1. Bacteriocin and its typing
Shyam Kumar Mishra
Shyam Kumar Mishra
Assistant Professor,
Department of Microbiology
Institute of Medicine
Tribhuvan University

2. Some definitions
Shyam Kumar Mishra

3. WORLD HEALTH ORGANIZATION-
Definition
• Epidemiology is the study of the distribution and
determinants of health-related states or events
(including disease), and the application of this study
to the control of diseases and other health problems.
• Various methods can be used to carry out
epidemiological investigations: surveillance and
descriptive studies can be used to study distribution;
analytical studies are used to study determinants.
Shyam Kumar Mishra

4. Endemic
• Constant or usual presence of an infection or
disease within a given geographic area or
population group, without importation from
outside
• Expected frequency of disease
E.g. Common cold
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5. Epidemic
• “Unusual” occurrence in a community or region, of
disease, specific health-related behavior or other
health-related events clearly in excess of “expected
occurrence”.
• An epidemic or outbreak is said to exist when an
unusual number of cases of a disease occurs in a
given time period and geographic area as compared
with the previous experience with that disease in
that area.
Shyam Kumar Mishra

6. Pandemic
• An epidemic usually affecting a large
population, occurring over a wide geographic
area such as a section of a nation, the entire
nation, a continent or the world
– E.g. influenza pandemics
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7. Index case
• Index or primary case of an illness in a family,
group, institution or community that may
serve as a source of infection to others.
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8. • Typing:Typing: characterization
• Properties:Properties: Typeability
ReproducibilityReproducibility
Discriminatory powerDiscriminatory power
PracticalityPracticality
Ease of interpretationEase of interpretation
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9. Shyam Kumar Mishra

10. Shyam Kumar Mishra

11. • Methods: Phenotypic / GenotypicMethods: Phenotypic / Genotypic
• Phenotypic methods:
 Characterize the products of gene expression to
differentiate strains
 Tendency to vary
– Biotyping
– Antibiogram
– Serotyping
– Bacteriophage typing
– Bacteriocin typing
– Dienes phenomenon
– Bettelheim reaction
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12. • Limitations:Limitations:
 Inability to grow certain fastidious pathogens
 Inability to maintain viability of certain pathogens in
specimens
 Extensive delay in cultivation and identification of
slowly growing pathogens
 Lack of reliable methods to identify certain organisms
grown in vitro
 Use of considerable time and resources in establishing
the presence and identify of pathogens in specimens
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13. Bacteriocin typing
Shyam Kumar Mishra
Bacteriocin
Typing

14. Introduction
Bacteriocins are bactericidal antibiotic like substances.
Apparently protein in nature which are produced by many
bacteria.
Have a killing action on strains of the same or closely
related species.
The narrow specificity of their action and their protein
nature distinguish them from other(classical) antibiotics.
The first discovery was reported by Gratia in 1925 of a
highly specific antibiotic produced by E. coli and active against
other strains of the same species.
Shyam Kumar Mishra

15. Since colicin like substances are produced by several other
bacteria also, the generic name bacteriocin was proposed by Jacob
et al (1952) for the group of highly specific antibiotic like substances
produced by certain strains of the bacteria which are active against
other strains.
Bacteriocins are given specific names based on the bacterial
species of origin, for example
Colicins - E. coli
Pyocins - P.aeruginosa
Diphthericins - C.diphtheriae
Shyam Kumar Mishra

16. Specific properties of bacteriocin
 They have a narrow inhibitory spectrum of activity
centered about the homologous species.
 They have an essential, biologically active protein
moiety.
 They have a bactericidal mode of action.
 There is attachment to specific cell receptors.
 The genetic determinants for bacteriocin production are
plasmid-borne.
Shyam Kumar Mishra

17. Reeves (1965) listed 16 classes of bacteriocins named on
the basis of the species they produced, e.g.,
-Colicins are bacteriocins of E.coli,
-Pyocins of P. aeruginosa,
-Cloacin of Enterobacter cloaceae,
-Pesticin of Y. pestis,
-Monocin of Listeria monocytogenes,
-Cerecin of Bacillus cereus
-Staphylococcin of Staphylococcus aureus.
Classification of Bacteriocins
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18. Medical significance of
Bacteriocins
- Bacteriocins produced by non-pathogenic bacteria kills
other pathogenic bacteria. (Normal flora vs. Pathogens)
- Bacteriocins have also been suggested for certain cancer
treatment.
- Bacteriocins have gained new attention particularly in the
epidemiology of nosocomial infections by bacterial typing.
Shyam Kumar Mishra

20. Methods of Bacteriocin Typing
By determining the bacteriocin production pattern of
a strain against a set of standard indicators.
By strain against a set of bacteriocin which areBy strain against a set of bacteriocin which are
applied to it.applied to it.
- Each method has been used in epidemiology to determine whether the
isolate from different sources are the same or not.
- If the isolates are same strains, their bacteriocin production and
susceptibility patterns will be identical.
- The bacteriocin typing technique has been widely used to answer the
epidemiological questions in the nosocomial infectionsepidemiological questions in the nosocomial infections..
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21. Contd….
Stab and overlay method (Fredricq)
-The test strains are stabbed in pre-poured BHI agar or nutrient agar
plates.
- Incubated at 37 C for 24 hours.ᴼ
- The plates are exposed to chloroform to kill the producing strain. For
this, a chloroform soaked filter paper is placed at the lid of the plate
and allowed to stand for 10-15 minutes.
- 3 ml of BHI soft agar or nutrient agar containing 0.1 ml of
standardized inoculum (2x108
cfu/ml) of sensitive (indicator strain)
culture is poured over the plates and incubated at 37 C for overnight.ᴼ
- After overnight incubation, zones of inhibition (ZOI) around producer
colonies are measured and documented.
Shyam Kumar Mishra

22. Contd….
Cross streak method
- The test strains are inoculated across the surface of BHI agar
plates.
- After overnight incubation at 37 C and exposing to chloroform,ᴼ
the test (producing) strains are scrapped-off from the plate.
- The sensitive strains (Indicator strains) are cross-streaked at
right angle to the test strains.
- Incubated at 37 C for 24 hours.ᴼ
- Observed for inhibition of growth at each side of the producing
strain.
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23. Contd….
Agar well diffusion method
- Pre-poured BHI agar plates are
overlaid with 3ml of BHI soft agar
containing 0.1 ml (2x108
CFU/ml) of the
sensitive (indicator) culture.
- Wells (5 mm in diameter) are cut into
these agar plates.
Shyam Kumar Mishra
- 100μl of the culture supernatants (Bacteriocin) are placed into each well and kept
at 4 C for 10-12 hours to allow the bacteriocin to diffuse into the agar.ᵒ
- The plates are then incubated at 37 C for 24 hours.ᵒ
- ZOI are measured in mm diameter.

24. Colicins
- Colicins are bacterial macromolecules which have narrow spectrum of
activity and they are produced by some strains of E. coli and related
Enterobacteriaceae.
- They are specified by col plasmid.
- They are synthesized in the cytoplasm of colicinogenic cells and released
into the extracellular medium.
- After being released into the medium, colicins kill sensitive bacteria in 3
defined steps:
1- Adsorption on to a specific receptors on the surface of the bacterium.
2- Translocation across the outer membrane.
3- Killing activity.
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25. Colicin typing (Djonne)
- Spot cultures are grown on plates of blood agar base at 37 C for 48ᵒ
hours and killed by exposure to chloroform vapour.
- Leave at room temperature (RT) for 30 minutes to remove residual
chloroform.
- One colony of universal indicator strain a1, grown on blood agar, is
mixed with 5 ml of 0.9% NaCl and then overlaid on sterilized (with
chloroform) plates.
- After leaving for 1-2 hours at RT, they are incubated for 24 hrs at
37 C.ᵒ
- If the inhibition zone is >1mm around the spot culture, colicin
production is considered to be positive.
Shyam Kumar Mishra

26. Pyocin Typing
- Several biological criteria have been assessed for typing
Pseudomonas aeruginosa, including
-pigmentation,
-antibiograms and
-phage sensitivity.
However the two most reliable and generally accepted methods
are serotyping and pyocin typing.
- In pyocin typing technique with cross streaking method, 105 main
types and 25 subtypes can be identified on the basis of pyocin
production by test strains using 13 indicator(1 to 8 and A to E).
Shyam Kumar Mishra

27.  48 hours required to obtain the result.
 The need to remove the producer strain growth before
application of the indicator strains, which is time consuming.
 The inability to reliably type mucoid strains of P. aeruginosa.
Cross-
streaking

28. FIG. 1. Examination for pyocin activity against indicator strain in four
strains of P. aeruginosa by the spotting method.
Strain A shows no pyocin activity (-);
Strains B, C and D show pyocin activity (+).
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29. FIG. 2. Typical routine typing plates obtained by the spotting
method. (A) Indicator strain; (B) indicator strain B.
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30. Thank
you