10_identification

• indentification w. high degree of accuracy

• needed for clinical microbiology to food production, phylogy/evolution

indirect / conventional methods

• isolation + culture of microorganism + determination of their various phenotypic characterstics

direct / culture-independent methods

• identification of specific microbes in a mixed population

• identification of non-culturable microbes

• Chromogenic Media

• Microscopy Techniques

• Biochemical Techniques (MS, Spectometry)

• Molecular Techniques (PCR, Ribotyping, WGS, MALDI-TOF MS)

=> chosing appropiate techniques is key

• developed by Fanny Hesse

• exponentially division -> colony building

• clonal colony -> derived from a single bacterium

• gain information by trained microbiologists

• important for diagnostics in clinic

• lot of wastage

• right medium for bacterium species growth necessary

• incubation under correct atmospheric conditions

• selectiv medium: grow + inhibit growth of other organisms

• differntial medium: differntiate closely related organisms/groups/organisms -> usage of dyes/chemicals to produce characteristic changes/growth patterns

• based on different cell wall constituents

• Gram positive bacteria stains: violet + chain like

  • thick peptidoglycan cell wall layers retaining crystal violet

• Gram negative bacteria stains: red + stick like

  • thinner peptidoglycan cell wall not retaining crystal violet during decoloring process

Cell Shapes

• Coccus

• Coccobacillus (only one Gram-positive)

• Vibrio: Gram negative (Vibrio cholerae)

• Bacillus

• Spirillum: Gram negative (H. pylori, Campylobacter jejuni)

• Spirochete: difficult to stain due to morphology (Borrelia burgdorferi, Treponema pallidum)

• can’t be used on all clinical material (e. g. not enough material available)

• needs sterile sites

• confirm identity of pathogen using another method -> poor sensitivity

• only part of identification process

• colony of unknown organism can be Gram stained

• identification of acid-fast microbes

  • Mycobacterium tuberculosis

  • Nocardia spp.

1. smear of smear

2. dry + heat fix

3. carbol fuschin over smear

4. gently heat until fumes appear + no overheating

5. 5 min wait

6. 20 % sulfuric acid + wait 1 min + repeat till light pink color

7. wash w. water

8. methylene blue + wait 2 min

9. wash w. water

10. Air dry slide + examine under oil immersion lens of light microscope

• cell wall constituents

• energy sources

• general nutritional type

• growth temperature optimum and range

• motility

• salt requirements + tolerance

• oxygen relationship

• Catalase test

• Coagulase test

• Oxidase test

• presence of enzyme catalase

  • aerobes + some facultative bacteria

  • positive for Gram-positives

• 2 H2O2 -> 2 H2O + O2

• quick

• cheap

• simple

• Staphylococcus

• looking for clots / thrombinase-like-action

• positive for Gram-positives

• Staphylococcus spp. / Staphylococcus aureus

• presence of cytochrome oxidase -> purple color

• ONLY Gram-negative bacteria

• Oxidase-negative: E. coli, Salmonella, Shigella

• Oxidase-positive: Neisseria, Pseudomonas, Vibrio, Helicobacter, Campylobacter

• usage of different kits for different organisms -> must know what you’re dealing with

• different colorings -> automated reading of results + comparision w. database

• rather expensive

• Vitek II : automated version

• primarely real-time PCR

• simultaneous detection + identification of pathogen

• from viable and non-viable cells

• knowledge of target genome required

  • design primers

  • suitable target: present in all strains of the species

    -> 16S rRNA

• translation of mRNA to protein

• present in cells of all living organisms

• coded by rRNA genes

• essential to functioning of cell -> slow change of sequence over time => detection of those changes

• identification depending on sequence approach

  • bad approach -> less resolution (missing species, genus level)

• good for mixed samples

• higher sensitivity and accuracy

• real-time DNA amplification through fluorescence intensity -> negating for post-PCR detection techniques

• (semi-) quantitative: amount of DNA quantified

• clinical infections, chronic wound tissues, GI mucosal biopsies

RAPD PCR (Random Amplification of Polymorphic DNA)

• universal random primers

• without any prior genetic information

• large number of samples

• poor reproducibility (requirement of consistent PCR amplification conditions)

RFLP (Restricion Fragment Length Polymorphism)

• unique fingerprints relying on polymorphisms

• restriction enzymes to cut DNA into fragments

• separation of different fragments to generate unique pattern

• slow + poor reproducibility

PFGE (Pulsed-Field Gel Electrophoresis)

• separation of large fragments of DNA

• for epidemiological studies

• “gold standard”

• restriction enymes targeting genomic sequence of interest

• southern blot transfer

• hybridization with probes

• analysis of ribotype RFLP bands

• requires prior knowledge of genome sequence

• fiddyl, difficult, semi-automatic

• Bavarian food diagnostic gold standrd

RiboPrinter System

• 5S, 16S, 23S sequnces w. spacer regions flanking genes on either side

• up to 8 bacterial isolates in 8 hours from sample input

• bacterial typing + evolutionary lineages

  • understanding AMR + outbreak investigations (e. g. extra toxin genes_

  • bottleneck: requires pure culture -> DNA extraction -> WGS

• increase in analysis speed + reduction of overall costs

Process

1. DNA extraction

2. DNA shearing

3. DNA Library Preparation

4. DNA library sequencing

5. DNA sequence Analysis

Platforms

• Sanger Sequncing/Dideoxy Nucleotides: mix of normal + terminator

• illumina/sequencing by synthesis: fluorescence read after each letter added

• PacBio/Single Molecule Real Time Sequencing (SMRT): read fluorescence as letter is added

• NanoPore: read changes in current as letters pass through pore

• ability of an antigen to react with an antibody

• AG: proteins/polysaccharides + stimulate immune system to produce antibodies directed against the anitgen

Antigen-antibody complexes

• Antibodies/immunoglobulins in 5 classes

• antibodies + specific antigen: inactive/destroy/remove antigen -> clumping

  => directly observed for bacterial identification purposes

• similar to Coagulase test

• need to know what it is

• visible clumping of bacterial cells -> happens = Gram positive

• initial confirmation of specific pathogens

• samples with low bacterial number -> specific + fast

ELISA (Enzyme-linked immunosorbent assay) in general

• presence of antigen/antibody

• unique antigens for all microbial species -> specific molecules of detection by ELISA

• either antigen or antibody detection identifying different bacteria strains at a time

1. direct ELISA: detection of antigen

2. indirect ELISA: detection of antibody

3. Sandwich ELISA: identification of different epitopes at a time

   • antibody-coated wall -> wash -> add antigen -> wash -> secondary Ab/enzyme-conjugated -> wash -> add substrate

4. Competitive ELISA: quantifying the antigen/antibody

   • antigen-coated wall -> wash -> add specific Ab + sample -> wash -> secondary Ab/enzyme-conjugated -> wash -> add substrate

   • high concentration Ag in sample -> low degree of coloring

   • low concentration Ag in sample -> high degree of coloring

5. Multiplex ELISA

• Lateral flow immunoassays known as immunochromatographic assay

• very useful for identification/diagnostics

  • rapid result (15 min)

  • easy operation

  • durable stability

  • low cost

• Double antibody sandwich assays / competitive assays

• fast

• reduced costs

• simplicity

• applicability for a wide range of bacteria

• automatation process

• several ionization + separation techniques coupled with MS

FTIR (Fourier transform infrared spectroscopy)

• minimal amount of sample needed

• chemical + label-free

• versatile

• fast

• non-invasive

• easy to perform

• in single measurement: detailed information about main biomolecules (lipids, proteins, carbohydrates, nucleic acids) -> stain level information

MALDI-TOF-MS (Matrix-assisted laser desorption ionization-time of lfight mass spectrometry)

• comparing to existing database by autmated program

  -> if spectrum not good enough: eyeball it

• rapid + robust identification

• identification of closely related bacterial species (at strain levels)

• done before WGS