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1. 1

2. A Potential Solution For Antibiotic Resistance Crisis
Khwaja Ali Hasan (Research Fellow HEJRIC)
SUPERVISOR
Dr. Syed Abid Ali Associate Professor (HEJRIC Lab# 205, 213, 307)
2

3. Learning Objective
• Anti-microbial Resistance
• Mechanisms of Anti-microbial Resistance
• Role of Antibiotic Selective Pressure on Acquired
Resistance
• Alternative Anti-bacterial Therapy
• Bacteriophages and their Role as an Anti-microbial
Alternative
3

4. Advent of Antibiotics and History of
Resistance
Antimicrobial Drug Resistance and
Resistance Mechanisms.
Antibiotic Selective Pressure and
Antimicrobial Resistance in Pakistan
(Facts and Figures)
Measures of Resistance Reduction
Alternative Antibacterial Therapy
Discovery of Bacteriophages and
their Structure
Phage families as Antimicrobials and
their Mode of Action
Bacterial Anti-phage Mechanisms
Remediation of Anti-phage Resistance
Phage Therapy and Pharmacology
Phage Encoded Proteins as Antibacterial
Design
Case Study
Summary and Conclusion
Outline
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5. 5

6. Introduction
to
Antibiotics
6

7.  Antibiotics: Treatment and Prevention of Infections.
• Natural Antimicrobial Substances.
• Bacteria: Streptomyces, Bacillus.
• Molds: Penicillium, Cephalosporium.
• Semi-synthetic Antibiotics: Chemically modified
derivatives of natural antibiotics.
• Synthetic antibiotics.
• Designed to achieve better bactericidal properties.
Introduction to Antibiotics
Drulis-Kawa et al, 2012; Current Protein and Peptide Science 7
Continue

8. • >200 different compounds.
• Each of the chemical class exhibits a
specific mode of action with a different
activity spectrum.
Introduction to Antibiotics
8

9. Cell wall synthesis inhibitor
Protein synthesis inhibitor
DNA synthesis inhibitor
Sosa et al, 2010; http://s.telegraph.co.uk/graphics/projects/antibiotic-resistance/
Cell membrane disruptor
Metabolite synthesis inhibitor
9
Discovery and Resistance Development

10. Antibiotic usage and
Selective Pressure
causes Antimicrobial
Resistance in Pakistan
(Facts and Figures)
10

11. Laxminarayan et al, 2013;
The Lancet Infectious Diseases Commission
Trends in retail sales of carbapenem
antibiotics for Gram-negative bacteria
Based on data obtained from IMS Health’s MIDAS™ database.
*An IMS grouping of Benin, Burkina Faso, Cameroon, Congo (Brazzaville), Gabon,
Guinea, Ivory Coast, Mali, Senegal, and Togo.
11
Continue

12. PARN: PAKISTAN ANTIMICROBIAL NETWORK (NIHP, PMRC)
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13. 13

14. Mechanisms
of
Antimicrobial
Resistance
14

15.  Acquire Antibiotic Resistance:
Presents a significant challenge for therapeutic measures.
• The most prevalent mode:
 Horizontal Gene Transfer
• The horizontal acquisition of resistance genes
from another organism is implemented by;
Antibiotic Resistance
Giedraitien et al, 2011 15

16. 16
Transformation Conjugation Transposition
Mutation (sporadic)
Transduction

17. Mechanisms of Antibiotics
Resistance
Denis K. Byarugaba, 2010 17

18. Measures of Antibiotic
Resistance Reduction
and
Alternative
Antibacterial
Therapies
18

19. To reduce the emergence of antibiotic resistance;
(i) Controlled or Cycle antibiotic usage.
(ii) Improvement of Hygiene.
(iii) Discovery or development of new antibiotics.
(iv) Modification of existing antibiotics.
(v) Development of inhibitors against antibiotic modifying or
hydrolysing enzymes.
(vi) Development of alternative antibacterial therapies for example;
Phage Therapy
Measures of Antibiotic Resistance Reduction
Drulis-Kawa et al, 2012 19

20. 20
Alternative Antibacterial Therapies
 Disease Prevention
 Vaccines
 Probiotics
 Prebiotics
 Synbiotics
 Competitive exclusion
 Disease treatment
 Endo- or exolysins
 Bacteriocins
 Predatory bacteria
 Phage Therapy
Allen et al, 2014; Annals of the New York Academy of Sciences

21. 21

22. Introduction
to
Bacteriophages
22

23. • Bacteriophages are viruses that infect
bacterial cells.
• Obligate parasites shows several life
cycles;
• Lytic
• Lysogenic
• Pseudo-lysogenic
• Chronic infections
Introduction to Bacteriophages
Golkar et al, 2014; J Infect Dev Ctries 23

24.  Bacterial viruses:
 Frederick Twort (1915) and Felix
d’Hérelle (1917).
 >5500 different bacteriophages: Each
of which being able to infect one or
several types of bacteria.
Discovery of Bacteriophage
Drulis-Kawa et al, 2012 24

25. Phage head: Coat protein and genome in
the core.
Genome: Encodes for enzymes and
proteins necessary to replicate more
viruses.
Tail Sheath: DNA travels from head to
bacteria through sheath.
Tail fiber: Helps to anchor the phage on the
cell membrane.
Structure of Bacteriophage
25

26. from the greek phagein, meaning "to eat“
Eaters or destroyers of bacteria
Transmission Electron
Micrographs of Bacteriophages
26

27. • Every environment, where their
bacterial host are present.
• Aquatic systems: 10 E4 – 10 E8 virions
per ml.
• Soil and Sediments: Approximately of
10 E9 virions.
Niche of Bacteriophage
27

28. 28
Antimicrobial
Phage Families
and their
Mode of Action

29. Filamentous phages
(M13 and Pf3)
• Phage as antimicrobials has focused on
lytic tailed phages representing three
families of Caudovirales order;
Phage Families as Antimicrobials
Cenens
et
al,
2013;
Bacteriophage
29
Siphoviridae
Small capsid head
(~50-60 nm)
long flexible,
noncontractile tail.
Podoviridae
Small capsid head
(~50-60 nm)
short tail
Cubic phages
(phiX174 and Qb)
Myoviridae
Biggest capsid head
(~150 nm)
contractile tail

30. 30
S. aureus bacteriophage (ISP),
Member of the Myoviridae family
Transmission Electron micrographs
Merabishvil
et
al,
2009;
PloS
ONE
P. aeruginosa bacteriophages (PNM),
Member of the Podoviridae family
Bacteriophage (PNM) attaching to
the P. aeruginosa cell wall
ISP bacteriophages (Myoviridae)
attached to S. aureus
1a
1b
2a
2b

31. 1. Adsorption to specific receptor 2. Nucleic Acid Injection
3. Redirection of host metabolism
4. Assembly and packing of phage particles
5. Bacterial cell lysis and phage progeny release
31
Mode of Action of Lytic-phages

32. Receptor Mediated Phage-Host
Interactions
32
Timothy
K
Lu1
and
Michael
S
Koeris,
2011;
Current
Opinion
in
Microbiology

33. 33
Bacterial Anti-Phage
Resistance Mechanisms
and
Its Remediation

34. 34
Timothy
K
Lu1
and
Michael
S
Koeris,
2011;
Current
Opinion
in
Microbiology
Bacterial Anti-Phage Resistance
Mechanisms
 Bacteria as phages prey have evolved several
adaptive mechanisms protecting the cell from
viral infection.
Continue

35. • Bacteria can inhibit the phage cycle on crucial steps of
propagation process by;
(i) Preventing phage adsorption.
(ii) Preventing DNA integration by Superinfection exclusion system
(Sie).
(iii) Degradation of phage DNA by Restriction-Modification (RM)
defense system and Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)
(iv) Blocking phage replication, transcription, translation or virions
assembly by Abortive Infection system (Abi).
Bacterial Anti-Phage Resistance
Mechanisms
Drulis-Kawa et al, 2012 35

36. • Unlike antibiotics, the remediation of the
anti-phage resistance problem is relatively
fast and easy.
• Isolation of novel active phages
• Progressive adaptation of viral parasite to
resistant host population.
• Cocktails of phage; To prevent possible
resistance development.
Remediation of the Anti-phage
Resistance
36

37. Phage therapy,
choice
and
Production
37

38. For therapy there must be enormous number of
phage variety as effective antimicrobials.
 Increase; Activity spectrum and may have
synergistic effect
Phage therapy and Phage choice
Abedon et al, 2010 38
Continue

39. The phage choice for application is usually
based on;
(i) Assortment of effective phages from an
available collection after phage typing of
isolated bacteria.
(ii) Selection of several most potent phages.
(iii) Direct isolation from environment. 39
Phage therapy and Phage Choice

40. • In phage choice and selection as potential safe
antimicrobials detailed comprehensive
characteristics such as;
• Phage genetics (genome sequencing) and
Phenotypic properties
• Presence of unfavorable features such as Lysogeny-
associated genes, Toxin or enzyme encoding
genes, could be detected and considered.
Prerequisites for Phage Selection
40

41. 41
The final product, a defined
bacterio-phage cocktail
Separation
Bacterial
Toxin
Removal
Formulation
Quality Control
Phage Production

42. Pharmacodynamics
and
Kinetics
42

43. • Antibacterial substances used in therapy
should exhibit desirable properties;
 Efficient Pharmacodynamics (PD)
 Pharmacokinetics (PK)
Desirable Antibacterial Properties of
Phages
43

44. • PD: Ability of a drug to eliminate bacterial
cells as well as the influence on host tissues
and organs.
• Antibacterial efficacy of phages;
• Phage Virulence
• Duration of phage generation time
• Efficient adhesion, latent period and virion
release
44
Pharmacodynamics

45. Phages are described as self-replicating
antibacterial agents.
 Efficient concentration can be
achieved at exact body/tissue location
where it is needed.
45
Pharmacodynamics

46.  Limited Side Effects
• Rapid lysis of a big number of cells and
release of LPS from bacteria in a short
period of time may cause serious side
effects on the host.
46
Pharmacodynamics
Timothy
K
Lu1
and
Michael
S
Koeris,
2011;
Current
Opinion
in
Microbiology

47. Phage therapy and
Immune Response
47

48. • Bacteriophages are seen by the immune
system as:
Potential invaders (viruses).
 Reticuloendothelial system
(RES) clearance
 Adoptive immune defense mechanisms
involving immunoglobulin.
Phage Therapy and Immune Response
48
Timothy
K
Lu1
and
Michael
S
Koeris,
2011;
Current
Opinion
in
Microbiology

49. 49
Phage Therapy
and
Pharmacokinetics

50. • PK Antimicrobial potency : refers to what the treated
organisms do with the drug.
The PK concerns:
(i) Absorption
(ii) Distribution (Administration efficiency, penetration to
particular tissues).
(iii) Metabolism (Shelf-life, mechanisms of metabolic
modification).
(iv) Excretion (Mechanism of elimination).
Phage therapy and Pharmacokinetics
50

51.  Delivery route
• Parenteral route (Systemic infections)
• Orally (GIT)
• Locally (Topical infections)
Possible Route of Phage Delivery
51
Merabishvil
et
al,
2009;
PloS
ONE
Application of BFC-1 on an
infected burn wound using a
syringe spray

52. Phage-encoded
Proteins and
Antimicrobial
design
52

53. • The major mechanisms of phage infection may
be used as a model for antimicrobials design.
 Destruct bacterial envelopes.
• Peptydoglycan, Cell membrane and Cell
capsule
 Affects bacterial
DNA replication, Transcription, Protein
synthesis
• Cell division by the protein-protein interactions.
Phage-encoded Proteins and
Antimicrobial design
Drulis-Kawa et al, 2012 53

54. Sanz-Gaitero, et al, 2014; Callewaert, et al, 2011
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55. Case Study
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56. Before treatment Treated with phage
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Wright et al, 2009

57. S.aureus infection
Treated with phage
impregnated pad Improvement in wound healing
Abedon et al, 2011 57

58. Polyvalent
Bacteriophage
Producers
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59. JSC “Biochim-pharm” 3 L. Gotua str., Tbilisi (0160), Georgia.
biochimpharm@geophage.ge; Web Site: http://www.biochimpharm.ge/
Polyvalent Bacteriophage Producers
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Treatment and prophylaxis of GIT
Treatment and prophylaxis of
Bacterial Inflammatory
Infections
Treatment and prophylaxis of
Dysentery caused by Shigella sp.
Treatment and prophylaxis of
Enteric fever

60. Advantages and
Limitations of Phage
Therapy
60

61.  The specificity of predator-prey interaction
ensures no influence on normal flora;
 No-Dysbiosis.
 Can synergize with Antibiotics
 Efficient concentration
Advantages of Phage Therapy
61

62.  Emergence of bacterial
strains resistant to particular
phages.
 Phage–neutralizing
antibodies.
Limitations of Phage Therapy
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63. Summery
and
Conclusion
63

64. Production Simple and cost effective.
Pharmacokinetics Initial dose increases exponentially if the
susceptible bacterial host is available.
Selectivity/Specificity Bacteriophages permits the target specific
killing of pathogens.
Cross Resistance Because of phages specificity, not likely to
develop phage resistance in other (non-
target) bacterial species
Side effects Humans are exposed to phages throughout
life so, can well tolerate them. No serious
side effects have been described.
Development of new drug Rapid process and frequently can be
accomplished in days. 64
Summery

65.  Phage therapy is a possible alternative for
eliminating multidrug resistant Pathogens.
 It is relatively simple task to isolate phages against
any given pathogen.
 The timescale and costs for the development of a
new phage(s) for therapy will be a fraction of those for
introducing a new antibiotic.
 Bacteriophages and phage-encoded proteins opens
new area of research and anti-bacterial development,
there by can reduce sufferings of Man kind.
Conclusion
65

66. • Zuzanna Drulis-Kawa., Grazyna Majkowska-Skrobek., Barbara
Maciejewska., Anne-Sophie Delattre and Rob Lavign (2012).
Learning from Bacteriophages - Advantages and Limitations
of Phage and Phage-Encoded Protein Applications. Current
Protein and Peptide Science. 13, 699-722.
• William Cenens., Angella Makumi., Mehari Tesfazgi Mebrhatu.,
Rob Lavigne and Abram Aertsen (2013). Phage–host
interactions during pseudolysogeny. Bacteriophage. 3; (1),
e25029.
• Marta Sanz-Gaitero., Ruth Keary., Carmela Garcia-Doval., Aidan
Coffey and Mark J van Raaij (2014). Crystal structure of the
lytic CHAPK domain of the endolysin LysK from
Staphylococcus aureus bacteriophage K. Virology Journal. 11,
133.
References
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68. 68