1

BIOT 309: ANTIMICROBIAL RESISTANCE

Jan, 2013

2http://courses.washington.edu/medch401/pdf_text/401_05_black_betalactams.ppt#5

3

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

4

We are running out of new classes of antimicrobials

Antimicrobial class Year of launch • Sulphonamides 1936 • Penicillins 1940 • Tetracyclines 1949 • Chloramphenicol 1949 • Aminoglycosides 1950 • Macrolides 1952 • Glycopeptides 1958 • Streptogramins 1962 • Quinolones 1962 • Oxazolidinones 2001• Cyclic lipopeptides 2003• Ketolides 2004• Glycylcyclines 2005

1969 – US Surgeon General said “It is time to close the book on infectious diseases.”

5

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

6MMWR Morb Mortal Wkly Rep 2002;51:565–567

Evolution of drug resistance in S. aureus

S. aureus Penicillin-resistantS. aureus

Penicillin Methicillin

1950s 1970s

Methicillin-resistantS. aureus (MRSA)

Vancomycin-resistantenterococci (VRE)

Vancomycin

Vancomycin-intermediate

S. aureus (VISA)

Vancomycin-resistant

S. aureus (VRSA)

1990s1997

2002

7

Emergence of resistance in Gram-negative 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

8

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

9

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

10

Resistant StrainsRare

xx

Resistant Strains Dominant

Antimicrobial Exposure

xxxx

xx

xx

xx

Selection for antimicrobial-resistant Strains

Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

• 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 19-year-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”

12

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– Horizontal transfer of resistance genes

13

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

14

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 thi-

mutants in S. aureus)

• Derepression of multidrug efflux systems

15

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)

16

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.

17

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 penicillin-binding 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.

18

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

19

Mosaic PBP Genes in penicillin-resistant 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

Czechoslovakia (1987)

USA (1983)

South Africa (1978)

S SXN

= pen-sensitive S. pneumoniae = Streptococcus ?

PBP 2B

http://www.uhmc.sunysb.edu/microbiology/35

Target modification

20

vanR vanYvanS vanH vanA vanX vanZ

Vancomycin resistance gene sequence

Detects glycopeptide; switches on other genes

CleavesD-Ala-D-Ala

Produces D-Lac *CleavesD-Ala and

D-Lac from end chain

ProducesD-Ala-D-Lac

Exact role?Teicoplaninresistance?

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

Target modification

21

Action of -lactamaseEnzyme modification of the antibiotic

22

Examples of -lactamasesGroup 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, PSE-4

*Plasmid encoded (TEM, PSE, OXA, SHV)

23

Enzyme modification of antibiotics

Inactivation of aminoglycosidesChemical 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.

24

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 pumpsExamples: tetracyclines, macrolides, quinolones

25

Many pathogens possess multiple mechanisms of antibacterial

resistance

+–Quinolones

–++Trimethoprim

–++Sulphonamide

++Macrolide

+–Chloramphenicol

+–Tetracycline

+++–Aminoglycoside

+Glycopeptide

++++-lactam

Modified target Altered uptake Drug inactivation

26

Transfer of antibiotic resistance (horizontal transfer of DNA)

• Transformation

• Conjugation

• Transduction

• Of these conjugation is the most important– R plasmids– Transposons & integrons

27

Plasmid carrying transposons & antibiotic-resistance genes

•From S. N. Cohen and J. A Shapiro, “Transposable Genetic Elements.” Copyright © 1980 by Scientific American, Inc.

28

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

29

Multi-resistance

• Multiresistance gene cluster on the chromosome of Salmonella typhimurium DT 104

http://www.irishscientist.ie/2001

30

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

Antibiotic resistance in food-borne organisms

• Salmonella, Shigella, Campylobacter, Enterococcus spp, multi-drug resistant E coli (and salmonella)

32

Common misuses of antibiotics

1. the patient does not have an infection2. the infection does not respond to antibiotics - eg viral

infections3. 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

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

34

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

35

Anti-protozoan resistance

Mechanisms• alteration in cell permeability• modifications of drug sensitive sites• increased quantities of the target enzymeMeans 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).

Pipeline is small and resistance is increasing – What can

biotechnology offer? • Sequencing – over pathogenic genomes

and multiple strains

36

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

38

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

39

What can biotechnology offer?

• Molecular biology– Clone and express amino acid sequences

WHAT ELSE?

40

• 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

41

• "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."

42

Role of molecular biology

• Genomics

• Proteomics

• Transcriptional profiling