with focus on genetic methods

1. Microbiological
identification
(Genetic Tools)
ALMA TAMMOUR
M.PHARM (PHARMACEUTICAL BIOTECHNOLOGY)
SPER, JAMIA HAMDARD

2. What does the term “Identification” mean?
Identification: matchning characteristics of an “unknown” to lists of known
organisms.
Classification: Placing organisms in groups of related species.

3. IMPORTANCE OF IDENTIFICATION
Determining the clinical significant of particular pathogen.
Guiding physician care of the patients.
Determining the laboratory testing for detection of antibacterial resistance is
warranted.
Determining the type of antibacterial therapy that is appropriate.
Determining whether infectious organisms are risk for other patients in the
hospital, the public and other laboratory workers.

4. Identification methods
Traditional methods / Phenotyping methods
Immunological methods / Serological methods
Genotypic methods / Molecular methods

5. Methods for identification of microorganisms
The methods can be devided to:
Classical methods
 In-house biochemical
Manual phenotyping methods (API Strips, BBL, etc)
Automated phenotyping methods (Vitek)
Cellular Fatty Acids (MIDI Sherlock System)
Carbon Source Utilization (Biolog)
Genetic (MiroSeq, Riboprinter)

6. Limitations of identification methods
 Phenotypic properties (biochemical, carbon source utilization) can be
variable, subjective, dependent on growth parameters and health of organism.
 Cellular fatty acid profiles change with temperature, age of culture and
growth medium.
 Some systems require subjective off-line testing such as gram stain, oxidase,
coagulase, etc, before determining the appropriate test card.
 DNA Sequencing system is not automated, numerous, somewhat complicated
lab procedures, and manual interpretation of results requires microbial
phylogenetic experience.

7. Method selection
Genotyping methods have been shown to be more accurate and
precise than traditional biochemical and phenotypic technique. These
methods are especially valuable for investigations into failures (e.g,
sterility test, media fill contamination). However, appropriate
biochemical and phenotypic methods can be used for the routine
identification of isolates.

8. Method selection
Specimens for microbial identifications originate from multiple areas and often have
different levels of criticality.
Air and surface monitoring
Personnel monitoring
Raw material and water testing
In-process and finished product
Bilogical indicators (Bis) growth
What is the most appropriate methosd to use ?

9. MICROBE IDENTIFICATION
The successful identification of microbe depends on:
 using the proper aseptic techniques
Correctly handling the specimen
Quickly transporting the specimen to the lab
Once the specimen reaches the lab it is cultured and identified
Use care and tact to avoid patient harm
The specimen is the beginning. All diagnostic information from the laboratory depends upon the knowledge by
which specimens are chosen and the care with which they are collected and transported.
_ Cynthia A. Needham

10. IDENTIFICATION USING GENOTYPING
METHODS

11. IDENTIFICATION USING GENOTYPIC METHODS
• identification of organisms based on analysis of the genetic properties of a
microorganism
•Genotypic methods are more stable because nucleic acid sequences are highly
conserved in most microbial species.
•Genotypic methods are technically more challenging and more time consuming
•Typically require investing in expensive equipment, reagents and supplies.

12. IDENTIFICATION USING GENOTYPIC METHODS
• Many labs opt for performing routine
identifications using a phenotypic system and
rely of genetic identification for critical and
investigational samples.
•DNA sequencing of the first 500 base pairs of the
16s sequence is a popular method used in
pharmaceutical identification labs and contract
laboratories.
• Strain typing is useful for tracking the source of
contamination.

13. Advantages and Limitations of Genotypic Systems

14. Genotypic methods
Genotypic methods of microbe identification include the use of:
 nucleic acid probs
 RFLP
 PCR
 Nucleic acid sequence analysis
 16s rRNA analysis
 Plasmid fingerprinting

15. NUCLEIC ACID HYBRIDIZATION
It is a technique involves using a
labelled nucleic acid probe, which is a
known DNA or RNA fragment, to bind
with the target nucleic acid, which is
usually a poorly understood,
heterogeneous population of nucleic
acids.
A probe labelled with detectable tracer
is prerequisite for determining a
specific DNA sequence or gene in a
sample.

17. ACCUPROBE SYSTEM – Nucleic Acid
Hybridization

18. ACCUPROBE SYSTEM – Nucleic Acid
Hybridization

19. Restriction fragment length polymorphism
(RFLP)
Restriction fragment length polymorphism (RFLP)
is a type of polymorphism that results from
variation in the DNA sequence recognized by
restriction enzymes.
These are bacterial enzymes used by scientists to
cut DNA molecules at known locations. RFLPs
(pronounced "rif lips") are used as markers on
genetic maps.
Typically, gel electrophoresis is used to visualize
RFLPs.

20. Restriction fragment length polymorphism
(RFLP)

22. Terminal Restriction fragment length
polymorphism (T-RFLP)

24. Restriction fragment length polymorphism
(RFLP)
LimitationsAdvantages
High quality and large amount od DNA
is required
Co-dominant
Radiolabelled probes are expensiveSpecific Sequence
Not automated, tidious, time
consuming
Genomic abundance
Reproducible

25. Polymerase Chain Reaction (PCR)
• PCR is widely used for the identification of microorganisms.
• Sequence specific primers are used in PCR for the amplification of DNA or RNA
of specific organisms.
• PCR allows for the detection even if only a few cells are present and can also be
used on viable nonculturables.
• The presence of the appropriate amplified PCR product confirms the presence
of the organisms.

26. Polymerase Chain Reaction (PCR)

27. EXAMPLES OF COMMERCIAL GENOTYPIC
METHODS

28. Why rRNA sequencing?
 Gold standard for bacterial identification
16S rRNA gene
 ~ 1500 bp
Small subunit of ribosome
Present in 1 or more copies
Critical to cell function

29. 16S rRNA
 Analogous to 18S – eukaryotes
 RNA gene product _ base-pairing forms a complex tertiary architecture
 Few genes are as highly relatively unchanged in evalution
 Nearly all bacteria share highly sequence conserved regions bracketing regions that are variable
species-specific manner

33. Advantages & Limitations of MicroSEQ

37. Advantages & Disadvantages of Riboprinter

38. REFERANCES
 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls.J.
Michael Janda* and Sharon L. Abbott
 Microbial Functional Genomics. By Jizhong Zhou, Dorothea K. Thompson, Ying Xu, James M. Tiedj
 Molecular methods to study the organization of microbial communities
Author links open overlay panel GerardMuyzer*Niels B.Ramsing**
 Identification and Characterization of Microorganisms Using Molecular Methods. Michael Waddington,
New England PDA, Burlington, MA, February 8, 2006