Biocontrol of cholera

1. BIOCONTROL OF CHOLERA

2. Contents
Introduction
Cholera disease
& Pathogenesis
Biocontrol agents
Bdellovibrio for cholera
Phages for cholera
FDA approved phages
Conclusion

3. Introduction
Biocontrol
 Biocontrol is the use of one living thing to control
another living thing OR
 The control of harmful organisms by the use of another
organisms

4. Biocontrol examples
1. Bacillus thuringiensis as insecticide
2. Agrobacterium tumefaciens against crown
gall disease
3.Bacillus papillae for Japense bettles
4.Trichoderma for soil born diseases.

5. Cholera is an infection of the small intestine by
the bacterium Vibrio cholerae.
 Symptoms
 Nausea
 Vomiting
 Watery diarrhea
 Dehydration

6. Vibrio cholerae
 Vibrio cholerae is a Gram-negative,
comma-shaped bacterium
 Polar flagella
 Optimum PH 8.5

7. Environment niche
 Vibrio cholerae, a bacterium
autochthonous to the aquatic
environment
 V. cholerae is present throughout
the year in and on its zooplankton
host
 V. cholerae, including both
serogroups O1 and O139, is found
in association with crustacean
zooplankton, mainly copepods,
and notably in ponds, rivers, and
estuarine systems globally

8. Phytoplankton bloom

11. Classification
 There are more than 200 serogroups in the
world
 Two serogroups are called serogroup O1 and
serogroup O139 (O139 is found only in Asia) and
can cause epidemic cholera
 The other serogroups are known collectively as
non-O1 and non-O139 Vibrio cholerae.

13. Worldwide cholera cases
 Each year there are 1.3 million to 4.0
million cases of cholera, and 21 000 to
143 000 deaths worldwide due to
cholera.
 Most of those infected will have no or
mild symptoms, and can be successfully
treated with oral rehydration solution
WHO(2016)

14. Total numberof cholera outbreaks,
1974–2005

17. Pathogenesis
 V. cholerae pathogenicity genes code for proteins
directly or indirectly involved in the virulence of the
bacteria.
 Colonization of the small intestine also requires the
toxin coregulated pilus (TCP), a thin, flexible,
filamentous appendage on the surface of bacterial cells
 Vibrio produces a enterotoxin CT toxin similar to LT
toxin of E.coli
 Toxin consist of 1A and 5B Subunits
 B subunit bind to intestinal receptors and A subunit
activates adenylate cyclase.

18. Pathogenesis

19. Vibrio cholerae genome
 .

20. Cholera treatment and
management
 Rehydration therapy
 Antibiotics
 Vaccines
1. Vaxchora(Paxvax)- FDA approved live oral vaccine
2. Shanchol (Shantha biotec)– Live Attenuated Oral

21.  As Antibiotics Fail, We Need More Vaccines
 Vaccines are still insufficient for cholera
 We need some more alternatives

22. Biocontrol agents for cholera

23. Bdellovibrio bacteriovorus
 Living antibiotic
 Gram Negative bacteria
 Obligate aerobic bacteria
 Comma shaped
 Motile predator

24. Bdellovibrio bacteriovorus
 Bacteria of the class delta-proteobacteria with the
ability to parasitize and kill other gram negative
bacteria
 This includes many pathogens Escherichia,
Pseudomonas, Salmonella, Legionella, and Shigella

25. Bdellovibrio attacking a prey

26. Bdellovibrio attacking other
bacterial cells

29. Method
 Suspensions of prey cells were placed on cover slips and
allowed to stand for 10 minutes to enable them to
attach to the surface of the covers slip,followed by the
transfer of one drop of a 24 - 48 hour suspension of
predator cell
 At four-hour intervals over72 hour,the cover glasses
were removed and acridine orange stained.
 Predators, infected prey (bdelloplasts), and uninfected
prey (i.e., a three-member population)were
enumerated.

34. The results of the present study showed for
the first time that Bdellovibrio could kill both
actively growing and VBNC cells of V. cholerae.
Bdellovibrios,the “living antibiotic” has great
potential as an agent in environmental
bioprotection water cleanup.
It can be used for environmental control of
cholera

35. Biocontrol of cholera by
Bacteriophages

36. Bacteriophages make Vibrio
cholerae deadly?

37. Bacteriophages
 Phage is a virus that infects and replicates
within Bacteria
 The term was derived from "bacteria" and the Greek
(phagein) , "to devour”
 Bacterial phages
 Archaeal phages?
 Fungal phages ?
 Viral phages ?

38. Discovery
Felix working at the Pasteur
Institute in Paris announced on 3 September
1917, that he had discovered "an invisible,
antagonistic microbe of the dysentery “.
It was then recognised that same
phenomenon was described by Twort in
1915 and by Hankin in 1896 against Vibrio
cholerae .

39. Structure and size
Size:-0.002-0.22 micrometer

40.  Types of phages against Vibrio choerae
Myoviridae
Siphoviridae
Podoviridae

41. Lytic cycle

42. Lysogenic cycle

43. Lysogenic cycle is
responsible for cholera toxin
 Vibrio cholerae is a non-toxic strain that
can become toxic, producing cholera
toxin, when it is infected with the phage
CTX φ due to lysogenic conversion

44. Lysogenic conversion
 Lysogenic growth results in integration of the phage DNA
into the host chromosome
 Most of the gene products of the lysogenic phage remains
dormant until it is induced to enter the lytic cycle.
 Some Lysogenic phage carry genes that can enhance the
virulence of the Vibrio bacteria

45. Phage inducing bacterial
toxins

46. Only lytic phages can be used
for biocontrol of Cholera

47. Video

48. How to isolate phages against Vibrio
cholerae?

49. Vibriophage sources
 Omnipresent
 Like bacteria, bacteriophages are found
everywhere
 In water the probable ratio of phages to
bacteria is 10 :1
 Phages have been found in the deserts as
well as simple top soil fields
 Sewage water :–Good source of Vibrio
phages

50. Vibriophage preparation
Water
sample
Water
sample
+
Vibrio
bacteria

51. Vibrio phage isolation
Filtration

52. Spot test

53. How do phages act on
bacterial cell wall?

54. Phages against forming Vibrio
cholerae

55. PPP
 Cocktail of three different phages isolated from surface waters in
Bangladesh and designated as JSF7, JSF4, and JSF3
 Vibriophages were tested for their host specificity towards V. cholerae O1
and O139, and the ability to disperse V. cholerae biofilms formed in the
laboratory
 A mixture of all three phages could effectively reduce both biofilm-
associated and planktonic V. cholerae strains .

56. Effect of phage JSF3 and
JSF4 on Vibrio cholerae.

57. Effect of JSF7 phage on
V.cholerae

58. Results
 Cells released from biofilms of the V. cholerae O139 strain were
resistant to JSF7 phage and were hence not killed by JSF7
 Instead of reducing the concentration of V. cholerae in water,
remarkably the action of JSF7 on V. cholerae O139 strain increased
the concentration of planktonic V. cholerae cells in water
 Biofilm associated V. cholerae cells were found to be mostly
resistant to phages JSF3 and JSF4 as compared to the
corresponding planktonic bacteria
 This resistance may be due to a lack of accessibility of phages to
the receptors on the bacterial surface or because biofilm
associated cells are metabolically less active and hence may not
support optimum phage-multiplication
 But mixture of all three phages could effectively reduce both
biofilm-associated and planktonic V. cholerae strains

59. Biocontrol of cholera by
using phages in waste water

60.  There has been less innovation in wastewater treatment
than in profit driven industries over the past 40 years.
 Phages have the potential to improve wastewater
treatment along with recreational and drinking water
sources.
 Incubating a mixture of this phage population with the
target cell sample so as to effect attachment to and
infection of target cells by test phage particles
 Incubating the target cells to complete infection by the
test phage and to cause phage progeny to be released
and recovered

61. Phages for biocontrol of cholera
in waste water

62. Challenges in utilizing phage
in waste water treatment
 High concentration of phages must be used for a
successful application.
 Use of polyvalent phages with broader host range could
lead to the degradation of useful bacteria
 Specific phages must be identified by the operator to
counter the foam forming bacteria without affecting other
bacteria
 The microbial analysis of the system is a prerequisite to
phage application as the bacterial population may vary
between wastewater treatment plants

63. Phages in
aquaculture

64. Vibrio phage application in
the aquaculture

65. FDA approved
phage
preparations

66. FDA approved phage preparations
 ListShield™
 EcoShield™
 SalmoFresh™

67. EcoShield™
 Natural product that contains three different
bacteriophages isolated from the environment
 The preparation specifically targets a foodborne
bacterium called Escherichia coli O157:H7
 Applying EcoShield™ to various ready-to-eat foods
significantly reduces (usually by 99-100%) their
contamination with this potentially deadly bacterium

68.  ListShield™ is a unique and proprietary blend of six
individual lytic phages that provides broad protection
against Listeria monocytogenes
 The active ingredients of ListShield™ are naturally
occurring bacteriophages that selectively and
specifically kill L. monocytogenes
 ListShield™ significantly reduces or eliminates
contamination levels of its targeted, susceptible L.
monocytogenes strains in various foods.

69. Salmofresh
 SalmoFresh™ is an all-natural product that contains six
different bacteriophages isolated from the environment
 The preparation specifically targets the foodborne
pathogen Salmonella enterica , which is responsible for
approximately 1.4 million infections each year in the
USA alone

70. Phage preparations against
Vibrio cholerae ?
 Cocktail of phages can be used in seafood to control
Vibrio cholerae

71. Phage approved in food, why
not as a therapeutic?

73. Immunogenicity
Phages are recognized as foreign antigens by mammalian hosts
Liver played the major role in phage elimination with over 99%
of the phages present in the circulatory system were
phagocytosed by the liver
Most studies to date were concerned with the treatment of
non-systemic diseases, such as ear and skin wound infections
, eye in the form of creams ,rinses, and as aerosol.
Very few studies in the favour of systemic usage
 No reports have been recorded of serious complications
accompanying phage therapy

74.  Each phage strain infects only one type of bacteria. The
downside of such narrow host range is the limited
applicability in cases where the identification of the
specific bacterial host cannot be achieved
 Bacteriophage cocktails are used to broaden the host
range and increase the overall therapeutic efficiency
Narrow host range

75. Regulatory approval
 Though successful application of phage therapy is in
Georgia,Russia and poland but their clinical data failed to
gain regulatory approval because such data were not
developed under the regulatory authorities frameworks
 Phages must undergo individual clinical trials and that the
composition of the approved phage cocktail cannot be
changed without re-approval. Such regulations do not take
into consideration the differences between phages and
antibiotics
 In 2009, Biocontrol Ltd Company (Nottingham, UK) has
completed randomized, double-blind, placebo-controlled,
fully regulated Phase II clinical trial for phage therapy
against Pseudomonas infections in the western world

76. Advantages Over
antibiotics
antibiotics Phage therapy
Specificity Effecting more
than targeted
oraganisms
Species or strain
specific
Side effects Allergic reactions
Intestinal disorder
etc.
No or minor side
effects
resistance Occurs and not
limited to targeted
organisms
Occurs but phage
can co –evolve
with host
production Time consuming
and expensive
Rapid

77. Phage therapy success story

78.  November 2017, a patient named Mallory Smith ,had cystic fibrosis
and chronic lung infections.
 Mallory Smith’s lung infection was caused by a strain of Burkholderia,
an opportunistic pathogen that resisted all attempted antibiotic
treatments.
 Her father desperate to save his daughter, Mallory Smith, from
an antibiotic-resistant bacterial infection sought an experimental
treatment
 He requested Dr. Steffanie Strathdee, Associate Dean of Global Health
at UC San Diego to try phage therapy.

80.  When Jessica Sacher, a University of
Alberta Ph.D. student
studying Campylobacter phages, saw the
request for phage,started working on
isolation of phages
 Finally phage therapy was given to the
patient and after removal of infection
successful lung transplant was performed

82. A scientific reason behind ganga
water purity?

83. Conclusion
 Multidrug-resistant bacteria have opened a second
window for phage therapy. Modern innovations,
combined with careful scientific methodology, can
enhance mankind’s ability to make it work this time
around.
 Phage therapy can serve as a stand-alone therapy for
infections that are fully resistant.
 It will also then be able to serve as a co-therapeutic
agent for infections that are still susceptible to antibiotics,
by helping to prevent the emergence of bacterial mutants
against either agent.

84. Infectious disease experts have warned
that there is now a immediate need to
develop totally new classes of antibacterial
agents, ones that cannot be resisted by the
same genes that render bacteria resistant
to antibiotics.