SEROTYPING, GENOTYPING & PHAGETYPING LECTURE

HOZA, A.S

BLS 206

•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 usingphenotypic 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 proceduree.g.

– Fungus Cryptococcus neoformans (capsule staining)

– Mycobacterium tuberculosis (acid-fast stain).

Identification of prokaryotes usingphenotypic 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 differencesUse 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 typingCommon 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.