Methods to find which types of bacteria are present in the

mouth of a patient
 Dr.Sonalee SHAH
Bacterial identification is the first step in establishing the bacterial etiology of a
particular disease. It includes the procedures and techniques used to correctly
identify the bacterial pathogens responsible for disease. Bacteriologist employs a
wide variety of techniques, based upon known characteristics of specific bacteria,
to arrive at the identity of a given specimen. The traditional way of detecting and
identifying bacteria from given specimen is based on :

culturing,

enumeration and

isolation of presumptive colonies for further identification analysis.

To summarize, identification is the process of determining that a new isolate

belongs to a particular ‘taxon’.

Principles of identification:

Identification of bacteria requires the knowledge of their morphological,

biochemical, physiological. and genetic characteristics.

Collectively, these characteristics can be grouped as phenotype and genotype.

The identification schemes start with broad categorization (e.g., Gram-staining)

and then progress to more specific tests.

Identification schemes can be classified into one of two categories:

a. those that are based on genotypic characteristics of bacteria and

b. those that are based on phenotypic characteristics –

Conventional methods for further subtyping of bacteria include - the study of the
phenotypic characteristics of the microorganisms.

These phenotypic methods include –

Biotyping : In biotyping, the biochemical growth requirements, environmental
conditions (pH, temperature, antibiotic resistance, bacteriocins susceptibility) and
physiological (colony and cell morphology, cell wall composition by microscopy
and membrane composition such as by fatty acid analysis) aspects of bacteria are
investigated.

Serotyping and Phage typing : serological and phage typing concentrate more on
the surface structure differences of bacteria.

c. Certain schemes rely on both genotypic and phenotypic characteristics.

Bacteria are usually identified using phenotype, immunological or molecular

characteristics.

Features of a Bacterial Habitat:

Most of the bacterial species found in the mouth belong to microbial communities,
called "biofilms", a feature of which has inter-bacterial communication, mediated
by two distinct phenomena.

The first is through direct cell-cell contact, which is mediated by specific protein
"adhesins" and often, as in the case of inter-species aggregation, also by
complementary polysaccharide receptors.Intra (auto-) and inter-species (co-)
aggregation both promote ordered successional integration of species into the
biofilm.

The second method of communication invoves cell-cell signalling molecules,

which are of two classes; those used for intra-species and those used for inter-
species signalling. An example of the intra-species cell-cell signalling molecules
is "quorum sensing", a process in which acyl-homoserine lactones (AHLs) induce
members of the same population to produce and release specific enzymes or to
initiate biofilm formation. As yet, no oral bacteria have been shown to produce
AHLs. However, they can produce small peptides, such as "competence
stimulating peptides", which have been shown to mediate intra-species signalling
and to promote single-species biofilm formation.
 A common form of inter-species signalling is mediated by 4, 5-dihydroxy-2, 3-
pentanedione (DPD). This spontaneously forms a family of inter-convertible
compounds, collectively called "Autoinducer-2" (Al-2). 

Fig-1:DIAGRAMMATIC REPRESENTATION OF BIOFILM DEVELOPMENT

Fig-2: BACTERIAL CO-AGGREGATION DURING PLAQUE MATURATION

The interaction between oral microflora and eucaryotic cells is highly complex and
involves active processes allowing both types of partners to co-exist. The microbial
encounter with the host is initiated by attachment of the organism to the host; and
bacterial surface proteins, termed "adhesins", that mediate this step. Bacteria also
secrete mediators that bind or invade the host.

a. Microbe identification using genotypic criteria:

It uses molecular techniques to identify bacteria by doing DNA or RNA
analysis of the bacterium’s genome .
This usually involves detecting the presence of a gene, or a part thereof, or
an RNA product that is specific for a particular organism.
In principle, the presence of a specific gene or a particular nucleic acid
sequence is interpreted as a definitive identification of the organism.
The genotypic approach is highly specific and often very sensitive.

The rapid development of molecular techniques during the past decade has
revolutionized the field of microbiology.

Two issues are of profound importance :-

 First, the discovery of phylogenetically informative DNA sequences, such

as the 16S rRNA gene, radically changed the concept of bacterial
relatedness and provided a universal system for bacterial identification and
categorization.
 Second, it became possible to detect, identify, and type bacteria independent
of their cultivability and, by these new means, to elucidate the diversity and
spatial organization of complex oral bacterial communities.

Of considerable benefit also, has been the fact that the same nucleic acid-based
molecular approaches can be applied in all microbial environments, ranging from
the oral cavity, to the surfaces of historical monuments, to the depths of open
oceans.

This has led to development of versatile PCR- and hybridization-based techniques

that allow a rapid and convenient analysis of the bacterial contents of oral samples
and offer previously unattainable possibilities for expanding studies on bacterial
epidemiology and characterization.
The simultaneous and inter-related rapid advances in genome sequencing, mass
spectrometry and computing power have provided the tools for examining oral
bacteria and their interactions with each other and the host at a global molecular
level. 

Analyses of gene phylogenies and DNA-composition reveal that an important

source of genetic innovation in prokaryotes is the transfer of sequences from other
bacteria. The advent of genomic data now allows determination of the importance
of horizontally-transferred genes on a genomic scale.

By comparative analysis of fully- sequenced oral bacteria and between them and
non-oral relatives, it is clear that horizontal gene transfer (HGT) is extremely
frequent in oral pathogens and that this process probably also takes place also
between bacteria and oral fungi. It is likely that HGT is facilitated, in oral biofilms
such as dental plaque, by the close physical contact between phylogenetically
distant bacteria. .

In some cases, certain sets of genes are shared between more than two species co-
inhabiting the mouth ecosystem, supporting the idea that there is an environment-
associated gene pool formed by sequences of special adaptive importance. This
habitat gene reservoir is also present in other natural ecosystems.

With the coming of  Human Microbiome Project (HMP) , a United

States National Institutes of Health (NIH) research initiative to improve
understanding of the microbial flora involved in human health and disease,
launched in 2008 & its second phase, known as the Integrative Human
Microbiome Project (iHMP) launched in 2014 it was possible to generate
resources to characterize the microbiome and elucidating the roles of microbes in
health and disease states.

Important components of the HMP were culture-independent methods of

microbial community characterization, such as metagenomics(which provides a
broad genetic perspective on a single microbial community), as well as
extensive whole genome sequencing (which provides a "deep" genetic perspective
on certain aspects of a given microbial community, i.e. of individual bacterial
species). The latter served as reference genomic sequences — 3000 such sequences
of individual bacterial isolates are currently planned — for comparison purposes
during subsequent metagenomic analysis.

Fig-3: METHODS EMPLOYED FOR BACTERIAL IDENTIFICATION

At present, 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 primers 2. Quantitative PCR 3. DNA hybridization assays

4. Ribosomal 16S cloning & sequence analysis 5. FISH and microscopy

They essentially require recovery of the bacterial DNA as an initial step of

analysis.

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.
Fig-4: METHODS OF DNA RECOVERY

1. PCR• Or “Polymerase Chain Reaction”

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’ & requires a Primer.

1a. 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
specificstrand of DNA from a specific specie & serves as a startingpoint for DNA
synthesis. They are required for DNA replicationbecause the enzymes that catalyze
this process. Primers are usually short, chemically synthesized oligonucleotides,
with a lengthof about twenty bases.

Almost every DNA based method uses PCR as it allows detection of DNA from as
low as one cell which is possible to do due to extensive, detailed analysis .Thus it
allows : Specific amplification of DNA from a target species even in the presence
of hundred of species.

Conventional PCR requires Gel-electrophoresis for amplification analysis.

Labelled probes are used.

Multiple amplifications can be analysed at specific time period during reaction

period.

PCR is widely used due to following reasons:

 Even a minuscule quantity of DNA can be studied, as a single DNA

molecule is adequate for amplification
 Rapid Clinical diagnostic procedures.
 Enables identification of different species.
 PCR allows researcher to identify uncultivable bacteria.

The general concept of PCR, includes primers, DNA polymerase, nucleotides,

specific ions, and DNA template, and consists of cycles that comprise steps of
DNA denaturation, primer annealing, and extension.

Variations of PCR :- The basic PCR methodology is modified to provide

sophisticated analytical tools.

Nested PCR

Multiplex PCR

2. Real Time PCR(quantitativePCR-qPCR):

In real time PCR (also denoted as quantitative PCR—qPCR), fluorescence is
measured after each cycle and the intensity of the fluorescent signal reflects
the momentary amount of DNA amplicons in the sample at that specific
time.
In initial cycles the fluorescence is too low to be distinguishable from the
background. However, the point at which the fluorescence intensity
increases above the detectable level corresponds proportionally to the initial
number of template DNA molecules in the sample. This point is called the
quantification cycle and allows determination of the absolute quantity of
target DNA in the sample according to a calibration curve constructed of
serially diluted standard samples (usually decimal dilutions) with known
concentrations or copy numbers .
The main advantages of qPCR are that
 It provides fast and high-throughput detection and quantification of
target DNA sequences in different matrices. The lower time of
amplification is facilitated by the simultaneous amplification and
visualization of newly formed DNA amplicons.
 qPCR is safer in terms of avoiding cross contaminations because no
further manipulation with samples is required after the amplification.
 Other advantages of qPCR include a wide dynamic range for
quantification (7–8 Log10) and the multiplexing of amplification of
several targets into a single reaction
 qPCR assays are used not only for the detection, but also to determine
the presence of specific genes and alleles, e.g., typing of strains and
isolates, antimicrobial resistance profiling, toxin production, etc.
 qPCR plays an important role in the detection, quantification, and
typing of viral pathogens & has become an indispensable tool in virus
diagnostics .
In cases when critical and timely intervention for infectious disease is
required, qPCR is capable of providing the required information in a
short time then the traditional, slow, and multistep culture techniques.
In such cases, however, results must be confirmed from, the
phenotypic and biochemical features of bacterial isolates .

Disadvantage of qPCR

 although qPCR-based typing tests are faster, their results should be

correlated with phenotypic and biochemical tests 
  it has an inherent incapability of distinguishing between live and
dead cells because samples for DNA isolation usually initially
contain very low numbers of target bacteria.

3. DNA Hybridization assay :

It measures-

a. the degree of genetic similarity between pools of DNA sequences

b. It is also possible to determine the genetic distance between two sequences

c. Because of the complexity of bacterial ecology, it helps in the necessity to

identify many species of bacteria from single sample

It  is the gold standard to distinguish bacterial species, with a similarity value
greater than 70% indicating that the compared strains belong to distinct species.

Thus, it is used to identify the bacterial species by the criteria that, every bacterial
species may have at least one or many unique sequences of DNA & so is the
superior method for establishing bacterial species.

Further identification & differentiation of the bacterial species is carried out by

the analysis or sequencing of the 16s rRNA.

4. Ribosomal 16S cloning & sequence analysis :

The gene target most commonly used for bacterial identification & classification
is the 16S rRNA gene, which is an approximately 1500-bp gene encoding a portion
of the 30S ribosomal subunit .

rRNA sequences are mainly used in ranking biological phylogenetic nomenclature

including that of microorganisms.

16S rRNA is a type of RNA that plays a major role in synthesis of protein. rRNA
contains very well-conserved regions among biological species, which makes the
comparison of 16S rRNA sequences possible in studies of molecular evolution.
16S rRNA sequences also enable the identification of microorganisms because the
16S rRNA contains variable sequences that change according to different species.

More than one 16S rRNA sequences may exist in a single bacterium.
It is considered to be fast and better alternative to other methods of bacterial
identification. Along with its use identifying the bacteria, 16S sequencing can also
be used to re-categorize the bacteria into new species.

The microflora within the oral biofilm is quiet complex & determination of the
microbial populations responsible for oral infections is even more difficult. Also,
Bacteria present in the oral cavity are both, gram positive and gram negative.
Therefore, knowledge of the ecological relationships among bacterial species in a
habitat of oral cavity can direct investigations on the critical bacterial interactions.

Hence,16S rRNA genes have been used to investigate the composition samples
from the human oral cavity. The 16S rRNA gene sequences are compared with
other known 16S rRNA sequences to identify the bacteria species. So far, around
700 orally derived 16S sequences are deposited in GenBank .

Advantages of 16SrRNA:

 16S rRNA gene sequence analysis is not only widely used as a taxonomic
tool but also recognized as an effective reference method for bacterial
identification .
 Samples for 16S rRNA sequence analysis have been expanded from the
bacteria in environments such as the oceans and soils to clinical settings.
 It allows to obtain accurate information regarding contaminated
environments within health care facilities, & comprehensive reevaluation of
microorganisms using culture-independent methods .
 16S rRNA gene sequence analysis shows higher sensitivity to detect
specific bacteria than the usual culture method
 16S rRNA gene sequence analysis has been used to identify novel and
emerging pathogens and to define complex microbial communities . This
analysis is especially valuable for detecting bacteria that are slow growing,
biochemically inert or variable, and fastidious.
 In addition, 16S rRNA gene sequence analysis has enhanced our
understanding of previously unrecognized, often opportunistic pathogens.
 According to recent studies, this molecular biology analytical method has
been used in the search of pathogens of infectious diseases, reporting a
higher rate of detection than the usual culture method .
 Indeed, 16S rRNA gene sequence analysis provides a comprehensive
assessment of microbial diversity compared with culturing alone, and is an
excellent complement to the culturing approaches.
 Limitations of 16SrRNA method:
Although 16S rRNA sequencing is being used widely for bacterial
identification, there are no specific guidelines for using the technique and
interpreting the sequence data. As , no threshold values are available, one
of the major drawback in the interpretation of sequence data is concerned
with the assigning the bacterial species in response to the likeness of search
results.
This technique may not be useful when two different bacterial species share
almost the same 16S rRNA sequence.
5. FISH & Microscopy :
The detection of whole-bacterial cells via the labeling of specific nucleic
acids with fluorescently labeled oligonucleotide probes is called
Fluorescence In Situ Hybridization (FISH).
The rRNA molecule, which exists in multiple copies in the cell (up to 104-
105), is an excellent target for fluorescently labeled oligonucleotide probes
which are directed against regions on the rRNA molecule specific for a
bacterial group, genus or species.
The small probes (16-20 nucleotides) cross the bacterial cell wall and
hybridize with their complementary target sequence.
Evaluation of the test result is done by epifluorescence microscopy.

b. Microbe identification using phenotypic criteria:

Phenotypic properties are expressed properties of the organism like shape,
size, staining properties and reactions in biochemical tests.
So, phenotypic criteria are based on observable physical or metabolic
characteristics of bacteria.
The phenotypic approach is the classic approach to bacterial identification,
and most identification strategies are still based on bacterial phenotype.
Most of the conventional bacterial identification methods are based upon the
phenotypic characteristics of bacteria which can be further classified as:
1. Identification by Bacterial cultivation.
2. Bacterial identification based upon their enzymatic capabilities.
 3. Tests for presence of metabolic pathways

The phenotypic characterizations used in diagnostic bacteriology based on

tests that establish a bacterial isolate’s morphology and metabolic
capabilities are commonly done to identify the phenotypic criteria from
among the following :
i) The range of temperatures within which the organism can grow.
ii) The requirement of specific nutrients in order to grow.
iii) The ability of the organism to make one or more specific chemicals
(such as indole).
iv) Production of specific enzymes by the organism (such as coagulase).
v) Fermentation of specific sugars such as lactose (sugar fermentation
Tests).
vi) Sensitivity or resistance to one or more specific antibacterial agents.
vii) Presence of one or more specific proteins or antigens on its surface.

1. Cultivation of bacteria:

Culture involves placing some of the specimen in conditions where the organism
or organisms of interest can grow and multiply. Once grown in culture, most
bacterial populations are easily observed without microscopy and are present in
sufficient quantities to allow laboratory testing to be performed. The successful
transition from the in vivo to the in vitro environment requires that the nutritional
and environmental growth requirements of bacterial pathogens be met.

The bacterial cultivation has three main purposes, which are:

■ To grow and isolate all bacteria present in an infection.

■ To determine which of the bacteria that grow are most likely causing infection
and which are likely contaminants or colonizers.

■ To obtain sufficient growth of clinically relevant bacteria to allow identification.

Growth media are used in either of the two phases: liquid (broth) or solid (agar)

In some instances, a biphasic medium that contains both a liquid and a solid phase
is used.
Liquid (broth) media : These are liquid media in which bacteria grow uniformly
producing general turbidity. Certain aerobic bacteria and those containing fimbriae
(Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on
the surface of undisturbed broth. Liquid media tend to be used when a large
number of bacteria have to be grown from an inoculum suspected to be low in
bacterial numbers.

Solid (agar) media: These are are made by adding a solidifying agent to the
nutrients and water. Agar agar (simply called agar) is the most commonly used
solidifying agent. Agar doesn’t contribute any nutritive property, it is not
hydrolyzed by most bacteria and is usually free from growth promoting or growth
retarding substances.

Various types of Culture media are used depending on requirements of Bacteria to

be grown. They are:

Enrichment media

Supportive media

Selective media

Differential media

The four most critical environmental factors affecting microbial growth in culture
They include :

1. oxygen and carbon dioxide (CO2 ) availability,

2. temperature,

3. pH, and

4. moisture content of medium and atmosphere

2. Bacterial identification based upon their enzymatic capabilities :

Staining provides valuable information about bacterial morphology, Gram

reaction, and presence of such structures as capsules and endospores.
Also, microscopic observation provides little additional information as to the genus
and species of a particular bacterium.

However, for identification of particular bacterial species we depend upon

biochemical testing.

The types of biochemical reactions each organism undergoes act as a “thumbprint”

for its identification. This identification scheme depends upon following facts,

 Each different species of bacterium has a different molecule of DNA (i.e.,

DNA with a unique series of nucleotide bases).
 Since DNA codes for protein synthesis, then different species of bacteria
must, by way of their unique DNA, be able to synthesize different protein
enzymes.
 Enzymes catalyze all of the various chemical reactions of which the
organism is capable. This, in turn, means that different species of bacteria
must carry out different and unique sets of biochemical reactions.

Principle of Enzyme based tests:

In diagnostic bacteriology enzyme-based tests are designed to measure the

presence of one specific enzyme or a complete metabolic pathway that may
contain several different enzymes.

These tests usually provide rapid results because they can be performed on
organisms already grown in culture & are commonly used to determine the
presence of a single enzyme.

These tests are easy to perform and interpret and often play a key role in the
identification scheme.

Although most single enzyme tests do not yield sufficient information to provide
species identification, they are used extensively to determine which subsequent
identification steps should be followed.

For example - the catalase test can provide pivotal information and is commonly
used in schemes for gram-positive identifications. The oxidise test is of
comparable importance in identification schemes for gram-negative bacteria.
Various EB tests are:

1. Catalase Test: Most bacteria are catalase-positive; however, certain genera

that don’t carry out aerobic respiration, such as Streptococcus, Lactobacillus,
and Clostridium, are catalase-negative
2. Oxidise Test: The test is initially used for differentiating between groups of
gram-negative bacteria. Among the commonly encountered gram-negative
bacilli Enterobacteriaceae, Stenotrophomonas maltophilia, and
Acinetobacter spp. are oxidase-negative, whereas many other bacilli, such as
Pseudomonas spp. and Aeromonas spp., are positive. The oxidase test is also
a key reaction for the identification of Neisseria spp. (oxidase-positive).
3. Indole Test: Indole production is a key test for the identification of
Escherichia coli.
4. Urease Test: The urease test helps to identify certain species of
Enterobacteriaceae, such as Proteus spp., and other important bacteria such
as Corynebacterium urealyticum and Helicobacter pylori.
5. PYR Test: This test is used for the detection of pyrolidonyl arylamidase
(also called pyrolidonyl aminopeptidase) activity in certain groups of
bacteria, such as Streptococcus pyogenes (group A strep), Enterococcus
spp., some coagulase-negative staphylococci, and some Enterobacteriaceae.
PYR detects PYRase activity in streptococcal organisms
6. Hippurate Hydrolysis: Hippurate hydrolysis test is used for detection of
hippurate hydrolyzing bacteria, mainly Streptococcal species.

8. Tests for presence of metabolic pathways :

Several identification schemes are based on determining what metabolic

pathways an organism uses and the substrates processed by these pathways.
In contrast to single enzyme tests, these pathways may involve several
interactive enzymes.
The presence of an end product resulting from these interactions is measured
in the testing system.
Assays for metabolic pathways can be classified into three general
categories:
a. Carbohydrate oxidation and fermentation -- In most instances the
presence of acid byproducts is detected by a change in the pH indicator
incorporated into the medium. Facultative anaerobic and anaerobic
bacteria are capable of fermentation .

b. Amino acid degradation and single substrate utilizations-- Determining

the ability of bacteria to produce enzymes that either deaminate,
dihydrolyze, or decarboxylate certain amino acids is often used in
identification schemes.

c. Single substrate utilizations-- Whether an organism can grow in the

presence of a single nutrient or carbon source provides useful
identification information. Growth is determined by observing the
presence of bacterial colonies or by using a pH indicator to detect end
products of metabolic activity.

To summarise :

In routine clinical dentistry, most infections are diagnosed on clinical

grounds alone. In rare situations, such as atypical presentations or in
ambiguous clinical circumstances, aid is sought from the laboratory.
Culture-based identification has been the traditional gold standard’
for the detection of most bacterial and fungal infections.
With the ever-expanding list of molecular techniques being
developed, the genetic approach to organism identification will
continue to grow and become more integrated into diagnostic
microbiology laboratory protocols.
Also, the emerging new microarray technology will facilitate great
strides in understanding the structure and dynamics of oral bacterial
communities and bacteria-host interactions, and will form the basis
for developing novel diagnostics for oral infections.