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ENVIRONMENTAL MICROBIAL BIOTECHNOLOGY (MICR 307)
INDICATOR
MICROORGANISMS
PROF A.O. OLANIRAN F3 03-028
Department of Microbiology
School of Life Sciences University of KwaZulu-Natal
Westville Campus
INDICATOR MICROORGANISMS
Monitoring and detection of indicator and disease-
causing microorganisms are a major part of sanitary
microbiology.
The routine examination of environmental samples
for the presence of intestinal pathogens is often a
tedious, difficult, and time-consuming task.
It is therefore customary to look for certain indicator
mos (IM) whose presence indicates that pathogenic
mos may be present.
Coliform bacteria normally occur in the intestines of
all warm-blooded animals and excreted in great no
in feces.
Their absence from water is an indication that the
water is bateriologically safe for human consumption.
Their presence is indicative that other kinds of mos
capable of causing disease may also be present and
that the water is potentially unsafe to drink.
U.S. Public Health service adopted the coliform group as an indicator of fecal contamination of drinking water in 1914.
Many countries have adopted coliforms and other groups of bacteria as official standards for drinking water, recreational bathing waters, wastewater discharges, and various foods.
Indicator Microorganisms are also used to assess the efficacy of food processing and water and wastewater treatment processes.
CRITERIA FOR AN IDEAL
INDICATOR ORGANISM
The organism should be;
useful for all types of water. It should be suitable for
the analysis of all types of water: tap, river, ground,
recreational, sea, waste etc.
be present whenever enteric pathogens are present.
have a reasonable longer survival time than the
hardiest enteric pathogen.
NOT grow in water - It should not reproduce in the contaminated water and produce an inflated value.
The testing method should be easy to perform. The assay procedure should have great specificity and high sensitivity.
The density of the indicator organism should have some direct relationship to the degree of fecal pollution.
The organism should be a member of the intestinal microflora of warm-blooded animals.
Unfortunately, no one indicator meets all
these criteria.
Thus, various groups of mos have been
suggested and used as indicator
organisms.
ESTIMATED LEVELS OF IM IN RAW SEWAGE
ORGANISM CFU PER 100 ML
Coliforms 107 – 109
Fecal coliforms 106 – 107
Fecal streptococci 105 – 106
Enterococci 104 – 105
Clostridium perfringe 104
Staphylococcus (Coagulase +ve) 103
Pseudomonas aeruginosa 105
Acid-fast bacteria 102
Coliphages 102 – 103
Bacteroides 107 - 1010
TOTAL COLIFORMS
All aerobic and facultative anaerobic, Gram
negative, non-spore-forming, rod-shaped bacteria
that ferment lactose with gas and acid formation
within 48 h at 35 oC (multiple-tube fermentation
technique) or
All aerobic and many facultative anaerobic, Gram-
negative, non-spore-forming, rod-shaped bacteria
that develop a red colony with a metallic sheen
within 24 h at 35 oC on an Endo-type medium
containing lactose (membrane filter technique).
The coliform group has been used as the standard for
assessing fecal contamination of recreational and
drinking waters for most of the century.
Absence in 100 ml of drinking water ensures the
prevention of bacterial waterborne disease outbreaks.
Relatively easy to detect
Includes Escherichia, Citrobacter, Enterobacter, and
Klebsiella species.
DEFICIENCIES WITH THE USE OF COLIFORM
BACTERIA AS INDICATORS OF WATER QUALITY
Regrowth in aquatic environments.
Regrowth in distribution systems.
Suppression by high background bacterial growth leading to underestimation of coliforms – especially when aerobic heterotrophic bacterial numbers exceed 500/ml
Not indicative of a health threat.
No relationship between enteric protozoan and viral concentration.
All members of the coliform group have been observed to regrow in natural surface and drinking water distribution systems.
The die-off rate of coliform bacteria depends on the amount and type of organic matter in the water and the temperature.
Of greatest concern is the growth or recovery of injured coliform bacteria in a distribution system because this may give a false indication of fecal contamination
Coliforms may colonize and grow in the biofilm found on the distribution system pipes, even in the presence of free chlorine. E.g. E. coli is 2400 times more resistant to free chlorine when attached to a surface than as free cells in water.
IDENTIFICATION OF
COLIFORMS IN WATER
There are three common methods used:
Most Probable Number (MPN) technique,
Membrane Filter (MF), and
the Presence-Absence (P-A) tests.
MPN Test
Allows detection of the presence of coliforms in a
sample and estimation of their numbers.
used for over 80 years as a water quality monitoring
method.
The method consists of inoculating a series of tubes
with appropriate decimal dilutions of the water
sample.
Production of gas, acid formation or abundant growth
in the test tubes after 48 h of incubation at 35 oC in
lactose broth constitutes a positive presumptive
reaction.
All tubes with a positive presumptive reaction are
subsequently subjected to a confirmation test.
The formation of gas in a brilliant green lactose bile
broth fermentation tube at any time within 48 h at
35oC constitutes a positive confirmation test.
Completed Test: Growth on EMB or LES Endo agar
plates, Gram reaction and spore staining
Once the positive tubes have been identified and
recorded, it is possible to estimate the total number of
coliforms in the original sample by using an MPN
table that gives numbers of coliforms per 100 ml.
Multiple Tube Water Testing Procedures
The fecal coliform test (using an EC medium).
The results are well authenticated and have been used as a basis for standards of bacteriological quality of water supplies.
The results of the MPN technique are expressed in terms of the most probable number (MPN) of microorganisms present.
This number is a statistical estimate of the mean number of coliforms in the sample.
MPN Index and 95% confidence limits for various combinations of positive results
when five tubes are used per dilution of the sample (10 ml, 1.0 ml, and 0.1 ml)
The MPN for combinations not appearing in
the Table, or for other combinations of tubes
or dilutions, may be estimated by Thomas’
simple formula:
MPN/100 ml = No of positive tubes x 100
√ (ml sample in negative tubes
x ml samples in all tubes)
E.g. 3-3-2
MPN offers a semi-quantitative enumeration of coliforms.
The precision of the estimation is low and depends on the number of tubes used for the analysis.
Many factors may significantly affect coliform bacteria detection by MPN, especially during the presumptive phase.
Interference by high numbers of non-coliform bacteria, as well as the inhibitory nature of the media.
The MPN technique lacks precision in qualitative and quantitative terms.
The time required to obtain results is higher than with the membrane filter technique.
However, this technique remains useful, especially when the conditions do not allow the use of the membrane filter technique, such as turbid or colored waters.
Multiple tube method for coliform detection
MF TEST
Allows for determination of the number of coliforms in
a sample.
Easier to perform than the MPN test because it
requires fewer test tubes and less labour (Fig 20.3,
pg 5).
A measured amount of water (usually 100 ml for
drinking water) is passed through a membrane filter
(pore size 0.45 μm) that traps bacteria on its surface.
The membrane is then placed on a thin absorbent pad that has been saturated with a specific medium designed to permit growth and differentiation of the organisms being sought. E.g. Endo medium for total coliforms and m-FC for fecal coliforms.
The filter is incubated appropriately. E.g. 35oC for 18-24 hours for total coliforms; 44.5oC for 24 hours for fecal coliforms.
To determine the no of coliform bacteria, the no of colonies having a green sheen are enumerated.
Typical colonies of fecal coliforms on mFC agar plates
The fecal colonies will appear as dark blue.
ADVANTAGES OF MF TEST
Good reproducibility.
Single-step results often possible.
Filters can be transferred between different media.
Large volumes can be processed to increase assay sensitivity.
Time savings are considerable.
Ability to complete filtrations on site.
Lower total costs in comparison with MPN procedure.
DISADVANTAGES OF MF TESTS
High-turbidity waters limit volumes sampled.
High populations of background bacteria
cause overgrowth.
Metals and phenols can adsorb to filters and
inhibit growth.
Inability to recover stressed or injured
coliforms (from water treatment processes).
A number of chemical and physical factors involved
in drinking water treatment, including disinfection,
can cause sublethal injury to coliform bacteria,
resulting in a damaged cell unable to form a colony
on a selective medium.
Exposure of bacteria to products like chlorine may
result in injury and increased sensitivity to bile
salts or to the replacement surface-active agents
(sodium desoxycholate or Tergitol 7) contained in
some selective media.
Some improvements in the method have
increased detection of injured coliform
bacteria.
E.g. the development of m-T7 medium
formulated specifically for the recovery of
stressed coliforms in drinking water.
P-A TEST
These are NOT quantitative tests.
Answer the simple question of whether the target organism is present in a sample or not.
A single tube of lauryl sulfate-tryptose-lactose broth as used in the MPN test, but without dilutions, would be used in a P-A test.
Enzymatic assays have recently been developed that allow the simultaneous detection of total coliform bacteria and E. coli in drinking water.
E.g. Colilert System
Based on the fact that total coliform bacteria
produce the enzyme β-galactosidase, which
hydrolyses the substrate o-nitrophenyl- β-D-
galactopyranoside (ONPG) to yellow nitrophenol.
E. coli can be detected by incorporation of a
fluorogenic substrate, 4-methylumbelliferone
glucuronide (MUG) which produces a fluorescent
end product after interaction with the enzyme β-
glucuronidase found in E. coli but not in other
coliforms.
The end product is detected with a long-wave
ultraviolet (UV) lamp.
The Colilert test is performed by adding the
sample to a single bottle (P-A test) or MPN
tubes that contain powdered ingredients
consisting of salts or specific enzyme
substrates that serve as the only carbon
source for the organisms.
After 24 hours of incubation;
samples positive for total coliforms turn
yellow
E. coli-positive samples fluoresce under
long-wave UV illuminator in the dark.
Indicator Selective media Incubation conditions
Total Heterotrophic
Bacteria (THB) Nutrient agar (NA) 48 hrs at 37°C
Total coliforms (TC) mEndo agar 24 hrs at 35°C
Faecal coliforms (FC) m-FC agar 24 hrs at 44.5°C
Faecal streptococci (FS)
Oxolinic acid
aesculin azide agar
(OAA)
48 hrs at 42°C
Presumptive Salmonella
spp. (SAL) S-S agar 24 hrs at 35°C
Presumptive Shigella
spp. (SHIG) S-S agar 24 hrs at 35°C
Total Coliforms & E. coli Chromocult agar 24 hrs at 37°C
MEDIA FOR FRIDAY’S PRACTICAL
FECAL COLIFORMS
Include the genera Escherichia and Klebsiella.
Differentiated in the laboratory by their ability to
ferment lactose with the production of acid and gas at
44.5oC within 24 hours.
The test indicates fecal coliforms but does not
distinguish between human and animal
contamination.
Fecal coliforms may be detected by methods similar
to those used for coliform bacteria.
For MPN method, EC broth is used
for MF method, m-FC agar is used.
FECAL STREPTOCOCCI
Group of Gram-positive Lancefield group D streptococci.
Belong to the genera Enterococcus and Streptococcus.
The genus Enterococcus includes all streptococci that share certain biochemical properties and have a wide range of tolerance of adverse growth conditions.
Differentiated from other streptococci by their ability to grow in 6.5% NaCl, pH 9.6, and 45oC.
Include Ent. avium, Ent. faecium, Ent. durans, Ent. facculis, and Ent. gallinarium.
Of the genus Streptococcus, only S. bovis and S. equinus are considered to be true fecal streptococci.
Fecal coliform/fecal streptococci (FC/FS) ratio of 4 or more has been suggested to indicate contamination of human origin, whereas a ratio below 0.7 is indicative of animal pollution.
Both MF and MPN method may also be used for the isolation of fecal streptococci.
The FC/FS Ratio
FC/FS Ratio Source of Pollution
>4.0 Strong evidence that pollution
is of human origin
2.0-4.0 Good evidence of the predominance of
human wastes in mixed population
0.7-2.0 Good evidence of the predominance of
domestic animal wastes in mixed
population
<0.7 Strong evidence that pollution is of
animal origin
The MF method uses fecal Streptococcus agar with incubation at 37oC for 24 hours.
All red, maroon, and pink colonies (due to reduction of 2,4,5-triphenyltetrazolium chloride to formazan, a red dye) are counted as presumptive fecal streptococci.
Confirmation of fecal streptococci is by subculture on bile aesculin agar and incubation for 18 hours at 44oC.
Fecal streptococci form discrete colonies surrounded by a brown or black halo due to aesculin hydrolysis.
ADVANTAGES OF FECAL STREPTOCOCCI OVER
THE COLIFORM AND FECAL COLIFORM
BACTERIA AS INDICATORS
They rarely multiply in water.
They are more resistant to environmental stress and chlorination than coliforms.
They generally persist longer in the environment.
The enterococci have been suggested as useful indicators of risk of gastroenteritis for recreational bathers and standards have been recommended.
They have been suggested as useful indicators of the presence of enteric viruses in the environment.
Clostridium perfringes
Sulfite-reducing anaerobic spore former.
Gram positive, rod shaped, and exclusively of
fecal origin.
The spores are very heat resistant (75oC for
15 min), persist for long periods in the
environment, and are very resistant to
disinfectants.
It has been suggested to be an indicator of;
past pollution
a tracer of less hardy indicators
and an indicator of removal of protozoan
parasites or viruses during drinking water
and wastewater treatment
The hardy spores of the organism limit its use
as an indicator
HETEROTROPHIC PLATE COUNT
(HPC)
Used for assessing the numbers of aerobic and
facultative anaerobic bacteria in water that derive
their carbon and energy source from organic
compounds.
Includes Gram-negative bacteria belonging to the
following genera: Pseudomonas, Aeromonas,
Klebsiella, Flavobacterium, Enterobacter, Citrobacter,
Serratia, Acinetobacter, Proteus, Alcaligenes, and
Moraxella.
Some members of this group are
opportunistic pathogens (e.g., Aeromonas,
Pseudomonas).
Although HPC is not a direct indicator of fecal
contamination, it does indicate variation in
water quality and potential for pathogen
survival and re-growth.
HPC are normally done by the spread plate method
using Plate count/Nutrient agar incubated at 35oC
for 48 h.
A low-nutrient medium, R2A has been recommended
for disinfectant damaged bacteria with an
incubation period of 5-7 days at 28oC.
HPC numbers can vary greatly depending on the
incubation temperature, growth medium, and
length of incubation.
BACTERIOPHAGE
Proposed as appropriate indicators of fecal
pollution because of their constant presence in
sewage and polluted waters.
Also suggested as indicators of viral pollution.
Closely resemble enteric viruses in terms of;
the structure, morphology, and size
behavior in the aquatic environment
They are therefore used;
To evaluate virus resistance to disinfectants
To evaluate virus fate during water and wastewater treatment
As surface and groundwater tracers
The use of bacteriophage as indicators of fecal pollution is based on the assumption that their presence in water samples denotes the presence of bacteria capable of supporting the replication of the phage
Two groups of phage studied
Somatic coliphage – infect E. coli host
strains through cell wall receptors
F-specific RNA coliphage – infect strains of
E. coli and related bacteria through the F+ or
sex pili
Somatic coliphages in water generally
outnumber F-RNA coliphage by a factor of 5
F-specific coliphage are similar in size and shape to
many of the pathogenic human enteric viruses
Coliphage f2, Ф174, MS-2, and PRD-1 are
commonly used as tracers and evaluation of
disinfectants
F-specific phage are not considered indicators of
fecal pollution
Infrequently detected in human fecal matter
Show no direct relationship to fecal pollution level
ADVANTAGES
They can be detected by simple and inexpensive techniques
Yield results in 8-18 hours
They can be detected by both plating method (the agar overlay method) and the MPN method in volumes ranging from 1 to 100 ml
Bacteriophage assay technique?
Bacteriophage of Bacteroides fragilis have been suggested as indicators of human viruses in the environment.
Bacteriophages that infect B. fragilis appear to be exclusively human in origin and appear to be present only in environmental samples contaminated with human fecal pollution.
Help differentiate human from animal contamination
They are absent from natural habitats
They are unable to multiply in the environment
They have decay rate similar to that of human viruses
OTHER INDICATOR ORGANISMS
A number of other organisms have also been
considered to have potential as alternative IO
or for use in certain applications (e.g.
recreational waters).
These include Pseudomonas spp., yeasts,
acid fast mycobacteria (M. fortuitum and M.
phlei), Aeromonas, and Staphylococcus.
Within the genus Pseudomonas, P. aeruginosa is of
significant public health concern.
It is associated with eye, ear, nose, and throat
infections.
It is also the most common opportunistic pathogen
causing life-threatening infections in burn patients and
immunocompromised individuals.
Numerous cases of folliculitis, dermatitis, and ear
(swimmer’s ear) and urinary tract infections are due to
P. aeruginosa associated with swimming in contaminated
or poorly maintained swimming pools and hot tubs.
Folliculitis is inflammation of one or more hair
follicles. It can occur anywhere on the skin. Common
symptoms include a rash, itching, and pimples or pustules
near a hair follicle in the neck, groin, or genital area
S. aureus and C. albicans have been proposed as better
indicators of respiratory tract, skin and eye infections
associated with swimming.
S. aureus have also been recommended as
an additional indicator of the sanitary quality
of recreational waters, because its presence
is associated with human activity in
recreational waters.
Aeromonas, especially A. hydrophila have
been associated with foodborne outbreaks.
It is also considered an opportunistic
pathogen in humans.
STANDARDS AND CRITERIA
FOR INDICATORS
Authority for setting drinking water standards was
given to the U.S. EPA in 1974 when Congress
passed the Safe Drinking Water Act.
Similarly, authority for setting standards for domestic
wastewater discharges is given under the Clean
Water Act.
A drinking water standard is legally enforceable in
the United States.
Criteria and guidelines are terms used to describe recommendations for acceptable levels of IM.
Ideally, all standards would indicate that an unacceptable public health threat exists or that some relationship exists between the amount of illness and the level of indicator organisms.
The use of microbial standards also requires the development of standard methods and quality assurance or quality control plans for the laboratories that will do the monitoring.
Knowledge of how to sample and how often to
sample is also important.
Sampling must proceed in some random fashion so
that the entire system is characterized.
Some positive samples may be allowed or tolerance
levels or averages may be allowed because of the
wide variability in numbers of indicators in water.
U.S. Federal and State Standards
for Microorganisms
AUTHORITY STANDARDS
U.S. EPA
Safe Drinking Water Act 0 coliforms/100 ml
Clean Water Act
Wastewater discharges 200 fecal coliforms/100 ml
Sewage sludge < 1000 fecal coliforms/4g
< 3 Salmonella/4g
< 1 enteric virus/4g
< 1 helminth oval/4g
CALIFORNIA
Wastewater reclamation
For irrigation ≤ 2.2 MPN coliforms
ARIZONA
Wastewater reclamation for 25 fecal coliforms/100 ml
Irrigation of golf courses 125 enteric virus/40 liters
No detectable Giardia/40 liters
Drinking Water Criteria of the
European Union
TAP WATER
Escherichia coli 0/100 ml
Fecal streptococci 0/100 ml
Sulfite-reducing clostridia 0/20 ml
BOTTLED WATER
Escherichia coli 0/250 ml
Fecal streptococci 0/250 ml
Sulfite-reducing clostridia 0/50 ml
Pseudomonas aeruginosa 0/250 ml
From European Union (1995)
Guidelines for Recreational Water
Quality Standards
Country Regime Criteria or
or agency (samples/time) standarda
U.S. EPA 5/30 days 200 FC/100ml
< 10% to exceed 400/ml
Fresh waterb
33 enterococci/100 ml
126 FC/100 ml
Marine watersb
35 enterococci/100 ml
EUROPEAN ECONOMIC 2/30 daysc 500 coliforms/100 ml
COMMUNITY 100 FC/100 ml
100 FS/100 ml
0 Salmonella/liter
0 Enteroviruses/10 L
ONTARIO, CANADA 10/30 days ≤ 1000 coliforms/100 ml
≤ 100 FC/100 ml
From Saliba, 1993; U.S. EPA, 1986.
a All bacterial numbers in geometric means.
b Proposed, 1986.
c Coliforms and fecal coliforms only.
Other current methods used for detecting
coliforms
1. Enzymatic Methods
1.1. General principles
The use of microbial enzyme profiles to detect indicator bacteria is an attractive alternative to classical methods.
Enzymatic reactions can be group-, genus- or species-specific, depending on the enzyme targeted.
Moreover, reactions are rapid and sensitive.
Thus, the possibility of detecting and enumerating coliforms through specific enzymatic activities has been under investigation for many years now.
β-D-glucuronidase is an enzyme which catalyzes the hydrolysis of β -D-glucopyranosiduronic derivatives into their corresponding aglycons and D-glucuronic acid.
1.2. P/A techniques and enumeration by multi-
tube techniques using enzymatic methods
The incorporation of MUGlu into lauryl tryptose broth
used as the medium for the multi-tube fermentation
(MTF) technique was first proposed for rapid
detection and immediate confirmation of E. coli in
food and water samples (Feng and Hartman, 1982).
The presence of methylumbelliferone due to the
hydrolysis of MUGlu (positive samples) was detected
by exposure to long-wave UV light and visualization
of blue-white fluorescence.
1.3. MF technique conjugated to enzymatic
detection of coliforms
Dahlen and Linde (1973) evaluated the incorporation of MUGlu into agar media to detect the presence of β-D-glucuronidase activity (E. coli).
Brenner et al. (1993) developed the MI agar medium, containing the fluorogenic MUGal and the chromogenic IBDG, to simultaneously detect Total Coliform and E. coli in waters.
The method was shown to be sensitive, selective, specific and rapid (results available in 24 h)
ChromoCult® Coliform Agar
Selective agar for the simultaneous detection of total
coliforms and E. coli in drinking water and processed
food samples.
US-EPA approved
Primarily the interaction of carefully selected
peptones in addition to pyruvate, sorbitol and a
phosphate buffer guarantees rapid colony growth,
even for the sublethally injured coliforms.
MODE OF ACTION
The growth of accompanying Gram-positive bacteria
flora as well as some Gram-negative bacteria flora is
largely inhibited by the use of Tergitol®7 which has
no negative effect on the growth of the coliform
bacteria.
A new combination of two chromogenic substrates
which allow for the simultaneous detection of total
coliforms and E. coli has been developed by Merck
Coliform identification
The characteristic enzyme for coliforms, ß-D-
galactosidase, cleaves the Salmon-GAL substrate
and resulting in a salmon to red colouration of the
coliform colonies.
E. coli identification
The substrate X-glucuronide is used for the identification of
ß-D-glucuronidase which is characteristic for E. coli.
E. coli cleaves both Salmon-GAL and X-glucuronide, so
that positive colonies take on a dark-blue to violet colour.
These are easily distinguished from the other coliform
colonies which have a salmon to red colour.
E. coli Citrobacter
1.4. Direct determination of enzymatic
activity by fluorimetry
More recently, the β-D-galactosidase and β -D-
glucuronidase properties of TC and E. coli have been exploited on seawater samples in rapid assays without any cultivation steps.
George et al. (2000) finalized a protocol based on the fluorogenic substrates MUGal and MUGlu for a direct enzymatic detection of FC in freshwaters in 30 min.
These methods allow a rapid and direct estimate of the level of microbiological contamination of surface water.
Their detection limits (20 CFU/100 ml for FC to about 340 CFU/100 ml for TC) preclude their use for the monitoring of drinking water.
An automated analyzer [Colifast CA-100 (Colifast
Systems, Oslo, Norway)] has been developed on the
basis of the enzymatic properties of coliforms.
The sample is incubated (after concentration by
filtration, if required) at 37oC for TC and 44oC for FC in
a growth medium selective for coliforms and containing
MUGal.
If the number of coliforms in the sample is sufficient, the
increase in fluorescence due to MUGal hydrolysis by
the coliforms present can be measured in less than 2h.
If not, the detection of a significant fluorescence signal
requires the prior growth of coliforms.
1.5. Detection of coliforms by enzymatic methods
using solid-phase cytometry
Recent method has been focused on the development of rapid enzymatic procedures applicable to drinking water with the objective of reaching results within a working day’s schedule.
Van Poucke and Nelis (1999a) evaluated instrumental detection of fluorescent signals for decreasing the time analysis of an enzymatic membrane filtration test.
They used a laser-scanning device which detects and enumerates low numbers of fluorescently labeled cells by means of a solid phase cytometry technique.
This system offers a very low quantitative direct detection limit allowing the detection of between 1 and 1000 fluorescently labeled cells distributed on a membrane.
Using this system, Van Poucke and Nelis (1999b) proposed a test allowing the detection of E. coli and TC within 3.5 h.
2. Molecular methods
Molecular methods have been developed to increase
the rapidity of analysis.
They are able to achieve a high degree of sensitivity
and specificity without the need for a complex
cultivation and additional confirmation steps.
Some of these methods permit the detection of
specific culturable and/or non-culturable bacteria
within hours, instead of the days required with the
traditional methods
2.1. Immunological methods
Immunological methods are based on the specific recognition
between antibodies and antigens and the high affinity that is characteristic of this recognition reaction.
Depending on the taxonomic level of the targeted antigens, immunological methods permit detection of antigens at family, genus, species or serotype levels.
Two types of antibodies can be produced: polyclonal and monoclonal antibodies, whichever are more specific to the target organism.
The properties of the antigen–antibody complex can be used: to perform an immunocapture of cells or antigens by enzyme-
linked immunosorbent assay (IMS or ELISA), or
to detect targeted cells by immunofluorescence assay (IFA) or immuno-enzyme assay (IEA).
2.2. Nucleic acid-based methods
Most of the nucleic acid methods use molecular hybridization properties, which involve the complementary sequence recognition between a nucleic probe and a nucleic target.
A hybridization reaction can be realized between a nucleic DNA probe and a chromosomic DNA sequence (DNA–DNA hybridization) or an rRNA or tRNA sequence (DNA–RNA hybridization).
Specificity, here, depends on the phylogenetic degree of conservation of the target within the taxonomic target group.
These methods offer taxonomic information at different levels, such as classes, genera, species or subspecies.
Some of them can be performed without the need for a complex cultivation step, thereby permitting the detection of specific bacteria within hours, instead of few days required with the cultivation-based methods.
The more frequently used nucleic-acid-based methods are the polymerase chain reaction (PCR) and the in situ hybridization (ISH) methods.
2.2.1. Polymerase chain reaction methods
PCR allows a DNA target fragment to be amplified by cycling replication. This replication, which can be performed in vitro or in situ, consists of a chain reaction catalyzed by a DNA polymerase (Taq polymerase) and the use of oligonucleotidic primers.
Detection specificity depends on the degree of homology
and complementarity between target and primer and on hybridization temperature.
The cycling of PCR results in an exponential amplification of the amount of the target sequence and significantly increases the probability of detecting a rare sequence or relatively low numbers of target microorganisms in a sample.
Figure 1. Agarose gel electrophoresis of PCR products of toxigenic V.
cholerae, S. Typhimurium, and E. coli O157:H7. Lane 1: 236-bp PCR product of
the STM4497 gene of S. typhimurium; lane 2: 360-bp PCR product of the ctx
gene of V. cholerae; lane 3: 432-bp PCR product of the rfbE gene of E. coli
O157:H7; lane 4: 100-bp DNA ladder.
Figure 2. Specificity of multiplex PCR. Lane 1: 100 DNA ladder; lane2:
positive control; lane 3: food sample contaminated with S. typhimurium,
toxigenic V. cholerae and E. coli O157:H7; lan4-10: Shigella dysenteriae,
Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhi,
Campylobacter jejuni, Klebsiella pneumoniae, Yersinia enterocolitica.
2.2.2. In situ hybridization techniques
ISH uses oligonucleotide probes to detect complementary nucleic
acids sequences.
This method exploits the ability of nucleic acids to anneal to one another in a very specific complementary way to form hybrids.
The probes are specific because they are built from, and are complementary to, selected nucleic acids sequences which are unique to a given microorganism, species or group.
The probes can target either DNA or RNA molecules.
Use of rRNA sequences (5S, 16S and 23S) to study phylogenetic relationships on the basis of their divergence and to develop determinative hybridization probes is now well established.
