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Recent advances in Microbiology

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Title: Recent advances in Microbiology


1
Recent advances in Microbiology
  • Dr.Mrs.N.ANBUMANI
  • M.B.B.S,M.D.,PG Dip in BI., Ph.D
  • ASSOCIATE PROFESSOR
  • DEPARTMENT OF MICROBIOLOGY
  • SRI RAMACHANDRA UNIVERSITY, PORUR,CHENNAI-600116.

2
Introduction
  • Clinical microbiologists have traditionally been
    concerned with the isolation and identification
    of pathogenic organisms from humans.
  • Conventional methods involve isolating the
    organism of interest in pure culture and
    performing predetermined biochemical or
    immunologic tests to identify it.

3
  • In many respects, cultures and the adjunct
    methods used for identification are limited in
    sensitivity, specificity, or both.

4
  • To improve test sensitivity, shorten detection
    times, and identify hard-to-culture
    microorganisms, immunoassays were developed.
  • These immunoassays allowed both large and small
    laboratories to expand services to meet
    diagnostic requirements in a timely fashion.
  • The outlook for even more sensitive, more
    specific, and more rapid testing is currently
    being founded in the recent advances in molecular
    biology methods.

5
  • Historically, this analysis of pathogens has
    relied on a comparison of phenotypic
    characteristics such as biotypes, serotypes,
    bacteriophage or bacteriocin types, and
    antimicrobial susceptibility profiles.

6
  • This approach has begun to change over the past 2
    decades, with the development and implementation
    of new technologies based on DNA, or molecular
    analysis.

7
  • These DNA-based molecular methodologies include
    pulsed-field gel electrophoresis (PFGE) and other
    restriction-based methods, plasmid analysis, and
    PCR-based typing methods.

8
  • The incorporation of molecular methods for typing
    of pathogens has assisted in efforts to obtain a
    more fundamental assessment of strain
    interrelationship.

9
CHARACTERISTICS OF TYPING METHODS
  • There are a number important attributes for
    successful typing schemes the methodologies
    should be standardized, sensitive, specific,
    objective, and subject to critical appraisal.

10
  • All typing systems can be characterized in terms
    of typeability, reproducibility, discriminatory
    power, ease of performance and interpretation,
    and cost (in terms of time and money).

11
Typeability
  • Typeability refers to the ability of a technique
    to assign an unambiguous result (type) to each
    isolate.
  • Nontypeable isolates are more common with
    phenotypic methods but can also occur with
    genotypic methods.

12
Reproducibility
  • The reproducibility of a method refers to the
    ability to yield the same result upon repeat
    testing of a bacterial strain.
  • Poor reproducibility may reflect technical
    variation in the method or biologic variation
    occurring during in vivo or in vitro passage of
    the organisms to be examined.

13
Discriminatory power
  • The discriminatory power of a technique refers to
    its ability to differentiate among
    epidemiologically unrelated isolates, ideally
    assigning each to a different type.
  • In general, phenotypic methods have lower
    discriminatory power than genotypic methods.

14
Cost Ease of interpretation
  • Most molecular methods require costly material
    and equipment but are relatively easy to learn
    and are applicable to variety of species.
  • On the other hand, phenotypic methods also
    involve costs in labour and material and are
    restricted to a few species for example,
    antisera for Salmonella serotyping will not work
    to type gram-positive organisms.

15
GENOTYPIC METHODS
  • The goal of genotyping studies is that
    epidemiologically related isolates collected
    during an outbreak of nosocomial disease are able
    to be linked to one another.
  • In other words, whether the isolates involved in
    a nosocomial outbreak are genetically related or
    not and thus originate from the same strain or
    otherwise

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  • Therefore, the use of strain typing in infection
    control decisions is based on several
    assumptions
  • (i) isolates associated with the outbreak are
    recent progeny of a single (common) precursor or
    clone,
  • (ii) such isolates will have the same genotype,
    and
  • (iii) epidemiologically unrelated isolates will
    have different genotypes

17
  • Typing of microorganisms classically involves
    the subdivision of a single or related species,
    using a set of defined characteristics

18
Bacterial typing systems
  • Typing methods fall into 2 broad categories
  • Phenotypic
  • Genotypic

19
Phenotypic methods
  • Biotype
  • Antibiogram
  • Serotyping
  • Bacteriocin typing
  • Phage typing

20
Biotyping
  • Based on subspecies diversity of
  • Colony morphology
  • Metabolic activity
  • Toxin production

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Biotyping
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Antibiogram
  • Changes in antibiograms may reflect spontaneous
    point mutations.
  • Thus,isolates that are epidemiologically related
    otherwise genetically indistinguishable may
    manifest different antimicrobial susceptibilities
    due to acquisition of new genetic material over
    time or loss of plasmids

24
Antibiogram
25
Serotyping
  • Uses a series of antibodies to detect different
    antigenic determinants on the surface of the
    bacterial cell
  • The classic strain typing techniques
  • It remains a key method for typing isolates of
    Salmonella,Shigella S.pneumococci

26
Serotyping
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Bacteriocin typing
  • Bacteriocin is protein products produced by other
    bacteria that inhibit growth of the test
    bacterium.
  • Classifies bacteria according to their
    susceptibility to bacteriocin.
  • Used in reference laboratories for typing
  • K.pneumoniae P. aeruginosa.

28
Bacteriocin typing
29
Phage typing
  • Classifies bacterial organisms according
  • to susceptibility of the bacteria to lysis
  • by the panel of bacteriophage.
  • Phage typing has played useful in
  • epidemiologic roles for S.aureus S.enterica
    serotype Typhi

30
Phage typing
31
Limitations of Phenotypic methods
  • Influenced by environmental selective pressure
    -unstable antigenic traits
  • -alterations in expression of
  • traits being assessed
  • Labour-intensive
  • Impractical
  • Slow
  • Lack discriminatory power

32
Genotypic methods
  • Procedures based on DNA analysis offer a more
    stable and universal approach to typing
    microorganisms, and are used increasingly in
    microbiology laboratories to supplement
    traditional typing methods.

33
Genotypic methods
  • DNA typing differentiates organisms on the basis
    of genetic variation at the level of chromosome,
    plasmid or gene.

34
Genotypic methods
  • The total (genomic) DNA of a bacterium consists
    of a single chromosome (typically 0.6-10 megabase
    pairs Mbp, together with any plasmid DNA.
  • In the case of fungi and protozoa, a number of
    chromosomes are present, in addition to
    mitochondrial DNA

35
Genotypic methods
  • Plasmid profile analysis PF
  • REA of Plasmid DNA
  • REA of Chromosomal DNA
  • RFLP
  • Pulse-Field Gel Electrophoresis (PFGE)
  • PCR Amplification methods

36
Plasmids
  • Extrachromosomal genetic elements
  • Number size of the plasmids are used as the
    basis of strain identification

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Plasmid profile analysisPF PLASMID FINGERPRINTING
  • First molecular method to be used as a bacterial
    typing tool

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PF
40
Pf
  • Plasmid strain typing technique is successfully
    used for analysis of outbreaks of nosocomial
    infections community acquired infections
    especially by gram negative rods.

41
Restriction Enzyme Analysis(REA)
  • Restriction enzymes
  • Makes double-stranded breaks in either Plasmid
  • or Chromosomal DNA at specific nucleotide
  • Locations Example
  • EcoRI of Escherichia coli
  • Recognizes GAATTC and cleaves between GA
  • 2. HhaI of Haemophilus influenza
  • GTPPyPuAC
  • Pyany pyrimidine base Puany purine base
  • Cleaves between PyPu

42
REA
  • Digest DNA using restriction enzyme(s)
  • Separate the digested chromosomal
  • DNA according to size in an agarose gel
  • using gel electrophoresis
  • Use molecular size marker
  • Small DNA fragments migrate faster than large DNA
    fragments

43
REA of Plasmid DNA
  • Enhanced discrimination between organisms can be
    achieved through the cleavage of the plasmid DNA
    using restriction enzymes.
  • These enzymes cut DNA at specific base sequences
    (restricted sites), and the frequency and
    location of the restricted sites determine the
    number of resulting fragments and their size.

44
REA OF PLASMID DNA

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Limitations of plasmid REA
  • 1.Plasmids can spread to multiple species of
    bacteria, causing a plasmid outbreak in which
    unusual antibiograms are recognised in multiple
    species.
  • 2.The structure of individual plasmid the
    plasmid content of the strain may vary over time.

47
REA of Chromosomal DNA
  • Two methods of typing microorganisms by REA of
    chromosomal DNA
  • Restriction enzyme that cuts the chromosome into
    hundreds of pieces (frequent cutter )followed by
    conventional electrophoresis
  • Fragments of 25-50 kb are resolved

48
REA of Chromosomal DNA (contd)
  • 2. Restriction Enzyme that cuts the chromosome
    infrequently generating 10 to 30 bands followed
    by novel form of electrophoresis

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RFLP
  • Chromosomal restriction digests produced by
    frequent cutting enzymes are separated by
    conventional agarose gel electrophoresis
  • The DNA fragments are transferred onto
    nitrocellulose or nylon membrane
  • The DNA on the membrane-is hybridized with a
    specific chemically or radioactively labeled
    piece of DNA or RNA( probe ) binds to few
    fragments complementary nucleic acid sequences.

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RFLP
  • Variation in the number size of the fragments
    detected by hybridization are referred to RFLP
  • VARIATION RIBOTYPING

56
Advantage of RFLP
  • Proportion of strains typable ALL
  • Reproducibility excellent
  • Discriminatory power moderate to excellent

57
Limitations of RFLP
  • Very difficult to interpret the complex profiles
    which consists of hundreds of bands that may be
    distinct or overlapping
  • Ease of interpretation-moderate
  • Ease of performance -difficult

58
Pulsed Field Gel Electrophoresis
  • PFGE first described in 1984 as tool for
    examining the chromosomal DNA
  • Bacterial genome (2,500-5,000kbpairs in size)
  • with rare restriction sites
  • Restriction
    enzyme
  • 10-30 restriction fragments (10-800kb)

59
  • Essentially all these fragments are resolved in
    to a pattern of distinct bands by PFGE
  • By a specially designed chamber that positions
    the agarose gel between three sets of electrodes
    that form a hexagon around the gel

60
PFGE
  • PFGE is gold standard for bacterial sub typing
  • Looks at whole genome of bacterial pathogens
    using rare cutting restriction enzymes
  • 10-30 fragments ranging in size from 10kb to
  • 800 kb in length are generated
  • Larger pieces of DNA are separated by
  • shifting direction of current frequently

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PFGE of E.fecalis
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PFGE of E.fecium
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PFGE of Ps. aeruginosa
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PFGE
  • Proportion of strains typable ALL
  • Reproducibility excellent
  • Discriminatory power excellent
  • Ease of interpretation-
  • difficult
  • Ease of performance -difficult

67
Detection of a specific bacterial pathogen
  • Cycling amplification technologies
  • 1.1. PCR, real-time PCR and RT-PCR
  • 1.2. Nested PCR
  • 1.3. PCR-ELISA
  • 1.4. Ligase chain reaction

68
Polymerase Chain Reaction Kary Mullis invented
PCR
69
DNA Between The Primers Doubles With Each Thermal
Cycle
70
Molecular Beacon
71
REAL TIME PCR
72
REAL TIME PCR
73
NESTED PCR
a modification of PCR intended to reduce the
contamination in products due to the
amplification of unexpected primer binding
sites.
74
PCR-ELISA
75
PCR-based Strain- typing methods
  • Based on random sequences AP-PCR
  • Based on heterogeneity restriction endonuclease
    sites AFLP
  • Based on interspersed DNA repetitive elements
    REP-PCR

76
Random Amplified Polymorphic DNA
  • Williams et al. (1990) developed Random Amplified
    Polymorphic DNA (RAPD)
  • Technique using very short 10 base primers to
    generate random fragments from template DNAs
  • RAPD fragments can be separated and used as
    genetic markers or a kind of DNA fingerprint

77
RAPD of E.faecalis
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RAPD
  • Proportion of strains typable ALL
  • Reproducibility GOOD
  • Discriminatory power GOOD-HIGH
  • Ease of interpretation-
  • MODERATE
  • Ease of performance - MODERATE

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AFLP
  • Proportion of strains typable ALL
  • Reproducibility GOOD
  • Discriminatory power GOOD-HIGH
  • Ease of interpretation-
  • MODERATE
  • Ease of performance - MODERATE

81
rep-PCR
  • This genomic fingerprinting method employed is
    based on the use of DNA primers corresponding to
    naturally occurring interspersed repetitive
    elements in bacteria, such as the REP, ERIC BOX
    elements, and the PCR reaction (rep-PCR).

82
  • Bacterial organisms carry certain genetic
    elements that jump, translocate, or transpose
    to new locations in the chromosome and called
    transposons or insertion sequences.
  • If the copy number of IS elements is high enough,
    and if they are randomly distributed in the
    chromosome, DNA sequence between these elements
    can be amplified by PCR

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REP PCR of S.pneumoniae
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Limitations
  • Patterns generated may be complex
  • and difficult to interpret
  • Technically demanding

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  • Isothermal amplification technologies
  • 2.1.Nucleic acid sequence-based amplification
  • 2.2. Transcription-mediated amplification
  • 2.3. Strand displacement amplification
  • 2.4. Rolling circle amplification
  • 2.5. Cycling probe technology
  • 2.6. Branch DNA.
  • 2.7. Hybrid capture.

88
Nucleic acid sequence-based amplification (NASBA)
  • an isothermal-based method of RNA amplification
    (Davey and Malek, 1989).
  • RNA is amplified by the action of an enzyme
    cocktail that includes AMV Reverse Transcriptase,
    T7 RNA polymerase and RNAse H at a fixed
    temperature (41C).
  • By coupling these technologies with a hand-held
    detection system, this method becomes a
    deployable monitoring device for onboard and
    remote sensing purposes.

89
NASBA
By pairing this technique with the ability to
monitor the fluorescence signal produced from
Molecular Beacon probes (Figure) in real time as
they hybridize to the amplicon, real time
analysis of samples performed data in obtained
a matter of minutes
Figure 1. NASBA amplification pathway. Target
ssRNA (in this case, Noroviral genome) binds to
Primer 1. An RNA/DNA hybrid is formed by the
action of reverse transcriptase. RNaseH then
degrades the RNA component of the hybrid and
reverse transcriptase using Primer 2 makes a cDNA
of the target region. Because Primer 1 contains a
T7 RNA polymerase promoter, many copies of the
target RNA are made. NASBA reagents are available
from Biomerieux
90
Transcriptionmediated amplification
TMA uses RNA transcription (RNA polymerase) and
DNA synthesis (reverse transcriptase) to produce
an RNA amplicon from a target nucleic acid.
Since RNA is more labile than DNA in the
laboratory environment, this feature diminishes
the possibility of carry-over contamination
91
Rolling circle amplification
In rolling circle amplification (RCA), a single
forward primer is extended by DNA polymerase
along a circular template for many rounds,
displacing upstream sequences and producing a
long single-stranded DNA of multiple repeats.
92
Cycling probe technology
Cycling probe technology (CPT) is an isothermal
probe amplification system for detection of
target DNA that utilizes a RNADNA chimeric probe
(RNA sequence flanked by two DNA sequences) to
hybridize to a specific region of an amplified
gene.
93
Branched DNA
Another technique that utilizes signal rather
than target amplification is called branched DNA
(bDNA).
94
HYBRID CAPTURE
Signal amplification is also the basis for some
commercial tests such as the Hybrid Capture
(HC2) assays from Digene (Gaithersburg, MD) The
chance of cross-contamination of reactions is
reduced.
95
Detection of bacterial pathogens by multiple
targets or universal targets
  • 3.1. Multiplex PCR
  • 3.2. Microarray
  • 3.3. Sequencing-based identification

96
Multiplex PCR
Multiplex PCR utilizes more than one set of
primers in a reaction and can be used for the
simultaneous detection of multiple bacterial
pathogens.
97
Multiplex PCR for Enterococcus
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Microarray
  • Microarray refers to a small, two-dimensional
    high density matrix of DNA fragments which are
    printed or synthesized on a glass or silicon
    slide (chip) in a specific order.
  • Hybridization of the DNA fragments to
    fluorescently labeled probes is detected by
    advanced instrumentation and software.

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MICROARRAY
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Pyrosequencing
Pyrosequencing ( AB, Uppsala, Sweden) is a
technology whereby a single-stranded DNA template
is prepared, a sequencing primer is hybridized to
a complimentary sequence on the template, and
enzymes catalyze a light reaction when each
nucleotide is incorporated into the growing DNA
strand .
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Sequencing-based identification
  • Universal targets such as the 16S rRNA genes or
    the
  • 16S23S rRNA gene interspacer region have been
    used extensively for bacterial identification,
    especially if bacteria are difficult to isolate
    by conventional methods
  • Other universal targets such as heat-shock
    proteins, like hsp65 or cold-shock proteins can
    also be used

106
Detection of bacterial pathogens by
non-amplification methods
  • 4.1. Fluorescence in situ hybridization
  • 4.2. Peptide nucleic acid-FISH
  • 4.3. Line probe assay
  • 4.4. Hybridization protection assay
  • 4.5. Mass spectrometry

107
Fluorescence in situ hybridization (FISH)
  • Fluorescence in situ hybridization (FISH)
    assays use fluorescently labeled 16S rRNA or 23S
    rRNA probes and fluorescent microscopy to detect
    intact bacteria directly in clinical specimens,
    such as blood or tissue, or after enrichment
    culture.

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Line probe assay
The line probe assay (LiPA) consists of a
nitrocellulose strip with specific
oligonucleotide probes attached as discreet
parallel lines along the strip. Hybridization
results in a color change that can be detected
visually or by an automated reader.
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Hybridization protection assay
  • Hybridization protection assays (HPA) utilize a
    chemiluminescent acridinium ester detector
    molecule on a DNA probe that targets the specific
    bacterial rRNA. The RNA/ DNA hybrid is detected
    in a luminometer.

112
Mass spectrometry
  • Mass spectrometry (MS) causes ionization and
    disintegration of a target molecule by bombarding
    it with electrons. The mass/charge ratio of the
    resulting molecular fragments is then analyzed to
    produce a molecular signature. MS has often been
    used to identify bacteria by protein signature

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Mass spectrometry
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Conclusion
  • Molecular techniques do not substitute for
    conventional methods
  • Each technique has its own unique advantages or
    disadvantages

116
  • The advantages of Nucliec acid detection tests
    over microbiologic methods include rapid results,
    low detection limits (theoretically a single
    cell), and specific organism detection.

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  • contamination must be minimized,
  • technicians must have proper training,
  • and quality control procedures must be
    incorporated into routine laboratory workflow.

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  • While the cost of some molecular diagnostic
    instruments is high,
  • the benefits of faster turnaround time, high
    throughput, and enhanced sensitivity over
    traditional methods may override this obstacle.
  • Another important consideration is that
    laboratory space must be allocated for
    instruments and dedicated as DNA-free areas.

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