SEROTYPING, GENOTYPING & PHAGETYPING

LECTURE

HOZA, A.S

BLS 209

•Identification of prokaryotes using phenotypic

characteristics

• Identification of prokaryotes using genotypic

characteristics

• Characterizing strain differences

Identification of prokaryotes

A.Identification of prokaryotes using phenotypic

characteristics

1. Microscopic Analysis

• An important step is to determine:

• size, shape and staining characteristics of a

microorganism.

• Microscopic examination sometime gives

information enough to make a presumptive

identification.

• Examples:

• Trichomonas (protozoa) in vaginal secretion

• Round worms eggs in stool can be

identified based on their shape and size

under the microscope.

Identification of prokaryotes using

phenotypic characteristics

•Gram stain is a differential method.

•Gram stain of a specimen by itself - generally not

sensitive and specific enough to diagnose the cause

of most infection,

•but very useful tool in narrowing the possible

identities of an organism.

• In certain cases, it gives enough information to

start appropriate antimicrobial therapy while waiting

more accurate identification.

•Certain microorganisms have unique characteristics

that can be detected with special staining procedure

e.g.

– Fungus Cryptococcus neoformans (capsule

staining)

– Mycobacterium tuberculosis (acid-fast stain).

Identification of prokaryotes using

phenotypic characteristics

Metabolic differences

Metabolic differences include

– culture characteristics

– selective media and

– biochemical tests.

1. Culture characteristics

•Colony morphology:

•can give initial clues for the identification of

certain microorganism.

– Colonies of Streptococci are generally

fairly small relative to many other bacteria.

– Pseudomonas aeruginosa often produces

a soluble green greenish pigment which

discolors the growth media and has a

distinct fruity odor.

Metabolic differences

Metabolic differences

Use of selective and differential media

•Blood agar media: differential media.

– Beta-hemolytic colonies are characteristics of

Streptococcus pyogenes.

•MacConkey agar: both selective and differential media.

– It inhibits the growth of most Gram positive bacteria

and Gram negative cocci.

– It has bile salts which inhibits the growth of most

nonintestinal organisms thus usually it is used to

select intestinal gram negative bacteria.

– It also differentiates lactose fermenting bacteria

from nonlactose fermenter

e.g. E. coli a lactose fermenter, forms

characteristic pink colonies on MacConkey

agar.

MacConkey agar:

Biochemical tests

•Culture characteristics can narrow the number of

possible identities of bacteria.

•but biochemical tests are generally necessary for a

more conclusive diagnosis.

Catalase:

• nearly all bacteria which grow in the presence of

oxygen are catalase positive.

• Catalase positive bacteria break down hydrogen

peroxide to release oxygen gas which cause

bubbling.

• Important exception are lactic acid bacteria which

include Streptococcus.

•Thus if a throat culture has beta-hemolytic

colonies on blood agar but are catalase positive,

then Streptococcus pyogenes is ruled out.

+ve -ve

Biochemical tests•Most biochemical tests rely on a pH

indicator or chemical reaction that results

in color change when a compound is

degraded e.g.

• Fermentation of sugar results in

acid production, which lowers the pH,

resulting in a color change from pink

to yellow and gas production.

• No color change (central tube)

indicate that sugar is not used.

• A medium designed to detect urease

enzyme that degrades urea to produce

carbon dioxide and ammonia, utilizes a

different pH indicator that turns bright

pink in alkaline conditions.

Biochemical tests

• The basic strategy for identifying

bacteria based on biochemical test

relies on the use of a dichotomous key,

which is a flow chart of tests that give

either a positive or negative result.

• The biochemical tests are usually

initiated simultaneously to speed

identification.

•In certain cases, biochemical test can be done

without culturing the organism e.g.

-Breath test which assays for the presence of

urease is done to detect Helicobacter pylori

•Commercial modifications of traditional biochemical

tests:

•e.g. API test strip, enterotube and Biolog

microtiter plate methods.

Biochemical tests

Serology•In some cases, proteins and polysaccharides present on the

surface of the bacterium are considered as identifying markers.

•The most useful of these are the molecules that make up

surface structures including the cell wall, glycocalyx, flagella

and pili.

•Antibodies directed against surface proteins and

polysaccharides are frequently used to identify various

bacteria.

•Methods which use antibodies for the detection of antigens are

called serology.

•Some serological tests such as used to identify Streptococcus

pyogenes are quite specific, simple and rapid.

Fatty acid analysis (FAME)

•Bacteria differ in the type and relative quantity of fatty acids

that make up their membranes. Thus cellular fatty acid

compositions can be used as an identification marker.

•The bacterial cells are grown under standardized conditions

and then chemically treated with sodium hydroxide and

methanol to release fatty acids and to convert those acids to

their more volatile methyl ester form (FAME stands for fatty

acid methyl ester).

•FAME are analyzed by gas chromatography.

•By comparing the pattern of peaks, or chromatogram, to

those of known species, an isolate can be identified.

Genotyping

•Identification of prokaryotes using genotypic characteristics

•Genotypic characteristics are used in the identification of

microorganisms particularly which are difficult to cultivate.

•Nucleic acid probes: are used to detect specific nucleotide

sequences that characterize a particular species of

microorganism.

•Fluorscence in situ hybridization (FISH) is increasingly being

used to identify intact microorganisms in environmental and

clinical samples.

•By using the rRNA specific probes, either specific species or

groups of related organisms can be identified

Polymerase chain reaction (PCR)

• PCR can be used to amplify specific nucleotide sequences of

microorganisms from samples such as body fluids, soil, food,

and water.

• This technique can be used to detect microorganisms that are

present in extremely low numbers as well as those can not be

grown in culture.

• In order to use PCR to detect microorganism of interest, a

sample should be first treated to release and denature DNA.

• All ingredients needed for PCR along with specific primers

known and designed for particular microbe are then added.

• After ~30 cycles of PCR, sufficiently amplified DNA fragment

is visualized as discrete band on an ethidium bromide stained

agarose gel.

• Alternatively, a DNA probe can be used to detect the amplified

DNA.

Sequencing ribosomal RNA genes

•Ribosomal RNA genes (DNA sequences) are highly

conserved, and can be used to identify organisms.

•This method is particularly useful for identification of those

prokaryotes which are difficult or currently impossible to grow

in culture.

•Three different rRNAs: 5S, 16S, and 23S.

•Some regions of 16S rRNA are virtually same in all

prokaryotes whereas others have quite variable sequence and

this variable region is used to identify an organism.

•In certain cases, 16S rDNA is used to identify uncultivable

organisms.

• In some situations it is useful to distinguish among different

stains of bacteria especially when only certain strains cause

disease.

• Example: only certain strains of E. coli cause intestinal disease as

only a few strains have the virulence factors such as toxin production

etc.

• Detecting strains differences is also helpful in tracing source

of an outbreak.

• The following methods are used for charactering various

strains

– Biochemical typing

– Serological typing

– Genomic typing

– Phage typing

– Antibiograms

Characterizing strain differences

Biochemical and serological typing

1. Biochemical typing

•Biochemical tests are mainly used to identify various

species of bacteria but they can also be used to distinguish

strains.

•A strain that has characteristic biochemical pattern is

called a biovar or biotype.

2. Serotyping

•Serological procedures used to differentiate strains

[serovars, serotypes] of microorganisms that differ in the

antigenic composition of a structure or product.

•Important in identifying a pathogen out of the group,

primarily using cell wall antigens.

Examples: Lancefield system to identify streptococci.

–Serotypes are identified by a letter A-O and is based on

specific antibody agglutination reactions with cell wall

carbohydrate [Polysaccharide O}.

–Further subdividing of Group A Strep based on specific

M protein antigens can also be performed.

•Other examples of serotyping:

– Identifying specific antigen-antibody reactions involving Flagella

[H] antigens,

–Capusular [K] antigens, and

–Cell wall [O] antigens.

2. Serotyping

2. Serological typing

•Proteins and carbohydrates that vary among strains can

be used to differentiate strains.

•Example: 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.

•A strain that varies serologically from other strains is

sometime called a serovar or a serotype.

3. Genomic typing

•Molecular methods can be used to detect genomic variations

that characterize certain strains.

•In some cases these differences include genes that encode

for toxins or other proteins related to disease.

•Example: E. coli O157:H7. The toxin gene can be detected

using a probe that consists of a specific nucleotide

sequence unique to that gene.

•Subtle differences in DNA sequences can be used to

distinguish among strains that are phenotypically identical.

This helps in tracing epidemics of foodborne illnesses.

•One method of genomic typing is to compare the pattern of

fragment sizes produced when the same restriction enzyme is

used to digest DNA from each organism.

•When the lengths of restriction fragments vary among

organisms, it is termed ‘restriction fragment length

polymorphisms’ (RPLFs).

3. Genomic typing

3. Genomic typing

Common methods used to look for RFLPs :

1. Pulse-field gel electrophoresis:

•The bands can be visualized by staining gel with ethidium

bromide.

2. Ribotyping:

•Uses a restriction enzyme that cuts genomic DNA into

many small fragments.

- As bacteria usually have several different rRNA

genes, the probe hybridizes to several different

restriction fragments, the pattern of which varies

among strains.

- Southern blot hybridization is then done using a probe

that hybridizes to only those fragments that have

sequences encoding ribosomal RNA.

Phage typing

•Strains of a given species sometimes differ in their

susceptibility to various types of bacteriophages.

•The susceptibility of an organism to a particular type of phage

can be readily demonstrated in the laboratory.

•The patterns of clearing around the bacteriophage spot

indicate the susceptibility of the test organism to different

phages.

•Different patterns are compared to determine strain

differences.

•Bacteriophage typing is largely replaced by molecular

methods.

Phage typing

plaque assay

Antibiograms

•Antibiotics susceptibility patterns or antibiograms, are also

used to distinguish among different strains.

•Again this method has largely been replaced by molecular

techniques.

•Different strains will have different patterns of clearing

around antibiotics disks.