Identification Methods of

Oral MicrobesDr. Ali Yaldrum

Faculty of DentistrySEGi 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 (classification) of Oral Microorganisms

• Describe how to obtain samples from Oral Cavity• Describe Molecular techniques of identification• Describe techniques that requires culture for identification

1

DiagnosticCycle

Infection?Virus? culture

2

Clinician request

*A-A-ah

3

collection

4 transportation

5

labortary analysis

6

data flow

What the!!!

7

interpretation

8diagnosis +treatment

1. prokaryotes VS eukaryotes

prokaroyte

cell wallpeptidoglycan singular supercoiled

circular chromosome

cytoplasm rich in ribosomes

plasmid cellmembrane

flagellum

(Fig.1)

eukaroyte

nuclear membrane

mitochondriacell membrane

cytoplasm

smooth endoplasmic reticulum

lysosome

rough endoplasmic reticulum

Golgi apparatus

(Fig.2)

2. Classification & Identification

classification

‘Classification is the arrangement of Organisms into groups (taxa) on the basis of their similarities and differences.’

The science of classification is called taxonomy

KINGDOM

PHYLUM

CLASS

ORDER

FAMILY

GENUS

SPECIES

SUB SPECIES

DIVISION

taxonomic hierarchy (Fig.3)

identification

‘is the process of determining that a new isolate belongs to particular taxon’

• Bacteria are identified 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 Classification

Shapes

Atmosphere

Obligate aerobesMicroaerophiles

Obligate anaerobesFacultative anaerobes

Capnophiles

requires O2

requires reduced O2

requires no O2

anaerobic or aerobic

requires increases CO2Spores

Key EnzymesBacteria lacking certain enzymes

Serological ReactionInteraction of antibodies with certain surface structures

DNA SEQUENCINGDNA sequencing of key genes ; Ribosomal 16S gene

peptidoglycan Teichoic acid

Plasma membrane

Outer membraneprotein

Thin peptidoglycan

layer

Cell wall

Gram +ve Gram -ve

GramReaction

(Fig.4)

Coccus

Bacillus

Coccobacillus

Fusiform bacillus

Vibrio

Spirochete

Spirillum

bacterial shapes

(Fig.5)

3. sampling Oral bacteria

• 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 profiles for different locations

• Tongue • Tooth surface• Gingival sulcus• Buccal mucosa• Gingival crevice

gingival sulcus1

2

3

4

5

6

1= Enamel2= Dentine3= Pulp

4= Free gingivae5= Cementum6= Alveolar bone

(Fig.6)

sampling saliva

• Easily sampled• Contains a mix of bacteria (planktonic)• Patient is asked to chew paraffin 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 plaque2.Subgingival plaque

Supragingival

• Curette is used to scrap the biofilm of the tooth surface (fig. 7)

• Can not be inserted more than 6mm

Periodontal Curette(Fig.7)

Subgingival

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

Endodontic Paper (paper point)(Fig.8)

4. Identifying Oral bacteria

Approaches to identifying bacteria can be grouped into 2major categories

• Techniques that do not require culture (molecular identification techniques)

• Techniques that require culture

*At present combination of both techniques is used to characterize the full compliment of organisms

molecular identification

• are most often based on sequence analysis of the ribosomal 16S genes

• Common techniques for molecular detection of bacteria:1. PCR with specific primers2. Quantitative PCR3. DNA hybridization assays4. Ribosomal 16S cloning & sequence analysis5. 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 specificity

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 amplification of target DNA using specific 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 specific to a particular lock.

So every primer is a strand of nucleic acid specific to a specific strand of DNA from a specific 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• Specific amplification of DNA from a target species even in

the presence of hundred of species

variations of PCR

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

Real-time PCR

• Conventional PCR requires Gel-electrophoresis for amplification analysis

• Labelled probes are used

• Multiple amplifications can be analysed at specific time period during reaction period

Watch Video ofPCR & 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 amplification

• Rapid Clinical diagnostic procedures. Sensitivity of PCR enables rapid diagnosis

• Enables identification of different species. PCR allowed researcher to identify uncultivable bacteria

DNA Hybridization

• Measures the degree of genetic similarity between pools of DNA sequences (fig. 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

Checkerboard hybridization

S. mutans

all streptococci

(Fig.9)

Watch video ofDNA 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

Sample & transport

1disperse, dilute & plateOnto selective or nonselective media

2pick individualcolonies & grow pure cultures

3characterize by morphology & bio-chemical tests

4

GenusXspecies 1species 2species 3

5classify

Bacterial identification process

(Fig.10)

O2 requirements

• Amount of O2 in the atmosphere is critical for bacterial growth

• Most Oral Bacteria are1. Facultative anaerobes or2. Anaerobes3. Capanophilic anaerobes (A. actinomysetemcomitans)

O2 requirements

• Facultative anaerobesa) 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 (fig.11)

O2 can be removed from the transport medium i. Boiling the media ii. Flushing with O2 free gasiii. Commercially available pre reduced media

(Fig.11)

Anaerobic Chamber

culture media

Non-selective media

• Blood agar supports growth of many oral species• Oral sample will produce diverse array of colony

morphologies• Difficult 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 specific nutritional requirements• Difficult to grow until those requirements are determined &

supplimented

Dispersion & Dilution

Non-selective & Selective agars

Incubate under appropriate atmospheric conditions

for various times

Colony count

Identification scheme

DNA extraction

16S rRNA gene amplification with universal primers

Cloning & partialsequencing

Search for homologyin database

Construction of specific probes for subsequent

analysis

references• Philip D. Marsh, Michael V Martin, “The Resident Oral Microflora” 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 Microflora” 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, classification and identification 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