Bacteriocin and its typing

Shyam Kumar Mishra

Shyam Kumar MishraAssistant Professor,

Department of MicrobiologyInstitute of MedicineTribhuvan University

Some definitions

Shyam Kumar Mishra

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

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 diseaseE.g. Common cold

Shyam Kumar Mishra

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

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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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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• Typing: Typing: characterization

• Properties: Properties: Typeability

ReproducibilityReproducibility

Discriminatory powerDiscriminatory power

PracticalityPracticality

Ease of interpretationEase of interpretation

Shyam Kumar Mishra

Shyam Kumar Mishra

Shyam Kumar Mishra

• 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

Shyam Kumar Mishra

• 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

Shyam Kumar Mishra

Bacteriocin typing

Shyam Kumar Mishra

Bacteriocin Typing

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

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

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

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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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

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 are By 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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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

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, ᴼthe 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.

Shyam Kumar Mishra

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.

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.

Shyam Kumar Mishra

Colicin typing (Djonne)

- Spot cultures are grown on plates of blood agar base at 37 C for 48 ᵒhours 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

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

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

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 (+).Shyam Kumar Mishra

FIG. 2. Typical routine typing plates obtained by the spotting

method. (A) Indicator strain; (B) indicator strain B.

Shyam Kumar Mishra

Thank you