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    PCR Based Microbial Typing

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    vg04
    This document discusses various molecular typing methods for microbial identification and classification, with a focus on PCR-based approaches. It describes using rRNA genes, especially the 16S rRNA gene, for bacterial phylogenetic analysis since they are universally present and conserved. It explains techniques like denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) analysis, and repetitive extragenic palindromic (REP)-PCR that can be used to generate DNA fingerprints for microbial strain typing and monitoring community shifts. The document also touches on challenges with culturing unculturable microbes and the potential of metagenomics to access their genetic diversity.

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    PCR Based Microbial Typing

    Uploaded by

    vg04
    25 views76 pages
    This document discusses various molecular typing methods for microbial identification and classification, with a focus on PCR-based approaches. It describes using rRNA genes, especially the 16S rRNA gene, for bacterial phylogenetic analysis since they are universally present and conserved. It explains techniques like denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) analysis, and repetitive extragenic palindromic (REP)-PCR that can be used to generate DNA fingerprints for microbial strain typing and monitoring community shifts. The document also touches on challenges with culturing unculturable microbes and the potential of metagenomics to access their genetic diversity.

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    PCR based microbial typing

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    This document discusses various molecular typing methods for microbial identification and classification, with a focus on PCR-based approaches. It describes using rRNA genes, especially the 16S rRNA gene, for bacterial phylogenetic analysis since they are universally present and conserved. It explains techniques like denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) analysis, and repetitive extragenic palindromic (REP)-PCR that can be used to generate DNA fingerprints for microbial strain typing and monitoring community shifts. The document also touches on challenges with culturing unculturable microbes and the potential of metagenomics to access their genetic diversity.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    25 views76 pages

    PCR Based Microbial Typing

    Uploaded by

    vg04
    This document discusses various molecular typing methods for microbial identification and classification, with a focus on PCR-based approaches. It describes using rRNA genes, especially the 16S rRNA gene, for bacterial phylogenetic analysis since they are universally present and conserved. It explains techniques like denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) analysis, and repetitive extragenic palindromic (REP)-PCR that can be used to generate DNA fingerprints for microbial strain typing and monitoring community shifts. The document also touches on challenges with culturing unculturable microbes and the potential of metagenomics to access their genetic diversity.

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    © All Rights Reserved
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    of 76

    PCR based microbial typing

    S. Karutha Pandian
    Department of Biotechnology
    Alagappa University
    Karaikudi – 630 003

    www.alagappabiotech.org
    Significance of Molecular typing
     Classification, Identification and typing of
    microbes are of crucial importance in hospital
    epidemiology, medical, environmental, industrial,
    agricultural microbiology and in microbial
    ecology studies.

     Successful microbial diagnostics and


    management of any outbreak organism in a
    hospital environment depends on quick and
    accurate detection, identification and typing
    methods
    Continue…
     Processing and maintenance of blood
    cultures are cumbersome

     Additionally, fastidious organisms may fail to


    grow on solid media or there may be
    insufficient growth for identification by
    standard methods
    Bacterial identification

    Phenotypic approaches

    Genomic approaches
    Phenotypic approaches
    The phenotype is the observable expression of the genotype

    Cellular (shape, endospore, flagella, inclusion bodies, Gram


    staining) and colonial (color, dimensions, form) characteristics

    The physiological and biochemical features include data on


    growth at different temperatures, pH values, salt
    concentrations, or atmospheric conditions, growth in the
    presence of various substances such as antimicrobial agents,
    and data on the presence or activity of various enzymes,
    metabolization of compounds, etc
    Biochemical Tests:
    •Gram Staining •Salt Concentration Test
    •Catalase •Spore Staining
    •Oxidase •Protease hydrolysis
    •Indole •β- galactosidase
    •Methyl Red
    •Voges Proskauer
    •Citrate
    •Nitrate Reduction
    •Carbohydrate Fermentation test
    Downside of biochemical identification

    Properties can be unstable at times and expression


    can be dependent upon changes in environmental
    conditions, e.g., growth, substrate, temperature, and
    pH levels. This can lead to less reliable identification
    results
    A huge battery of tests done at once to identify an
    unknown organism would result in a lot of media
    and time being wasted dealing with irrelevant tests
    Phenotypic identification of bacteria is inferred
    based on the phenotypic profile generated for the
    known, reference strains. However these phenotypic
    characteristics are not static and can change due to
    stress or evolution. Hence as common microorganism
    having unusual phenotypic profile or as phenotypic
    profile of unusual microorganism are not present in
    the reference database or as databases are out of date,
    reliance on phenotypes can be inaccurate
    Genomic approaches

    DNA or RNA?
    DNA is the premier choice of biomarker
    for molecular systematics because the
    Chromosomal DNA is unaffected by
    growth conditions
    Ribosomal RNA
    The prokaryotic ribosome is 70S
    The 70S ribosome comprises two subunits: a
    large 50S subunit, and a small 30S subunit.
    The 50S subunit contains a 23S and a 5S rRNA
    plus 34 proteins
    The 30S subunit contains a 16S rRNA plus 21
    proteins
    Use of ribosomal RNA in bacterial phylogenetic
    analysis

    Woese

    The most useful molecules for phylogenetic


    measurement are ribosomal RNAs, particularly the
    genes that code for them. Woese described the first use
    of ribosomal 16S rRNA gene homology in bacterial
    taxonomy in 1977
    Reasons to choose rRNA genes
     Ribosomal RNAs are essential elements in protein synthesis.
    So they present in all organisms. Hence the genes coding for
    rRNAs are conserved

     The variation in the rRNA sequence of a given pair of


    organisms is representative of the dissimilarity shown by the
    corresponding genome

     Even taxonomically distinct organisms share a degree of rRNA


    homology because rRNA gene sequences have changed very
    little over time and therefore relatedness can be assessed.
    Species level identification of closely
    related bacteria is possible because
    certain sections of rRNA evolve more
    rapidly than others

    Lateral gene transfer of rRNA is believed


    to be rare
    rRNA gene sequencing
    5S, 16S, 23S rRNA
    The information content of 5S rRNA gene is
    approximately 120-nucleotide, which is relatively
    small
    23S rRNA molecules are around 3,000 nucleotides

    For a reliable phylogenetic analysis, 1000 nucleotides


    are sufficient
    An average bacterial 16S rRNA molecule has a length
    of 1,500 nucleotides. So 16S rRNA gene is preferred
    for bacterial phylogenetic analysis
    Denaturing Gradient Gel Electrophoresis
    (DGGE)

    The method separates double stranded DNA


    based on their melting characteristics.

    When the temperature is right DNA will “melt”,


    i.e. the double helix “unzips” and becomes
    single stranded.
    The temperature at which DNA unzips
    depends on the DNA sequence - more Gs and
    Cs the higher the temperature at which the
    DNA unzips.

    To help unzip the DNA a denaturing


    agent/helix destabiliser can be added, this
    lowers the temperature at which the DNA
    melts.
    The fact the DNA would still migrate when it is
    single stranded is useless, especially because we will
    be running a PCR product of the one size. There
    would be no separation of the different PCR
    products in the mixture. So we add an extra piece
    of DNA to one end of the PCR product which
    prevents the double stranded DNA from fully
    separating. This has the effect of stopping the DNA
    from moving in the gel when it has partially
    unzipped.
    How to use DGGE in microbial community analysis ?

    Environmental DNA

    Amplification of 16S rRNA gene (Approximately 500bp) (Forward


    primer with GC Clamp)

    Because the high degree of conservation in the SSU gene PCR


    products will all have approximately the same size, but they more
    likely will differ in GC content.

    DGGE

    Individual PCR products can be separated


    Advantages of DGGE in microbial
    community analysis

    Making it feasible to monitor shifts in populations


    over periods of time or different environmental
    conditions

    It allows the simultaneous analysis of multiple


    samples

    Before the arrival of DGGE this was more commonly


    achieved by cloning and sequencing - an approach
    that is both labour intensive and relatively costly,
    especially when dealing with numerous samples.
    Advantages of DGGE in microbial
    community analysis

    A few examples include profiling of microbial soil


    communities, marine environments, hydrothermal vents,
    and the gastrointestinal tract of humans and even the
    study of a microbial community resident in a medieval
    wall
    T-RFLP (Terminal RFLP analysis)

    This profiling method is essentially an RFLP


    analysis, which uses the restriction enzymes to
    differentiate the 16S rRNA gene of microbial
    community
    How to use T-RFLP in microbial community analysis
    P
    FL
    Environmental DNA
    R
    T-

    Contain mixture of 16S


    rRNA genes
    Multiplex T-RFLP for the diagnosis of eye pathogens
    Infected sample
    (Blood, tissue, corneal scrapings etc.,)
    Can be used to detect polybacterial infections caused by different
    pathogenic bacteria in a single stretch.

    Can also be used to detect fungal pathogens since fluorescently


    labelled 18S is also used.

    The resulting peaks can be compared with the existing database to


    identify the bacterial and fungal pathogens.

    The T-RFs generated can be used to construct a database specifically


    for eye pathogens frequently occurring in South India.

    This effort will be the first of its kind at the National level.
    Advantages of M-T-RFLP:
    Detection of polybacterial infections will help in administration of
    antibiotics.
    M-T-RFLP circumvents the use of many biochemical test required
    to identify different bacteria.
    In a single experiment we can detect both bacterial and fungal
    pathogens.
    Identification of fungal pathogens is usually cumbersome.
    Comparison of resulting peaks from 18S T- RFs with the RDB
    database helps in accurate detection of fungal pathogens.
    Repetitive Extragenic
    Palindromic (REP) – PCR
    • Epidemiological tool for strain typing
    • Primers target noncoding repetitive sequences
    interspersed throughout the bacterial genome
    • Well established approach for subspecies
    classification and strain delineation of bacteria
    • Automated rep-PCR systems are more convenient
    and user-friendly
    • Consistent and reproducible DNA profiles are generated
    with multiple laboratories, personnel, laboratory
    equipment, various template DNA concentrations, multiple
    microfluidics instruments, and different culture conditions.
    Genomic DNA concentration and technical
    reproducibility by automated rep-PCR.
    Dendrogram and virtual gel images of E. coli and S. aureus isolates
    processed by three laboratory personnel using three DiversiLab systems.
    Microbial Metagenomics
    Unculturables are expected to be a vast repertoire of new
    genetic and molecular information.
    In the quest to explore valuable resources, it is important
    to assess the unexplored ‘unculturable’ microbial wealth.
    It has been estimated that 1 g of soil might contain
    between 1000 and 10000 species of unknown prokaryotes,
    and there is likely to be further diversity within each
    species.
    Epifluorescence micrography of soil microorganisms stained with
    4′,6diamidino-2-phenylindole (DAPI).

    The total bacterial count was 4.2 x 1010 cells gram–1 soil (dry weight) by
    fluorescent microscopy, and 4.2 x 106 colony-forming units gram–1 soil (dry
    weight) by plating.
    Important features of ‘unculturables’:
    Unculturables microorganisms are phylogenetically
    similar or identical to the culturable minority but remain
    in a physiological state that makes them recalcitrant to
    standard culturing methods.
    The remaining microbes represent novel lineages that are
    phylogenetically distinct from the culturables and cannot
    be cultivated in standard media.
    Cultivation techniques underestimate the great microbial
    diversity of environmental samples.
    The tremendous chemical virtuosity and use of
    unculturables have not been harnessed to their
    maximum.
    A new science has evolved that aims to characterize the
    ‘unculturable’ organisms through the use of phylogenetic
    analysis.
    For phylogenetic analysis, genes encoding the small
    subunit (SSU) rRNA molecule (16S rRNA or SSU rRNA)
    have been widely used.
    Microorganisms as a chemical repository

    Antibiotics - Tetracycline, Erythromycin,


    Vancomycin, β -lactams,

    Cephalosporins and Rifampicin.

    Industrially important - Cellulases, Amylases,


    enzymes Proteases, and Lipases

    Restriction enzymes, DNA polymerase


    Conventional procedure for screening
    industrially important microorganisms

    Growing microorganisms

    Harvesting

    Extracting cultures with solvents

    Testing the extracts


    Only a small fraction of microorganisms in any
    environment sample can be cultured by standard
    techniques.

    The methods and media available in a standard


    microbiology laboratory simply cannot reproduce the
    conditions necessary to recover the vast majority of
    bacteria in a particular environment.

    The number of species currently cultivatable


    from environment is thought to represent 1%
    or less of the total population
    Large number of unculturable and
    largely unknown prokaryotic species in the
    environment provides a promising source of
    untapped genetic diversity, with the potential
    of yielding useful natural products. These
    remarkable observations are encouraging to
    develop alternative approaches to exploit
    these resources.
    METAGENOMICS
    Meta - analysis (the process of
    statistically combining
    separate analysis)
    Genomics :Comprehensive
    analysis of an organism’s

    genetic material
    What is metagenomics?
     Culture independent genomic
    analysis of microbial communities

    Environmental genomics
    Community genomics
    Microbial population genomics
    Until now, scientists have started with a
    known species, be it human or anthrax
    bacteria, and sequenced the genome to
    learn more about it. But metagenomics
    works in reverse. It starts with the DNA
    of unknown organisms and then tries to
    figure out what the organisms are……
    Construction of Metagenomic
    library
    Analysis of Metagenomic library
     To increase the proportion of active clones in a
    Metagenomic library, several strategies have been
    designed to enrich for the sequences of interest before
    cloning.

    1.BrdU-enriched Metagenomic library construction


    2.Stable isotope probe enriched Metagenomic
    library construction
    3.Metagenomic clone library enrichment
    BrdU - Bromodeoxyuridine
    PCB - Polychlorinated biphenyls
    Equilibrium centrifugation of
    isotopically labelled DNA in
    CsCl/ethidium bromide density
    gradients. a, Pure fractions and
    a mixture of the DNA extracted
    PROVEN EXAMPLE FOR
    METAGENOMICS
    Bermuda
    Sargasso Sea
    • This site was selected because it represents an
    ecosystem with relatively low microbial diversity.

    • The site has a long history of physical and biological


    oceanography data which is all freely available on the
    World Wide Web.
    Microbial Collection from the
    Ocean
    Sargasso sea water 20um nytex netting

    0.1um filter 0.8um filter 3.0um filter

    Tangential flow filtration


    system with 50kd cartridge Viral concentrate
    Extraction of metagenome

    Construct Metagenomic library in


    pBR322 derivative vectors

    Sequence all the Metagenomic insert


    100 Million letters of genetic code every 24 hours

    J. Craig Venter Science Foundation Joint Technology


    Center
    ABI 3730XL DNA sequencers
    Gene predictions in the
    Sargasso
    • Minimum of 1800 new species

    • 1,280,663 new genes


    “In one drop of water are found all the secrets of all the
    oceans."
    Kahlil Gibran
    Opportunity to create a one time database of
    marine genomes and genes

    Sargasso samples generated over 1 billion bp of ocean


    genomes and 1.3 million new genes

    Sampling from 300 additional sites can provide


    hundreds of billions of base pairs of DNA sequence
    and up to 1 billion new genes that will permit cost
    effective analysis of ocean microbial diversity and
    environmental impact

    One time cost for DNA sequencing and database $50-


    70 Million ($15 million in hand)
    Exploiting microbial biodiversity for enhanced
    bioremediation and restoration involves the elucidation
    of :
    (i) efficient or novel catabolic pathways for improved
    detoxification,
    (ii) Suitable chemotactic or biosurfactant-producing
    strains to access insoluble or ‘aged’ pollutants easily for
    microbial degradation,
    (iii) regulatory circuits and genes for constructing suicidal
    systems.
    Microbial metabolism is now accepted as a safer and
    efficient tool for the removal of many such organic
    pollutants.
    Xenobiotics remain a challenge for environmental
    biotechnologists because they are not efficiently degraded
    by culturable microorganisms.
    The remaining unculturable microbial diversity can be
    exploited to identify desirable degradative genes and/or
    properties via the construction of metagenomic libraries
    and to transfer them subsequently to culturable bacteria
    for enhanced bioremediation.
    THANKS

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