Chapter 10Prokaryote Identification

• Microscopy– Chapter 3

• Other Phenotypic Characteristics– Chapters 4, 17

• Genomic Identification

• Strain Differences

Prokaryote Identification

Microscopy

1670sAntony van Leeuwenhoek

Microscopy

Microscopy: Stains

Gram Stain

Gram Stain: Clinical Specimen

Acid-Fast StainMycobacterium species

Cell wall composition prevents dye uptake

Red dye added heated over boiling water or concentrated dye added

Rinse

Decolorizing acid-alcohol wash

Blue counterstain

Capsule Stain / Negative Stain

Cryptococcus neoformans

Capsule (glycocalyx)Gel-like layer for protection or attachment

Distinct and gelatinousStains poorly :

Endospore Stain

Green dye added heated gently

Rinse

Red counterstain

Bacillus and Clostridium species

Endospores resistant to Gram stain

Fluorescent Dyes and Tags

Living: greenDead: red

AuramineMycobacterium cell wall

(a) Metabolic activity alters fluorescent properties of a dye (b) Fluorescent dyes bind to pathogen-specific compound (c) Dye-conjugated antibodies: recognize pathogen-specific antigens

S. pyogenes protein

Identification by Microscopy

Prokaryote Identification

Enrichment Culture

Streak Plate

Types of Growth Media

Complex vs. Defined Media

Selective & Differential Media

Selective MediaInhibits growth of all but target organism

Differential MediaTarget bacteria changes in a recognizable way

MacConkey AgarIsolate/identify Gram negative rods from the intestine

Selective: bile salts/crystal violet dye inhibit growth of all but Gram negative rodsDifferential: lactose fermenting bacteria produce pink colonies (pH indicator)

Differential Media: Blood Agar

Differential media detects bacteria that lyse red blood cells (hemolysin production)Red blood cell lysis generates a zone of clearing (a) clear zone = beta hemolysis = S. pyogenes (b) greenish zone = alpha hemolysis = non-pathogenic Streptococcus

Prokaryote Identification

Biochemical Tests

Biochemical Tests

Catalase testbubbles = catalase activity

Sugar fermentation testGas and acid production = fermentation activity

Urease testGas and ammonia (base) production = urease activity

Dichotomous Key

Biochemical Tests:Commercial Arrays

API strip testadd liquid culture to dehydrated media

EnterotubeInnoculation from rod drawn through liquid compartments

Other formats: 96 well plates, Miniaturized systems

Prokaryote Identification

Serology:1. Recognition of host antibody produced in response to bacterial antigen2. Direct recognition of bacterial antigen

SerologySeronegativeNo specific antibodies to a pathogenbecause no prior exposure

SeropositiveProduction of specific antibodiesto a pathogen

SeroconversionSwitch from seronegative to positive

SerologyIn vitro study of antibody-antigeninteractions

SerumFluid portion of the blood that remains after blood clots

Serology: Indirect ELISAELISAEnzyme-Linked Immunosorbent Assay

Bacterial antigen affixed to substratePatient serum added if antibody vs. antigen present, it will stay in the wellPeroxidase-conjugated anti-human IgG antibody added color reaction = antibody in serum recognizes antigen

Testing for antibody production in response to antigen

Often used as HIV test (anti-gp120, anti-p24 antibodies)

“Indirect” because second antibody required for signal

Western Blot Analysis

To confirm positive HIV ELISA result

HIV proteins run on SDS-PAGE gel protein separation based on sizeSeparated proteins transferred to membrane Test serum addedEnzyme-labeled secondary antibody added

Direct ELISA

Known antibodies vs. antigen attached to substratePatient sample added if bacterial antigen is present, it will stay in the wellPeroxidase-conjugated antibody added

Direct testing for presence of antigen

“Direct” because antibody directly conjugated to peroxidase

ELISA plates

96 well format for ELISA, other high-throughput protocols

Antibody titer (concentration): serial dilutions of serum testedreciprocal of last dilution with positive reaction = titer (1:256 dilution = 256 titer)

Fatty Acid Analysis

Bacteria differ in the type and quantity of membrane fatty acids

Fatty acids removed and converted to methyl ester form (Fatty Acid Methyl Ester)Gas chromatography analysis

Prokaryote Identification

Nucleic Acid Hybridization

Polymerase Chain Reaction

Nucleotide sequence must be known for primer design

Polymerase Chain Reaction

Polymerase Chain Reaction

30 cycles = billion-fold amplification

Prokaryote Identification: PCR

Could also use DNA probe instead of gel

Requires knowledge of a specific nucleotide sequence from the target pathogen

rRNA Sequencing

Ribosomes protein + rRNA components S = Svedberg unit measure of sedimentation

mRNA translation & protein synthesis important / conserved process

Sequence rRNA directlySequence DNA that codes for rRNA

Usually 16S rRNA sequenced

Strain Differences

E. coli strainsK-12

EPECEHECSTECETECEIEC

EAggEC

Strains: related, but not identical, isolates of a species

Detecting Strain Differences

Biochemical Typing

• Some bacterial strains can be differentiated with biochemical tests

• Biovar/Biotype:strain with a characteristic biochemical pattern

• Vibrio cholerae:– Pandemics 1-6 = Classical Biotype– Pandemic 7 = El Tor Biotype

Serological Typing

• Strain differentiation based upon antigenic (protein/carbohydrate) differences

• Serovar/serotype: strain with characteristic antigen pattern

• V. cholerae O1 and O139 (LPS O region)

• E. coli O157:H7– 1st antigen = carbohydrate (LPS O region)– 2nd antigen = protein (flagella)

Genomic Typing

RFLP: Restriction Fragment Length Polymorphism

Strain differences based on subtle differences in DNA sequence

Genomic Typing

PulseNet: national database of RFLPs from foodborne pathogens

Phage Typing

Strain differences based on bacteriophage susceptibility patterns

Antibiograms

Strain differences based on antibiotic susceptibility patterns

SummaryProkaryote Identification / Classification

• Microscopy– Chapter 3

• Other Phenotypic Characteristics– Chapters 4, 17

• Genomic Identification

• Strain Differences