteristics, Resistance Factors, Resistance Transfer Factors
Drug Resistance Mechanisms
Penicillin Resistance, Penicillinase, Beta-lactamase
Multiple Drug Resistance
Transformation by Plasmids
OTHER MOBILE GENETIC ELEMENTS
Insertion Sequences
Transposons
Integrons
Superintegrons
Conjugative Transposons
Genomic Islands
The Busy Genome
2
3
PLASMIDS:
SMALL, CIRCULAR, DOUBLE-STRAND DNA
MOLECULES
~ 5 - 50 GENES,
CYTOPLASMIC LOCATION,
NOT ESSENTIAL (NORMALLY)
REPLICATION GENES AND SITES
1 - 20 COPIES EACH / CELL
SEVERAL DIFFERENT PLASMIDS / CELL
RESISTANCE FACTORS - PLASMIDS WHICH CARRY GENES
WHICH ENCODE PROTEINS WHICH MAKE THE BACTERIAL
HOST RESISTANT TO ANTIBIOTIC - CALLED DRUG
RESISTANCE
RESISTANCE TRANSFER FACTORS - ALL ABOVE PLUS
ABILITY TO TRANSFER PLASMID IN MATING
[CONJUGATION]
OTHER PLASMID GENES:
HYDROCARBON CATABOLISM
TOXIN PRODUCTION
MINERAL UPTAKE
4
MECHANISMS OF DRUG RESISTANCE
1. MUTATION RESULTS IN ALTERED BACTERIAL PROTEIN.
IT NO LONGER RECOGNIZES ANTIBIOTIC BUT
CONTINUES TO PERFORM NORMAL FUNCTION
IN BACTERIAL GROWTH
EX: STREPTOMYCIN
2. BACTERIA ACQUIRE NEW GENE WHICH CODES FOR
ENZYME WHICH DESTROYS ANTIBIOTIC
EX: PENICILLINASE DESTROYS PENICILLIN
3. BACTERIA ACQUIRE NEW GENE WHICH CODES FOR
ENZYME WHICH PUMPS ANTIBIOTIC BACK OUTSIDE
CELL
EX: TETRACYCLINE RESISTANCE
PENICILLIN CLEAVAGE [INACTIVATION]
BY PENICILLINASE
b - LACTAM RING
PENICILLINASE [b - LACTAMASE]
INACTIVE
5
Mechanisms of resistance
Imipenem resistant
Pseudomonas
aeruginosae
Streptococcus
pneumoniae
resistance to
penicillins
Tetracycline
MRSA
penicillin
binding protein
PBP2A
Penicillins,
Cephalosporins
Hawkey, P. M BMJ 1998;317:657-660
Evolution of resistance
•Antibiotic use represents a strong selection
pressure
•If a population of bacteria with a few
resistant individuals is exposed to a lethal
antibiotic, the susceptible bacteria will die,
but the resistant bacteria will survive
•In an environment with a lot of antibiotic
use, resistance alleles spread rapidly
•The problem is compounded by horizontal
gene transfer and by cross-resistance
Horizontal transfer
•Simple selection isn’t the only means for
resistance alleles to spread
•Bacteria can acquire resistance genes by
transformation, when they pick up DNA
from the environment
•They can also get resistance genes by
conjugation: bacterial sex, when they
exchange plasmids
•Plasmids can have multiple resistance
genes, conferring multiresistance
Cross-resistance
•Resistance to one antibiotic can confer
resistance to others
•Resistance to cephalosporins gives
resistance to methicillin, even in bacteria
that have never been exposed to methicillin
Managing resistance
•There are two different approaches to
managing antibiotic resistance:
1.Managing existing resistant pathogens
2.Avoiding future evolution of more resistance
•The first can be done by, in the case of
MRSA, improving hygiene in hospitals,
screening hospital visitors and isolating
patients
•The second can be done by changing
selection on bacteria
Selection and resistance
•Reduce inappropriate prescription of antibiotics
– Increase public awareness that many diseases
cannot be cured with antibiotics
•Reduce use of agricultural antibiotics
•Increase the number of patients who finish their
courses of antibiotics
•Restrict the use of new antibiotics
•Where possible, use other treatments:
– Vaccines
– Phage treatment?
Mechanisms of resistance
• 1. Antibiotic modification: some bacteria have
enzymes that cleave or modify antibiotics: e.g. b
lactamase inactivates penicillin
• 2. Denied access: membrane becomes impermeable
for antibiotic: e.g. imipenem
• 3. Pumping out the antibiotic faster than it gets in: e.g.
tetracyclines
• 4. Altered target site: antibiotic cannot bind to its
intended target because the target itself has been
modified
• 5. production of alternative target (typically enzyme):
e.g. Alternative penicillin binding protein (PBP2a) in
Antimicrobial Resistance
Mechanisms
1.
2.
3.
4.
5.
6.
7.
8.
9.
Resistance to aminoglycoside antibiotics
Resistance to b-lactams
Resistance to glycopeptides
Resistance to quinolones
Resistance to macrolides and streptogramins
Resistance to sulphonamides
Resistance to trimethoprim
Resistance to tetracyclines
Resistance to chloramphenicol
Antimicrobial Resistance
Mechanisms
1. Resistance to aminoglycoside antibiotics
- Enzymatic modification
• Aminoglycoside-modifying enzymes
(AMEs) : acetylating enzymes AAC,
phosphorylating enzymes APH,
adenylating enzymes AAD or ANT
• Usually plasmid-encoded
Antimicrobial Resistance
Mechanisms
1. Resistance to aminoglycoside antibiotics
(Cont’d)
- Ribosomal resistance
• Rare
• Mutations in ribosomal genes
• High-level streptomycin resistance in
enterococci
• Streptomycin resistance in M tuberculosis
- Ineffective transport
• Oxygen transport system
Antimicrobial Resistance
Mechanisms
2. Resistance to b-lactams
- Enzymatic modification - b-lactamases
- Modification of penicillin binding proteins
(PBPs)
- “Bypass resistance” : alternative routes
of peptidoglycan synthesis (PBP2’ in
MRSA, PBP5 in Enterococcus faecium)
- Impermeability : porin loss
Antimicrobial Resistance
Mechanisms
3. Resistance to glycopeptides
- GNB are naturally resistant
because glycopeptides are large
molecules that cannot pass
through the outer membrane
porins
Antimicrobial Resistance
Mechanisms
3. Resistance to
glycopeptides (Cont’d)
- Acquired vancomycin
resistance : genes
encoding ligases that
incorporate D-lactate
in place of D-alanine
as the terminal amino acid of the peptide chain 
reduced affinity for glycopeptide antibiotics
Antimicrobial Resistance
Mechanisms
3. Resistance to glycopeptides (Cont’d)
- VanA : high-level resistance to
vancomycin and teicoplanin, inducible
- VanB : moderate- to high-level
resistance to vancomycin, susceptible
to teicoplanin, constitutive VanB strains
resistant to teicoplanin
Antimicrobial Resistance
Mechanisms
3. Resistance to glycopeptides (Cont’d)
- VanC : low-level resistance to vancomycin
only, not inducible, intrinsic in
Enterococcus casseliflavus, E gallinarum
- VanD : resistance to vancomycin (MIC = 64
mg/l), low-level resistance to teicoplanin,
not inducible, not transferable
- VanE : low-level resistance to vancomycin
(MIC = 16 mg/l), susceptible to teicoplanin,
inducible, not transferable
Antimicrobial Resistance
Mechanisms
4. Resistance to quinolones
- Mutations in genes coding for DNA gyrase
(gyrA, gyrB) and type IV DNA
topoisomerase (parC, parE)
- Altered permeability : decreased quantities of
OmpF outer membrane protein
- Efflux : efflux pumps (acrB, tolC, …)
- qnr
- Ciprofloxacin-modifying enzyme: aac(6)-Ib-cr
(KRAkRTbR)
Antimicrobial Resistance
Mechanisms
5. Resistance to macrolides and streptogramins
- Target site alteration : modification of 23S
rRNA by methylase, encoded by a class of
genes erm (erythromycin ribosome
methylation)
- Drug modification : macrolide-modifying
enzymes (esterase, phosphotransferase)
- Altered transport (efflux)
- Decreased permeability
Antimicrobial Resistance
Mechanisms
6. Resistance to sulphonamides
- Overproduction of paraaminobenzoic acid (PABA)
or a change in
dihydropteroic acid
synthetase enzyme,
mutations in chromosomal
genes, plasmid-mediated
- Decreased cell permeability
pteridine + PABA
dihydropteroic
acid synthetase
dihydropteroic acid
Antimicrobial Resistance
Mechanisms
7. Resistance to trimethoprim
- Mutation in gene coding for
production of dihydrofolate
reductase (DHFR), and/or
- Increased production of DHFR
- Thymine auxotrophy
- Decreased cell permeability
(loss of OMP)
- Resistance genes integrated
into transposons, on plasmid
or chromosome
dihydropteroic acid
dihydrofolic acid
dihydrofolate
reductase
(DHFR)
tetrahydrofolic acid
thymidine
Antimicrobial Resistance
Mechanisms
8. Resistance to tetracyclines
- Energy-dependent efflux
- Enzymatic modification : Bacteroides
- Ribosomal protection proteins
Antimicrobial Resistance
Mechanisms
9. Resistance to chloramphenicol
-
Chloramphenicol
acetyltransferase (CAT) :
acetylated chloramphenicol
cannot bind to ribosome
-
Active efflux : Pseudomonas
aeruginosa
Antimicrobial Resistance
Detection of resistance genes
- Hybridization
- Oligotyping
- Polymerase Chain Reaction
(PCR)
- Nucleotide sequencing