medical Microbiology lecture power point ppt drug resistance

1. Drug-Resistance and nosocomial
Infection

2. Section 1 Drug-Resistance
Antibiotics :powerful medicines that fight
bacterial infection
Literal translation
• anti – against
• biotic – living things

3. Discovery
Alexander Fleming in 1928
– He was an extremely messy
scientist
– Came back from holiday to see
a mould growing on his
Staphylococcus agar plates
– Noticed that the
Staphylococcus couldn’t grow
anywhere near the mould
– The mould prevented bacterial
growth!

4. How antibiotics work
Antibiotics can be either
• Broad Spectrum
– Kill a wide range of bacteria e.g.
Penicillin
• Narrow Spectrum
– Kill a specific type or group of
bacteria e.g. Isoniazid
Antibiotics work in one of two ways
• Bactericidal
– Kills the bacteria
• Bacteriostatic
– Prevents the bacteria from dividing

5. Miracle Cure?
– Before the 1930s there were no treatments
for bacterial infections
– Following the discovery of penicillin
industry started searching for more
antibiotics in nature
– Streptomycin was the first drug to have an
effect on tuberculosis – a condition
previously untreatable
– Surgeons could attempt more dangerous
operations

6. Sources of Antibiotics
[ v 'mekt n]ɪ ə ɪ 伊维菌素
利福霉素类
[ p li 'm ks n]ˌ ɒ ː ɪ ɪ 多粘菌素 [ bˌ æs 'tre s n]ɪ ɪ ɪ 杆菌肽素
双效菌素
[ ks z 'la d nw nz]ɒ ə ɒ ɪ ɪ ʌ 恶唑烷酮类 [la 'nez l d]ɪ ɒ ɪ 利奈唑胺

7. Miracle Cure?
– Many antibiotics prescribed
by the doctor are broad
spectrum
– These kill the body’s good
bacteria as well as the bad
– With the good bacteria gone
there is more room for bad
microbes to invade!
Overuse of antibiotics can damage our
normal/good bacteria.

8. Miracle Cure?
Antibiotics resistance
– Many bacteria have
developed the ability to
become resistant to
antibiotics.
– These bacteria are now a
major threat in our hospitals.
– Antibiotic resistant bacteria
include Methicillin 甲氧苯青
霉素 Resistant
Staphylococcus aureus
(MRSA)

9. Antibiotic Resistance
The Causes
– Overuse
• Antibiotics used to treat infections when they are
not needed or not effective i.e. for the flu
– Misuse
• Not completing a prescribed course
• Using antibiotics not prescribed for you

10. How antibiotic resistance can be
prevented
– Antibiotics should be the last line of defence NOT the first
• Most common infections will get better by themselves through
time, bed rest, liquid intake and healthy living.
– Only take antibiotics prescribed by a doctor
– If prescribed antibiotics, finish the course.
– Do not use other peoples or leftover antibiotics
• they be specific for some other infection

11. Antibiotic Resistance
APPEARANCE
DRUG INTRODUCTION OF RESISTANCE
Penicillin 1943 1946
Streptomycin 1945 1959
Tetracycline 1948 1953
Erythromycin 1952 1988
Vancomycin 1956 1988
Methicillin 1960 1961
Ampicillin 1961 1973
Cephalosporins 1964 late 1960’s

12. Origins of Resistance
Drug resistance is a natural by-product of the evolutionary process:
natural selection acting on pre-existing genetic variation
400 microbial strains were isolated from natural sources and sealed
into vials in 1917, long before the clinical introduction of antibiotics
- Recent analysis: 11 of these 400 strains had antibiotic resistance
(at a low level)
-

13. Origins of Resistance
Nearly all clinically useful antibiotics are natural products, or their
synthetic derivatives; most were isolated from other microbes
- Fungi (penicillins, cephalosporins)
- Soil bacteria of genus Streptomyces (erythromycin, streptomycin,
tetracycline, vancomycin)
In 1999, only 1 class of antibiotic was totally synthetic (Ciprofloxacin)
Antibiotics are an ancient weapon...
[d 'rev t vz]ə ə ɪ
环丙沙星

14. Origins of Resistance
What does the antiquity [æn't kw ti]ɪ ə 古代 of antibiotic resistance tell
us?
- There is likely to be considerable genetic variation in natural
populations for genes that can potentially confer drug resistance
(i.e., the raw genetic material is there)
- Strong selection will quickly lead to the explosive growth of
resistant individuals, especially when most cells are susceptible
Widespread antibiotic use =
- nukes their competition[nu k]ː 以核武器攻击
- the fittest survive and reproduce, passing on their resistance both
to clonal offspring and to other unrelated bacteria

15. Inherent Resistance
• Darwinian evolution:
– Bacteria that resist an antibiotic's effects are better
suited to survive in an environment that contains
the antibiotic.
– Genes that confer resistance are transferred to the
bacterial progeny.
• Bacteria naturally resistant (e.g., Gram-
negative bacteria resistant to penicillins).
• Bacteria may be resistant because
– They have no mechanism to transport the drug into
the cell.
– they do not contain or rely on the antibiotic’s target
process or protein.

16. Acquired Resistance
• Bacteria that don’t begin life resistant to a
certain antibiotic can acquire that resistance.
• Horizontal evolution:
– Resistance genes pass from a resistant strain to a
nonresistant strain, conferring resistance on the
latter.
– Presence of an antibiotic is a selective pressure.
• Gene transfer mechanisms:
– Conjugation.
– Transduction.
– Transformation.

17. Conjugation
• Transmission of
resistance genes via
plasmid exchange.
• Resistance spreads
much faster than
simple mutation and
vertical evolution
would permit.

18. Transduction and Transformation
• Transduction:
Virus transfers
gene.
• Transformation:
DNA released
from a bacterium
is picked up by a
new cell.

19. Antibiotic Mechanisms

20. Antibiotic Targets
The major classes of antibiotics affect 1 of 3 targets in bacteria cells:
(1) Cell wall biosynthesis
penicillins
cephalosporins
vancomycin (non-ribosomal peptide)
(2) Protein synthesis
erythromycin (macrolide ['mækro la d]ʊ ɪ 大环内酯物 polyketides)
tetracycline (aromatic [ˌær 'mə æt k]ɪ 芳香的 polyketides 聚酮化合物 )
streptomycin, kanamycin (aminoglycosides)
(3) DNA replication
quinolones (Cipro) 盐酸环丙沙星制剂
(β-lactams)

21. Antibiotic Targets
Antibiotics work by exploiting biochemical differences between
our eukaryotic cells and the prokaryotic cells of bacteria
(1) Cell wall biosynthesis
- block synthesis of peptidoglycan, the covalently cross-linked
peptide/glycan network, which imparts osmotic resistance to cell
(2) Protein synthesis
- target 23S rRNA + associated proteins in peptidyl transferase
center of bacterial ribosome
-
(3) DNA replication
- inhibit gyrase, essential enzyme that uncoils intertwined circles of
DNA after replication of the circular bacterial chromosome

22. Antibiotic Target 1: Cell Wall
Cell wall is peptidoglycan, a repeating polymer of di-saccharide,
tetra-peptide repeats cross-linked into a 3D matrix
β-lactam antibiotics interfere with cell wall biosynthesis of
Gram-positive bacteria (Staphylococci, Streptococci)
-

23. Antibiotic Target 1: Cell Wall
Bacterial transpeptidase enzyme forms crosslinking amide bonds
between #3 L-Lysine and #4 D-Alanine residues
TPase cuts off #5
D-Ala residue,
then links L-Lys
side chain to the
remaining D-Ala

24. Antibiotic Target 1: Cell Wall
Catalytic Serine -OH forms a temporary bond to the substrate
- when Lysine side-chain attacks the temp. ester linkage,
the Serine is restored to normal
-

25. Antibiotic Target 1: Cell Wall

26. β-lactams: Mechanism of Action
β-lactams inhibit transpeptidase by mimicking its substrate,
the terminal D-Ala—D-Ala
Transpeptidase attacks the β-lactam ring of penicillin, forms a
covalent bond that is slow to hydrolyze; enzyme is deactivated
Normally, the enzyme forms a temporary bond with D-Ala that
is rapidly broken by the side chain of Lysine

28. Resistance: β-lactamase Enzymes
Bacteria produce enzymes to hydrolyze the β-lactam ring before
drugs can reach inner membrane where PG synthesis occcurs

29. Overcoming β-lactam Resistance
Augmentin combines β-lactam antibiotic w/ clavulanate 克拉
维酸钾 , a
“suicide” β-lactam that occupies the β-lactamase enzymes
- Allows active drug (amoxacillin, [ m ks 's l n]ə ɒ ɪ ɪ ɪ 阿莫西林 )
(resistance) slow to
hydrolyze
(cell wall enz.)

30. Vancomycin: Mechanism of Action
Vancomycin, the crucial “drug of last resort,” inhibits PG synth
by binding directly to the D-Ala—D-Ala end of the peptide
- forms a cap over the end of the chain; blocks cross-linking

31. Vancomycin: Mechanism of Action
Completely surrounds its target peptide, preventing enzymes
from reacting with the end of the peptidoglycan chain
3D model of Vancomycin in
complex with D-Ala—D-Ala
note “cup-like”
shape of Van

32. A cell may produce 100,000 lactamase enzymes, each of which
can destroy 1,000 penicillins per second
100 million molecules of drug destroyed per second
Resistance: β-lactamase Enzymes

33. Vancomycin makes 5 H-bonds with the
D-Ala—D-Ala cap of the PG peptide
-
-
Vancomycin
D-Ala D-Ala

34. Van Resistance: D-Ala-D-Lactate
Vancomycin-resistant bacteria have peptidoglycan chains that end
in D-Ala—D-Lactate, instead of the usual D-Ala—D-Ala
(A) What genes are necessary to make this change?
(B) How does this confer resistance?
D-Ala—D-Ala
D-Ala—D-Lactate

35. Genetics of Van Resistance
VanAVanH
VanX
5 gene products are required to produce Lac-terminal PG
- 2 “sensor” genes detect Van, turn on other 3 genes
- 2 synthesize the critical D-Ala—D-Lactate piece
- 1 destroys the pool of D-Ala—D-Ala in the cell (equilibrium)
reduction
hydrolysis 1,000 fold lower
affinity for Van

36. Vancomycin: Mechanism of Action
D-Ala—D-Ala cap makes 5 H-bonds with Vancomycin
D-Ala—D-Lac makes 1 less H-bond Resistance You die

37. Genetics of Van Resistance
Why did penicillin resistance appear in 2 years, but Van resistance
take 30 years to become a major health hazzard?
One answer: genetic complexity of resistance mechanism
Penicillin resistance requires the activity of one gene product
(β-lactamase enzyme)
- usually 2-4 year lag when only 1 gene is involved
Van resistance takes 5 gene products
- apparently delays development of infectious, highly resistant
strains when multiple gene products are involved

38. Overcoming Van Resistance
Approach #1: Screening of semi-synthetic analogues of Van
found that hydrophobic derivatives restore potentcy 100-fold
- Partitions drug to membrane surface, thus altering activity
and availability to target enzymes
chlorinated
bi-phenyl
substituent

39. Overcoming Van Resistance
Approach #2: Screening combinatorial libraries for novel small
molecules that cleave the D-Ala—D-Lac depsipeptide
[dep'saipepta d]ɪ 缩酚酸肽
- Look for drugs that can effectively function like an enzyme
Combinatorial library of 300,000 tripeptide derivatives yielded
3 hits, all w/ an N-terminal serine & an intramolecular H-bond
Pharmacophore deduced from computer modeling studies
N
HO
NH2
O
SProC5 “resensitized” bacteria
with Van-resistance, by cleaving
their D-Ala—D-Lac depsipeptide
SProC5
Chiosis & Boneca, Science 2001

40. Protein Synthesis Inhibitors
Erythromycin
(macrolide polyketide)
Tetracycline
(aromatic polyketide)
Kanamycin
(aminoglycoside)

41. Resistance to Aminoglycosides
Chemical modification of the drug lowers its
binding affinity for RNA target in the ribosome
-
(formerly a
protein kinase?)

42. MultiDrug Resistance Pumps
Bacteria use ATP-powered membrane proteins to pump any
lipophilic molecule out of the cell
- common in antibiotic-producing bacteria, to get drugs out
of their cells without poisoning themselves
Powerful method of resistance, because many different drugs
will be equally affected by these efflux pumps

43. MultiDrug Resistance Pumps
(1) substrate binding:
lipophilic drug binds
inside cone-shaped
chamber; triggers
ATP hydrolysis
(2) chamber
then closes,
substrate flips
to opposite
orientation
(3) chamber then
opens, substrate
is expelled to
outer face of
membrane
outside cell

44. Erythromycin Resistance
In addition to efflux pumps, erthyromycin resistance can arise
from reprogramming the target (akin[ 'k n]ə ɪ 近似的 to Van
resistance)
Methylation of a specific adenine ['æd n n]ə ɪ on the 23S rRNA
component of the ribosome
- decreases binding affinity of erythromycin-class drugs
- does not impair protein synthesis
- present as a self-immunity mechanism in erythromycin-
producing bacteria

45. Overcoming Erythromycin Resistance
Introduction of a 3-keto ['ki to ]ː ʊ 氧化 group into macrolide
ring of erythromycin class antibiotics alters conformation
- no induction of ribosome-methylating genes
- lower susceptibility to efflux by pumps
Erythromycin

46. Selection favoring Resistance
What causes the rapid occurrence of widespread resistance?
(1) Incomplete treatment: people fail to finish the full course of their
medication
- in the 1980’s, tuberculosis was almost wiped out w/ antibiotics
- in 1990’s, came back with a vengence['vend ns]ʒə 复仇 , due to
resistant strains
- 25% of previously-treated tuberculosis patients relapsed with drug
resistant strains; most had failed to complete their initial course
(2) Livestock doping['do p ]ʊ ɪŋ 服用禁药 : 50% of antibiotics used
by livestock farmers to increase yield of chicken, beef, pork
- high levels of antibiotics used in livestock result in strongly
resistant bacterial strains, which can then infect humans

47. Selection favoring Resistance
What causes the rapid occurrence of widespread resistance?
(3) Mis-prescription: my mom demands antibiotics for a cold
- widespread inappropriate use: up to 50% of prescriptions in
developing countries are for viral infections that cannot respond
(4) Gene transfer & multi-drug resistance
(a) genes encoding resistance accumulate on plasmids, transposons
confer simultaneous resistance to multiple drugs
(b) DNA is easily exchanged between unrelated bacteria
- vancomycin-resistant gut bacteria known since 1987
- resistance genes finally transferred to deadly infectious
Staphylococcus aureus in a Michigan hospital in 2002

48. Loss of Resistance...?
Resistance carries a cost: resistant bacteria grow more slowly under
normal conditions, pay a 10-20% fitness cost
- Replicating extra plasmid DNA is costly to the cell
- Ribosomal mutations that confer resistance slow protein production
When we stop using an antibiotic, does resistance go away?
- Can we reverse selection, and favor the vulnerable bacteria instead
Experiments show bacteria quickly evolve compensatory mutations
that lower the costs of resistance, instead of just losing resistance
-
Levin et al. 2000, Genetics 154: 985-997

49. Conclusion
• We overuse antibiotics and often neglect to
complete a full course of antibiotics once it
has been prescribed, leading to the spread of
antibiotic resistance.
• Resistance can disappear if there is no
selective pressure to maintain resistance.

50. Section 2 Nosocomial infections
• The word derives from the Greek
nosokomeian, meaning hospital
• These days the terms hospital acquired –
and healthcare associated – are used
• A very emotive subject with the public,
driven by the press
• Do hospitals really deserve to be blamed for
all cases of hospital infection?

51. Nosocomial infections are…
• Infections that are acquired in hospital (48
hours or more after admission)
• Approx 7% of patients will suffer from an
infection whilst in hospital – the risk increases
with length of stay
• A significant financial burden on NHS

52. Impact of nosocomial infections
• Possibly up to100,000 infections per year in
UK
• A cause of ~5,000 deaths with nosocomial
infections playing a role in ~15,000 others
• Costs the NHS £1 billion – 9% of its in-
patient budget
• Cannot be eradicated but it’s thought they
could be reduced by up to 30%

53. Impact of nosocomial infections
• Longer stays in hospital – bed occupancy
• Outbreaks leading to ward closures
especially norovirus and C. difficile
• Pain and anxiety for patients and families
• Loss of earnings

54. Why are we more likely to get an
infection in hospital?
Consider 4 important factors…
1. The host
2. The microbes
3. The environment
4. Treatment

55. The host 1
• People in hospital are already sick!
• They may have poor general resistance to
infection
• Lack of immunity
– Extremes of age
– Immunocompromised (eg cancer
chemotherapy)

56. The host 2
• Reduced immunity
– Diabetes, severe burns
• Poor local resistance
– Poor blood supply to tissues
• Surgery
– Wounds, sutures
• Medical devices
– Catheters, prostheses, tubing etc

57. The microbes
• Virtually any infection can be acquired in
hospital
• However a number of “usual suspects”
predominate
• What are they, where do they come from and
why do they cause nosocomial infection?

58. Opportunistic infections
• Nosocomial infections are often caused by
opportunistic pathogens i.e. those which
do not normally cause infection in healthy
people
• May be a reflection of reduced defences of
host or access to sites not normally
colonised by organisms
• May be from normal flora or environment
• Antibiotic resistance is a problem

59. Opportunistic pathogens
• Pseudomonas aeruginosa
• staphylococci
• E. coli and other coliforms
• streptococci and enterococci
• Bacteroides fragilis
• Candida albicans
• Herpes simplex virus
• Cytomegalovirus

60. Biofilms
• Biofilms are microbial communities (cities)
living attached to a solid support eg
catheters/ other medical devices
• Biofilms are involved in up to 60% of
nosocomial infections
• Antibiotics are less effective at killing
bacteria when part of a biofilm

61. The Environment
• There are many different sources of
pathogens when in hospital
– Our own normal flora (endogenous infection)
– Infected patients
– Movement of staff and visitors
– Environment e.g. fungi, Legionella
– Blood products (v. rare)
– Surgical instruments eg vCJD (v. rare)

62. ENVIRONMENTAL SOURCES OF PATHOGENS IN THE HEALTHCARE SETTING
Source Bacteria Viruses Fungi
Air Gram-positive cocci (originating from skin)
Tuberculosis
Varicella zoster
(chickenpox),
Influenza
Aspergillus
Water (tap
and bath)
Gram-negative bacteria (Pseudomonas
aeruginosa, Aeromonas hydrophilia, Burkholderia
cepacia, Stenotrophomonas maltophilia, Serratia
marcescens, Flavobacterium meningosepticum,
Acinetobacter calcoaceticus, and Legionella
pneumophila) Mycobacteria (Mycobacterium
xenopi, Mycobacterium chelonae, or
Mycobacterium avium-intracellularae)
Molluscum contagiosum
Human papillomavirus
(bath water)
Noroviruses
Aspergillus
Exophiala
jeanselmei
Food Salmonella species, Staphylococcus aureus,
Clostridium perfringens,
Clostridium botulinum, Bacilluscereus and other
aerobic spore-forming bacilli Escherichia coli
Campylobacter jejuni ,Yersinia enterocolitica,
Vibrio parahaemolyticus, Vibrio cholerae,
Rotavirus
Caliciviruses

63. Treatment
• There is continuous usage of antibiotics in
hospitals especially in ICU
• As a result there will be a natural selection for
strains that are antibiotic resistant – infections are
getting harder to treat
• This has led to problems with multi-resistant
bacteria e.g. MRSA, VRE, ESBLs
• Antibiotic treatment can also lead to alterations in
normal flora and allow pathogens cause infection
eg C. difficile

64. Summary
Definitions:
Inherent Resistance
Acquired Resistance
MultiDrug Resistance Pumps
Briefly describe the main antibiotic mechanisms?