BIOT 309: ANTIMICROBIAL
RESISTANCE
Jan, 2013
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http://courses.washington.edu/medch401/pdf_text/401_05_black_betalactams.ppt#5
Antibiotic Resistance
• Major health challenge
• WHY: inappropriate use of antibiotics in
hospitals and the community
– Treating patients with viral infections with antibiotics
(common cold, flu, viral pneumonia, viral
gastroenteritis)
– Using broad spectrum rather than narrow spectrum
antibiotics
– Using new, special antibiotics to treat infections when
an older antibiotic would be effective
• Use of antibiotics to improve growth &
production in animals
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We are running out of new classes
of antimicrobials
Antimicrobial class
• Sulphonamides
• Penicillins
• Tetracyclines
• Chloramphenicol
• Aminoglycosides
• Macrolides
• Glycopeptides
• Streptogramins
• Quinolones
• Oxazolidinones
• Cyclic lipopeptides
• Ketolides
• Glycylcyclines
Year of launch
1936
1940
1949
1949
1950
1952
1958
1962
1962
2001
2003
2004
2005
1969 – US Surgeon General said “It is time to close the book on infectious
diseases.”
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The resistance “crisis”
• No new classes of antibiotics in the pipeline
• Already resistance emerging to newly released
antimicrobials
• Community acquired MRSA an emerging problem
• Now facing untreatable Gram-positive & Gram-negative
infections
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Evolution of drug resistance in
S.
aureus
Penicillin
Methicillin
S. aureus
1950s
Penicillin-resistant
S. aureus
1970s
Methicillin-resistant
S. aureus (MRSA)
Vancomycin
1997
Vancomycin
-resistant
S. aureus (VRSA)
2002
Vancomycinintermediate
S. aureus (VISA)
1990s
Vancomycin-resistant
enterococci (VRE)
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MMWR Morb Mortal Wkly Rep 2002;51:565–567
Emergence of resistance in Gramnegative bacteria
• 1960s – Resistance in E coli and its relatives started to
emerge
• These rapidly developed resistance to ampicillin, early
cephalosporins, aminoglycosides
• Multi-drug resistant (MDR) G-ves major problem in
hospitals in 1970s and 1980s
• More antibiotics (aminoglycosides, extended spectrum lactamases, -lactam/ -lactamase-inhibitor
combinations, fluoroquinolones) – worked for awhile
• 2000 - extended spectrum -lactamase producing
Gram-negatives (ESBLs) – untreatable infections
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What enables a bacterium to
become resistant to an antibiotic?
• Some organisms are naturally resistant to some classes of
antibiotics (natural or intrinsic resistance)
• Mutation continually occurring in bacterial genomes (all genomes!)
• Mutation of key genes important to the action of an antimicrobial can
result in resistance in that organism to that antimicrobial
• This resistance may evolve before the organism has been exposed
to that antimicrobial
• So, resistance determinants that have always been present in
bacteria
– Antibiotic producing strains of bacteria (note that resistance
genes have been found in antibiotic preparations …)
– Soil and gut organisms
– Bacterial housekeeping proteins, e.g., efflux pumps
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How does antibiotic resistance
come about?
• Use of antibiotics selects out the (low) number of
resistant strains – they multiply – the sensitive
strains die out – the population of bacteria is
resistant …….
• So, antibiotic resistant strains of bacteria emerge
under the selection pressure from use of
antibiotics
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Campaign to Prevent Antimicrobial Resistance in Healthcare Settings
Selection for antimicrobialresistant Strains
Resistant Strains
Rare
Antimicrobial
Exposure
Resistant Strains
Dominant
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• Bacteria lurking in soil in the 1960s and 70s resist an antibiotic that didn't exist then.
• Three strains of what amount to future-predicting bacteria showed extreme resistance
to six common antibiotics, including ciprofloxacin, which was first sold in 1989.
• One strain of soil bacteria was even able to fend off a dose of ciprofloxacin that would
be lethal to humans.
• Developed such defenses as part of the evolutionary arms race that has been going
on for billions of years between soil-dwelling microbes.
•Many antibiotics drugs come from naturally occurring molecules produced by soil bacteria
and fungi,
• Some drugs, such as Cipro (the brand name of ciprofloxacin), developed in lab.
Scientists examined 3 strains from company that stocks thousands of frozen bacteria
•two Klebsiella pneuomoniae, an opportuniztic pathogen, were isolated from dirt in
1973 and 1974
•Alcaligenes, last tasted agar in 1963
• grew well in wide range of antibiotics – they were resistant to all
• resistant to lethal dose of rifampicin, an antibiotic introduced in 1967 and Cipro, a 19year-old drug that resembles nothing seen in nature
"You can pretty safely say that there is no way
these bacteria have seen these antibiotics
before”
Types of antibiotic resistance
• Natural resistance - particular microbes are inherently
resistant to particular agents – eg
– multi-drug efflux pumps in Pseudomonas aeruginosa
– aminoglycoside resistance in strict anaerobes
– inability of penicillin G to penetrate Gram-negative cell wall
• Acquired resistance involves bacteria becoming resistant to a
drug that was previously effective. eg
–
–
–
–
multi-drug resistance in Mycobacterium tuberculosis
fluoroquinolone resistance in Neisseria gonorrhoeae
methicillin resistance in Staphylococcus aureus
penicillin resistance in Streptococcus pneumoniae
• Multiple resistance of particular concern
Acquired resistance occurs in response to exposure of
bacteria to antibiotics
– Mutational change and resistance passed to progeny
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Antibiotic resistance
Examples of natural or intrinsic resistance
• Inaccessibility of the target (i.e. impermeability
resistance due to the absence of an adequate
transporter: aminoglycoside resistance in strict
anaerobes)
• Multidrug efflux systems: i.e. AcrE in E. coli,
MexB in P. aeruginosa
• Drug inactivation: i.e. AmpC cephalosporinase in
Klebsiella
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Antibiotic resistance
Examples of acquired resistance
• Target site modification (i.e. Streptomycin resistance:
mutations in rRNA genes (rpsL), ß-lactam resistance:
change in PBPs (penicillin binding proteins))
• Reduced permeability or uptake
• Metabolic by-pass (i.e trimethoprim resistance:
overproduction of DHF (dihydrofolate) reductase or thimutants in S. aureus)
• Derepression of multidrug efflux systems
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Antibiotic resistance
Examples of horizontal transfer of resistance genes
• Mobile genetic elements – transposons & plasmids)
• Drug inactivation (i.e. aminoglycoside-modifying
enzymes, ß-lactamases, chloramphenicol
acetyltransferase)
• Efflux system (i.e. tetracycline efflux)
• Target site modification (i.e. methylation in the 23S
component of the 50S ribosomal subunit: Erm
methylases)
• Metabolic by-pass (i.e trimethoprim resistance: resistant
DHF reductase)
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Five strategies of antimicrobial
resistance
1. Antibiotic modification - the bacteria avoids the
antibiotic's deleterious affects by inactivating the
antibiotic.
eg production of B lactamases
2. Prevention of antibiotic entry into the cell - Gram –ve
bacteria - porins are transmembrane proteins that allow
for the diffusion of antibiotics through their highly
impermeable outer membrane. Modification of the porins
can bring about antibiotic resistance,
eg Pseudomonas aeruginosa resistance to imipenem.
3. Active efflux of antibiotic - Bacteria can actively pump
out the antibiotic from the cell.
eg energy dependent efflux of tetracyclines widely seen
in Enterobacteriaceae.
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Five strategies of antimicrobial
resistance
4. Alteration of drug target - Bacteria can also evade
antibiotic action through the alteration of the compound's
target.
eg Streptococcus pneumoniae modified penicillinbinding protein (PBP) which renders them resistant to
penicillins.
5. Bypassing drug's action - bacteria can bypass the
deleterious effect of the drug while not stopping the
production of the original sensitive target.
eg alternative PBP produced by MRSA in addition to the
normal PBP;
sulfonamide-resistant bacteria that have become able to
use environmental folic acid like mammalian cells, and in
this way bypass the sulfonamide inhibition of folic acid
synthesis.
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Resistant mechanisms against the
major classes of antibiotics
Mechanism of action
Major resistance
mechanisms
-lactams
Inactivate PBPs
(peptidoglycan
synthesis)
•-lactamases
•Low affinity PBPs
•Decreased transport
Glycopeptides
Bind to precursor of
peptidoglycan
•Modification of
precursor
Aminoglycosides
Inhibit protein synthesis
(bind to 30S subunit)
•Modifying enzymes
(add adenyl or PO4)
Macrolides
Inhibit protein synthesis
(bind to 50S subunit)
•Methylation of rRNA
•Efflux pumps
Quinolones
Inhibit topoisomerases
(DNA synthesis)
•Altered target enzyme
•Efflux pumps
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Target modification
Mosaic PBP Genes in penicillinresistant Strep pneumoniae
• Resistance is due to alterations in endogenous PBPs
– Resistant PBP genes exhibit 20-30% divergence from sensitive
isolates (Science 1994;264:388-393)
– DNA from related streptococci taken up and incorporated into S.
pneumoniae genes
S SXN
PBP 2B
Czechoslovakia (1987)
South Africa (1978)
USA (1983)
= pen-sensitive S. pneumoniae
= Streptococcus ?
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http://www.uhmc.sunysb.edu/microbiology/35
Target modification
Resistance to vancomycin
• Seven-step gene co-operation
• Involves activity of resolvase, transposase and ligase enzymes
• Alters pentapeptide precursor end sequence from
D-alanyl-D-alanine to D-alanyl-D-x, where x is lactate, serine or
other amino acid
• Or produces (vanY) tetrapeptide* that cannot bind vancomycin
Vancomycin resistance gene sequence
vanR
vanS
vanH
Detects
Produces D-Lac
glycopeptide;
switches on other genes
vanA
Produces
D-Ala-D-Lac
vanX
Cleaves
D-Ala-D-Ala
vanY
vanZ
*Cleaves
D-Ala and
D-Lac from
end chain
Exact role?
Teicoplanin
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resistance?
Enzyme modification of the antibiotic
Action of -lactamase
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Examples of -lactamases
Group of
enzyme
Preferred
substrate
Inhibited by
clavulanate
Representative
enzymes
1
cephalosporin
-
AmpC (G-ves)
2a
penicillins
+
Penicillinases from
G+ves
2b
Penicillins,
cephalosporins
+
TEM-1, TEM-2,
SHV-1 (G-ves)*
2be
Penicillins,
cephalosporins,
monobactams
+
TEM-3 to TEM 26
2br
penicillins
+/-
TEM-30 to TEM-36
2c
Penicillins,
carbenicillin
+
PSE-1, PSE-3, PSE4
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*Plasmid encoded (TEM, PSE, OXA, SHV)
Enzyme modification of
antibiotics
Inactivation of aminoglycosides
Chemical inactivation
• performed by enzymes produced by the bacteria
• three distinct classes based upon the reactions
that they catalyse:
(i) acetyltransferases which acetylate amino
groups on the aminoglycoside;
(ii) nucleotidyltransferases which transfer a
nucleotide moiety onto the drug, and
(iii) phosphotransferases which phosphorylate one
or more hydroxyl groups on the antibiotic.
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Multi-drug Efflux Pumps
Bacteria use ATP-powered membrane proteins to pump
foreign molecules out of the cell
- common in antibiotic-producing bacteria, to get substances out
of their cells without poisoning themselves
Powerful method of resistance, because many different drugs
will be equally affected by these efflux pumps
Examples: tetracyclines, macrolides, quinolones
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Many pathogens possess multiple
mechanisms of antibacterial
resistance
Altered uptake Drug inactivation
Modified target
-lactam
+
+
++
++
Glycopeptide
+
Aminoglycoside
–
+
Tetracycline
–
+
Chloramphenicol
–
Macrolide
++
Sulphonamide
++
–
Trimethoprim
++
–
Quinolones
–
+
+
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Transfer of antibiotic resistance
(horizontal transfer of DNA)
• Transformation
• Conjugation
• Transduction
• Of these conjugation is the most important
– R plasmids
– Transposons & integrons
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Plasmid carrying transposons &
antibiotic-resistance genes
•From S. N. Cohen and J. A Shapiro, “Transposable Genetic Elements.” Copyright © 1980 by Scientific American, Inc.
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Examples of transposons carrying antibiotic
resistance genes.
http://www.uhmc.sunysb.edu/microbiology/12
Tn5397
http://www.eastman.ucl.ac.uk/~microb/gene_transfer.html
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Multi-resistance
• Multiresistance gene
cluster on the
chromosome of
Salmonella
typhimurium DT 104
http://www.irishscientist.ie/2001
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Examples of major antibiotic resistance
problems
Hospital
• Methicillin resistant Staphylococcus aureus (MRSA) –
hospital and community acquired
• Vancomycin resistant enterococci (VRE)
• Multi-resistant Gram-negative bacteria (eg Acinetobacter
baumannii, many others
Community
• Community acquired MRSA
• Penicillin-resistant Streptococcus pneumoniae
• Multi-drug resistant Mycobacterium tuberculosis
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Antibiotic resistance in food-borne
organisms
• Salmonella, Shigella, Campylobacter,
Enterococcus spp, multi-drug resistant E
coli (and salmonella)
Common misuses of antibiotics
1. the patient does not have an infection
2. the infection does not respond to antibiotics - eg viral
infections
3. the latest "wonder drug" is used when an older product
would be effective
– protecting the new product for situations where it is really
needed
4. the patient "prescribes" for him/herself - using antibiotics
left over from a previous illness
5. in countries with poor health care services antibiotics are
sold without prescription
6. use of antibiotics for non-therapeutic purposes – eg
growth promotion or improved production in livestock
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Alternatives to antibiotics
• Probiotics, prebiotics & competitive exclusion
organisms
– Reduce pathogenic microorganisms in animal
GIT
• Bacteriophages
– Potential to use to control campylobacter,
salmonella
• Natural products – eg tea tree or eucalyptus
oils
• Bacteriocins
• Vaccines
Resistance to antiviral drugs
• This is often a big problem – especially with RNA viruses
• Resistant mutants arise spontaneously (even in the
absence of drug) and are selected,
– eg acyclovir-resistant mutants are unable to phosphorylate the
drug (TK mutants) or,
– do not incorporate the phosphorylated drug into DNA (pol
mutants)
• To overcome resistance it is crucial to use drugs at
sufficient concentration to completely block replication
• The use of more than one drug, with more than one
target, reduces significantly the emergence of resistant
mutants
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Anti-protozoan resistance
Mechanisms
•
alteration in cell permeability
•
modifications of drug sensitive sites
•
increased quantities of the target enzyme
Means of development in protozoa
•
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).
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Pipeline is small and resistance
is increasing – What can
biotechnology offer?
• Sequencing – over pathogenic genomes
and multiple strains
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What can biotechnology offer?
• Bioinformatics – use of computer
programs
– From both strands of DNA, predict open
reading frames (ORFs), i.e., what mRNAs can
be made
– From both strands of DNA and ORFs, predict
amino acid sequences, i.e., which proteins
can be made
37
What can biotechnology offer?
• Bioinformatics – use of computer
programs
– From amino acid sequences, perform
functional analyses, i.e., database
comparisons to help identify similarity with
proteins whose function is known
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What can biotechnology offer?
• Bioinformatics – use of computer
programs
– From amino acid sequences, predict protein
tertiary structures to identify potential sites
(pockets) where drugs can bind
– From pockets build/predict chemical
structures that possibly bind and inhibit
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What can biotechnology offer?
• Molecular biology
– Clone and express amino acid sequences
WHAT ELSE?
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• For example, open reading frames
(ORFs)—long sequences that begin with a
start codon (three adjacent nucleotides;
the sequence of a codon dictates amino
acid production) and are uninterrupted by
stop codons (except for one at their
termination) —suggest a protein coding
region
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• "Knowing the bases that make up a gene and where it's
located on a chromosome doesn't tell you what the gene
does. After sequencing, we still need to determine what
proteins the genes produce, and what those proteins do
in the cell.”
• "So, the sequence is really a starting point we still need
to know the structure and function of the protein
produced by the gene, and how that protein interacts in
the environment of the cell. The sequence, you might
say, is the detailed map we need to help us find the
buried treasure."
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Role of molecular biology
• Genomics
• Proteomics
• Transcriptional profiling