Identication Methods of Oral Microbes

Dr. Ali Yaldrum Faculty of Dentistry SEGi University, Kota Damansara, Malaysia
18-06-12

Learning Objectives
At the end of this session, the student should be able to: Develop an understanding of Taxonomy (classication) of Oral Microorganisms Describe how to obtain samples from Oral Cavity Describe Molecular techniques of identication Describe techniques that requires culture for identication

Infection? Virus? culture

2 3
*A-A-ah

1
diagnosis + treatment

Clinician request

What the!!!

Diagnostic Cycle
collection

interpretation

data ow

transportation

labortary analysis

VS eukaryotes
1. prokaryotes

prokaroyte
cell wall peptidoglycan singular supercoiled circular chromosome

agellum cytoplasm rich in ribosomes plasmid cellmembrane

(Fig.1)

eukaroyte
mitochondria cell membrane nuclear membrane lysosome

cytoplasm

rough endoplasmic reticulum Golgi apparatus

smooth endoplasmic reticulum

(Fig.2)

2. Classication

Identication

&

classication

Classication is the arrangement of Organisms into groups (taxa) on the basis of their similarities and differences. ! ! The science of classication is called taxonomy

taxonomic hierarchy
KINGDOM PHYLUM DIVISION CLASS ORDER FAMILY GENUS SPECIES SUB SPECIES

(Fig.3)

identication
is the process of determining that a new isolate belongs to particular taxon Bacteria are identied using phenotype, immunological or molecular characteristics

why this is important

Revealing their identity Behavior and likely response to treatment Also to predict their pathogenicity Isolate microorganisms that spread in community & cause serious disease

Bacterial Classication

(Fig.4)

Cell wall peptidoglycan Teichoic acid Outer membrane protein

Shapes

Thin peptidoglycan layer Gram +ve Gram -ve

Gram Reaction
Obligate aerobes requires O2 requires reduced O2 requires no O2 anaerobic or aerobic requires increases CO2

Plasma membrane

Atmosphere

Microaerophiles Obligate anaerobes Facultative anaerobes Capnophiles

Spores

Bacteria lacking certain enzymes

Key Enzymes
Interaction of antibodies with certain surface structures

Serological Reaction

DNA sequencing of key genes ; Ribosomal 16S gene

DNA SEQUENCING

bacterial shapes
Coccus Bacillus Coccobacillus Fusiform bacillus
(Fig.5)

Vibrio Spirillum Spirochete

3. sampling

bacteria

Oral

 Oral Cavity contains a variety of different niches that harbour distinctive communities of bacteria. Location and environment determines the diversity of eco system.

Studies of various niches have shown distinctive microbial proles for different locations Tongue Tooth surface Gingival sulcus Buccal mucosa Gingival crevice

gingival sulcus
1 2 3 4 5 6

1= Enamel 2= Dentine 3= Pulp

4= Free gingivae 5= Cementum 6= Alveolar bone

(Fig.6)

sampling saliva

Easily sampled Contains a mix of bacteria (planktonic) Patient is asked to chew parafn prior to collecting saliva Results in enriched tooth derived saliva Used for collection of large population samples

sampling plaque

2 approaches can be used 1.Supragingival plaque 2.Subgingival plaque

Supragingival Curette is used to scrap the biolm of the tooth surface (g. 7) Can not be inserted more than 6mm

Periodontal Curette
(Fig.7)

Subgingival Endodontic paper (paper point) can be used (g. 8) For pockets deeper than 6mm Wicks up uid containing bacteria Large number of bacteria can be obtained

Endodontic Paper
(Fig.8)

(paper point)

4. Identifying

bacteria

Oral

Approaches to identifying bacteria can be grouped into 2major categories Techniques that do not require culture (molecular identication techniques) Techniques that require culture
*At present combination of both techniques is used to characterize the full compliment of organisms

molecular identication
are most often based on sequence analysis of the ribosomal 16S genes Common techniques for molecular detection of bacteria: 1. PCR with specic primers 2. Quantitative PCR 3. DNA hybridization assays 4. Ribosomal 16S cloning & sequence analysis 5. FISH and microscopy

bacterial DNA recovery

To extract the bacterial DNA, the bacterial cell wall must be lysed with out damage to the DNA Several methods are available Methods that yield high recovery of DNA in one organism might not yield same amount in another

dna recovery

COMMERCIAL KITS
Target one type of bacteria Variable intensity High specicity

Detergents & Proteinase K

Lyse a wide spectrum of bacteria Unable to lyse Gram-ve bacteria

Bead Beating
Bacteria are mixed with small slurry of tiny glass beads Vile is placed in a vibrating apparatus Will lyse the most sturdy bacteria's Not used for fragile bacteria

what is PCR

Or Polymerase Chain Reaction It is the process which results in cyclic amplication of target DNA using specic primers, theoretically from one single cell

what is a Primer
The simplest explanation of a primer is to consider it as a key, as every key is specic to a particular lock. So every primer is a strand of nucleic acid specic to a specic strand of DNA from a specic specie

what is a Primer
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process
*Primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases

PCR

Almost every DNA based method uses PCR Allows detection of DNA from as low as one cell Possible to do extensive, detailed analysis Specic amplication of DNA from a target species even in the presence of hundred of species

variations of PCR

The basic PCR methodology is modied to provide sophisticated analytical tools Nested PCR Multiplex PCR Real Time PCR

Real-time PCR
Conventional PCR requires Gel-electrophoresis for amplication analysis Labelled probes are used Multiple amplications can be analysed at specic time period during reaction period

Watch Video of PCR & Gel Electrophoresis

https://www.youtube.com/watch?v=GLgt-EGkhZs&feature=related

why is PCR widely used

Even a minuscule quantity of DNA can be studied, as a single DNA molecule is adequate for amplication Rapid Clinical diagnostic procedures. Sensitivity of PCR enables rapid diagnosis Enables identication of different species. PCR allowed researcher to identify uncultivable bacteria

DNA Hybridization
Measures the degree of genetic similarity between pools of DNA sequences (g. 9) Possible to determine the genetic distance between two sequences Because of the complexity of bacterial ecology, necessary to identify many species of bacteria from single sample

S. mutans

all streptococci

Checkerboard hybridization
(Fig.9)

Watch video of DNA Hybridization

! http://www.phgfoundation.org/tutorials/dna/2.html

Watch video of DNA Microarrays

http://www.phgfoundation.org/tutorials/dna/6.html

cultivation of bacteria

Consist of diverse group of bacteria Requires a spectrum of physical & chemical for successful growth Laboratory cultivation conditions must be adjusted

Bacterial identication process

GenusX species 1 species 2 species 3

1
Sample & transport

2
disperse, dilute & plate Onto selective or non selective media

3
pick individual colonies & grow pure cultures

4
characterize by morphology & biochemical tests

5
classify

(Fig.10)

O2 requirements
Amount of O2 in the atmosphere is critical for bacterial growth Most Oral Bacteria are 1. Facultative anaerobes or 2. Anaerobes 3. Capanophilic anaerobes (A. actinomysetemcomitans)

O2 requirements

Facultative anaerobes a) Streptococcus mutans b) Lactobcillus Both cause caries and can be grown in environment rich in O2

CO2 requirements
Subgingival species are exclusively anaerobic Must be grown in special chambers containing low levels of CO2 (g.11) O2 can be removed from the transport medium i. Boiling the media ii. Flushing with O2 free gas iii. Commercially available pre reduced media

Anaerobic Chamber
(Fig.11)

culture media
Non-selective media Blood agar supports growth of many oral species Oral sample will produce diverse array of colony morphologies Difcult to sort out individual species Species comprising of small percentage might not be seen

culture media

Selective media Contains ingredients that inhibit growth of all but a few species Useful in isolating individual species Enables detection of bacteria that are present in low levels

culture media

Special requirements Some bacteria have specic nutritional requirements Difcult to grow until those requirements are determined & supplimented

Dispersion & Dilution Non-selective & Selective agars Incubate under appropriate atmospheric conditions for various times Colony count Identication scheme

DNA extraction 16S rRNA gene amplication with universal primers Cloning & partial sequencing Search for homology in database Construction of specic probes for subsequent analysis

references
Philip D. Marsh, Michael V Martin, The Resident Oral Microora in Oral Microbiology, 5th Edition, Churchil Livingstone, 2009, pp 24-29 Philip D. Marsh, Michael V Martin, Methods of Determining Composition of the resident oral Microora in Oral Microbiology, 5th Edition, Churchil Livingstone, 2009, pp 50-54 " Eugene J. Leys, Ann L. Griffen, Purnima S. Kumar and Mark F. Maiden, Isolation, classication and identication of Oral Microorganisms in oral Microbiology and Immunology, ASM Press pp 73-88. PCR - DNA Fingerprinting https://www.youtube.com/watch?v=GLgt-EGkhZs&feature=related DNA Microaarays http://www.phgfoundation.org/tutorials/dna/6.html

Hybridization ! http://www.phgfoundation.org/tutorials/dna/2.html FISH ! http://www.phgfoundation.org/tutorials/dna/3.html ! http://www.dnalc.org/view/15924-Making-many-copies-of-DNA.html