Epidemiological marker (serotyping and bacteriocin typing)

Santosh Kumar Yadav
Dept. of Clinical Microbiology (M.Sc.)
IOM, TUTH
Epidemiological Marker
(Serotyping & Bacteriocin typing)

Introduction
 Important to analyse multiple isolates within a given
species to determine whether they represent a single
strain or multiple strains.
 If a species of bacteria is isolated and cultivated in the
laboratory it is known as a strain.
 A single isolate with distinctive characteristic[s] may also
represent a strain.
 Members of the same species that have small differences
between them can be distinguished by additional
methods. These species is then subdivided into
subspecies, subgroups, biotypes, serotypes, variants etc.
 The process of differentiating strains based on their
phenotypic and genotypic differences is known as
'typing'.

 These typing methods are useful in hospital infection
control, epidemiological studies, and understanding the
pathogenesis of infection.
 In hospital settings they may be used to:
 determine whether a set of isolates obtained from one
patient represents a single infecting strain or multiple
contaminants.
 determine whether a series of isolates obtained over
time represents relapse of an infection due to single
strain or separate episodes of disease due to different
strains.

Criteria for evaluating typing systems:
1. Typeability: It is the ability to obtain unambiguous
positive result for each isolate analysed. Non-
typeable isolates are those that give either a null or
an uninterpretable result.
2. Reproducibility: It is the ability of the technique to
yield the same result when same strain is tested
repeatedly.
3. Discriminatory Power: It is the ability to
differentiate among unrelated strains. Ideally, each
unrelated isolate is detected as unique.
4. Ease of performance: To be widely useful, a typing
method should be applicable to a broad range of
microorganisms as well as inexpensive and
technically easy.
5. Ease of interpretation: The typings tests must
produce reproducible and unambiguous results that

Types of typing methods:
1. Phenotypic techniques, those that detect
characteristics expressed by the microorganism and
2. Genotypic techniques, those that involve direct DNA-
based analysis of chromosomal or extrachromosomal
genetic elements.

Serotyping
 Based on fact that strains of same species can differ in
the antigenic determinants expressed on the cell
surface.
 Surface structures such as lipopolysaccharides,
membrane proteins, capsular polysaccharides, flagella
and fimbriae exhibit antigenic variations and antibodies
raised against them can be used to group isolates into
defined serotypes.
 Strains differentiated by antigenic differences are known
as 'serotypes'. Serotyping is used in several gram
negative and gram positive bacteria.
 Some species are characterized by numerous antigenic
types and serotyping for these species is highly
discriminatory,

 Serotyping is performed using several serologic tests
such as bacterial agglutination, latex agglutination,
co-agglutination, fluorescent and enzyme labelling
assays.
 The following are the antigens used for developing
antibodies for serotyping.
 "O" antigen- cell wall lipopolysaccharide antigen
 Vi antigen
 "H" antigen- protein antigen of flagella
 "K" antigen- capsular polysaccharide antigen

For example,
 Salmonella enterica - more than 2300 serotype based
on "O" antigen, Hantigen and Vi antigen (in Kauffman-
White scheme)
 E. coli vary in the antigenic structure of certain parts of
the LPS portion of the cell wall, the O antigen. The
composition of the flagella, the H antigen, can also
vary..The strain designation of E. coli O157:H7 refers to the
structure of LPS and its flagella
 Campylobacter jejuni- 55"O" antigens, 56"H" antigens,
80 "K" antigens
 Klebsiella pneumoniae- 75 "K" antigens
 Yersinia enterocolitica- 34 "O" antigens
 Citrobacter freundii- 32 "O" antigens, 75 "H" antigens
 Capsules, M and T proteins- Streptococcus pyogenes
 Toxins- Clostridium spp.

Advantages:
 Most strains are typeable. They have good
reproducibility and ease of interpretation though some
have ease of performance.
Disadvantages:
 Some autoagglutinable (rough) strains are untypeable.
Some methods of serotyping are technically demanding.
 There is dependency on good quality reagent from
commercial sources.
 In-house preparation of reagents is difficult process.
 Serotyping has poor discriminatory power due to large
number of serotypes, crossreaction of antigens and
untypeable nature of some strains.

BACTERIOCIN
11
 Produced by many bacteria, protein in nature and
have bactericidal activity.
 Have killing action on strains of same or closely
related species.
 First reported by Gratia in 1925, Escherichia coli
producing a substances which is active against other
strains of the same species.
 Colicin – Gratia and Frederique in 1946.
 Bacteriocin- Jacob and Woolman in 1953.

contd…
12
 Are named on the basis of their bacterial species of
origin . Some of them are
i) Colicins are bacteriocins of E.coli,
ii) Aeruginocin of P. aeruginosa,
iii) Diphthericins - C. diphtheriae
iv) Cloacin of Enterobacter cloaceae,
v) Pesticin of Y. pestis,
vi) Monocin of Listeria monocytogenes,
vii) Cerecin of Bacillus cereus
viii) Staphylococcin of Staphylococcus aureus,
ix) Warnerin of et S. warneri

contd…
13
 Target cells have specific receptor for
attachment of bacteriocin( same as
bacterophage)
 Bacteria producing bacteriocin also carry
gene for immunity to them on chromosome or
plasmid.
 Many have narrow inhibitory spectrum of
activity ( but some have activity on broad
class of bacteria)
 Are plasmid or chromosomal mediated.

Importance
14
 Bacteriocins produced by non-pathogenic
bacteria kills other pathogenic
bacteria(Normal flora vs. Pathogens).
 Bacteriocins have also been suggested for
certain cancer treatment.
 Used for food presrvation in food industry (
eg..nisin produced by Lactococcus lactis is
active against many food spoiling bacteria)
 Used for bacteriocin typing of clinical strains
to aid in their identification and
characterization.

Bacteriocin typing
15
 A strain may be typed by:
1) Activity of their bacteriocin against a set of indicator
strains of same or closely related species, or
2) By strain against a set of bacteriocin which are
applied to it.
 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 infections.

Stab and overlay method
(Fredricq)
The test strains are stabbed in pre-poured BHI agar
or nutrient agar plates.
 Incubated at 37ᴼC for 24 hours.
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 37ᴼC for overnight.
After overnight incubation, zones of inhibition (ZOI)
around producer colonies are measured and

Cross streak method
The test strains are inoculated across the surface of
BHI agar plates.
After overnight incubation at 37ᴼC and exposing to
chloroform, 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 37ᴼC for 24 hours.
Observed for inhibition of growth at each side of the
producing strain.

Preparation of cell free neutralized supernatant
(bacteriocin)
 Producer strain is grown overnight at 37ᵒC in trypticase
soya broth.
 0.25 ml of chloroform (for each 5ml of broth) is added to
kill the organisms.
 1ml of mitomycin C can be added to induce the
production of bacteriocins.
 Supernatant fluid is obtained by centrifuging at 10,000g
for 10 minutes.
 pH of supernatant is adjusted to 7.0 with sterile 1N
NaOH.

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.
 100μl of the culture
supernatants (Bacteriocin) are
placed into each well and kept
at 4ᵒC for 10-12 hours to allow
the bacteriocin to diffuse into
the agar.
 The plates are then incubated
at 37ᵒC for 24 hours.

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.

Colicin typing (Djonne)
 Spot cultures are grown on plates of blood agar
base at 37ᵒC for 48 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 37ᵒC.
If the inhibition zone is >1mm around the spot
culture, colicin production is considered to be
positive.

 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.
Pyocin typing

23
Gillies and Govan(1966)- cross streaking
method
The test strains are inoculated across the
surface of BHI agar plates.
After overnight incubation at 37ᴼC , culture is
exposed to chloroform to kill the test strains and
then the test (producing) strains are scrapped-
off from the plate by using slide.
 The sensitive strains (Indicator strains) are
cross-streaked at right angle to the test strains.
Incubated at 37ᴼC for 24 hours.
Observed for inhibition of growth at each side of
the producing strain.

Contd…
24
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 indicators(1 to 8 and A to E).
Disadvantages of streak method
 To remove test strains growth before application
of indicator strain.
 Not reliable for mucoid colony of P. aeruginosa.
 48 hour period is needed to obtain result.

Klebocin typing
 Spot inoculation method as described by Chugh et al(1980)
using 6 producer strains 153,154,155,156, US-5 and US-6.
 Each producer strain is cultured in a broth.
 To each broth culture(0.7ml) of producer strains, 3.3 ml of
trypticase soya broth and 1ml of mitomycin C(0.5µgm/ml) is
added and incubated at 37C for 5 hours with intermittent
shaking.
 The cells are killed by addition of 0.25 ml of chloroform.
 The suspension is centrifuged at 3000rpm for 10 minutes in
cold centrifuge.
 Supernatent containing respective klebocin is aliquoted in
screw cap vials and stored at -20C.

 The test culture is swabbed over a nutrient agar plate.
 12 µl of each klebocin is spot inoculated on marked
sectors with a micropipette.
 A positive control plate streaked with strain WC-50 is
also put up with every set of test.
 The negative and positive reactions to 6 klebocins are
recorded.

Advantages:
 This technique has fair amount of reproducibility,
discriminatory power and ease of interpretation.
Disadvantages:
 This technique is available only at reference centres.
 Even for the experienced worker, the technique is
demanding
 Many strains are non-typeable.

Epidemiological marker (serotyping and bacteriocin typing)

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