Faecal indicator organsim

Problem: wastewater discharges are made into

receiving streams or rivers which serve as the
drinking water source for other communities
downstream
• Potable water must be free of chemical and
biological agents harmful to health.
• Water may be perfectly clear and still be
contaminated with pathogenic organisms.
 Indicator organsim
• Indicator organisms are microorganisms
whose presence in water indicates probable
presence of pathogens (disease-causing
organisms)
• Microbiological indicator organisms can be
used to monitor hygienic conditions in food
production.
• Coliforms
• Fecal coliforms
• Fecal streptococci
• Enterococci
• Clostridium perfringens
• Staphylococcus (coagulasepositive)
• Pseudomonas aeruginosa
• Acid-fast bacteria
• Coliphages
• Sanitary survey of drinking water and drinking
water supplies:-by sanitarians and engineers-
inspection of
1) source of raw water
2) operation of water purification plant or
construction of well
3) mechanism of water distribution to
consumers
• HACCP systems are implemented in most
companies to certify the safety and
consistency of products.
 Total coliforms

• Gram-negative, rod-shaped bacteria .

• It includes thermotolerant coliforms and bacteria of faecal
origin, as well as some bacteria that may be isolated from
environmental sources.
• Its presence may or may not indicate faecal contamination.
(ex)
• It might be caused by entry of soil or organic matter into
the water or by conditions suitable for the growth of other
types of coliform.
• In the laboratory total coliforms are grown in or on a
medium containing lactose, at a temperature of 35 or 37
°C. They are provisionally identified by the production of
acid and gas from the fermentation of lactose.
 Thermotolerant (faecal) coliforms

• The term “faecal coliform” has been used in water

microbiology to denote coliform organisms which grow at
44 or 44.5 C and ferment lactose to produce acid and gas.
• the presence of thermotolerant coliforms nearly always
indicates faecal contamination.
• Usually, more than 95 per cent of thermotolerant coliforms
isolated from water are the gut organism Escherichia coli,
the presence of which is definitive proof of faecal
contamination.
• In the laboratory thermotolerant coliforms are grown on
media containing lactose, at a temperature of 44 or 44.5 °C.
They are provisionally identified by the production of acid
and gas from the fermentation of lactose.
• determination of the more specific indicator, E. coli
 Faecal streptococci
• The presence of faecal streptococci is evidence
of faecal contamination.
• It tend to persist longer in the environment
than thermotolerant or total coliforms and are
highly resistant to drying.
• Faecal streptococci grow in a medium
containing sodium azide, at a temperature of
37-44 °C. They are usually detected by the
reduction of a dye (generally a tetrazolium-
containing compound) or the hydrolysis of
aesculin.
 Coliform Bacteria
• Indicators of Fecal Contamination bacteria in
the family Enterobacteriaceae generally
include the genera Enterobacter, Klebsiella,
Citrobacter, and Escherichia present in the
intestinal tract of numerous organisms,gram
negative, non-spore forming rods,ferment
lactose with acid and gas production in 48
hours at 35 °C.
• Found in intestinal tracts of cold and warm-
blooded animals
 Fecal coliforms
• Present in sewage and indicate possibility of human
pathogens
•Distinguished from Total coliform by ability to ferment
lactose at 44.5°C
•Group members: E. coliand Klebsiella(not always fecal
often associated with paper, textile & pulp waste)
•If fecal coliforms are present it is presumed that human
or animal excrement is present
•Diseases such as typhoid fever, hepatitis, gastroenteritis,
dysentery and ear infections can be contracted in
water with high Fecal coliform level
E. Coli
•Escherichia coli is a specific species within the Fecal
coliform group
•Survives in salt water
•Used in some states as indicator organism in estuarine and
marine waters
• These bacteria are easily to identify using nutrient media
which contain chromogenic substrates for their enzyme
β-galactosidase (e.g.: X-GAL).
• Some genera are pathogenic and can cause serious
diseases. Genera of Enterobacteriaceae are Cedecea,
Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella,
Kluyvera, Morganella, Proteus, Rahnella, Salmonella,
Serratia, Shigella and Yersinia.
 Faecal enterococci
• Enterococci are found in high concentrations in human
feces, usually between 104 and 106 bacteria per gram
wet weight
• Due to their ubiquity in human feces and persistence in
the environment, enterococci have been adopted as
indicators of human fecal pollution in water.
• More recently, their densities on human hands have
been used as indicators of hand hygiene.
• The use of enterococci as indicators of human fecal
pollution or contamination can be problematic,
however, because enterococci are also found in animal
feces
• Fecal enterococci from the GI tract consortia
of healthy humans are generally not virulent.
Nevertheless, multidrug-resistant
Enterococcus strains have emerged as leading
causes of hospital-acquired infections
• Vancomycin-resistant enterococci are
particularly important pathogens
• as are esp-containing E. faecalis and E.
faecium
• Salinity.The ability of enterococci to grow in the
presence of salt (6.5% NaCl) is one of the distinguishing
characteristics of the genus (see Enterococci and the
Genus Enterococcus).
• the survival of enterococci against disinfection is a
better predictor of the fate of viruses than are coliforms
• Starvation. The transition from the animal
gastrointestinal tract, a nutrient-rich environment, to
oligotrophic environmental waters exposes enterococci
to nutrient starvation, one of the abiotic factors
detrimental to their survival
• Predation.Grazing by bacterivorous protozoa,
bacteriophage infection followed by virus-mediated
lysis, and predation control the abundance of
prokaryotic organisms in the environment.
• presence of pathogens in a waterway can cause cloudy
water, unpleasant odors, and decreased levels of
dissolved oxygen.
• Enterococci levels should be measured in marine and
fresh waters while E. coli should only be measured in
fresh waters. Acceptable levels of E. coli are measured
in cfu (colony forming units) and commonly include
both a 30 day mean (126 cfu/100mL) and a single
sample number (235 cfu/100mL – 575 cfu/100mL).
• Suitable levels for enterococci in marine waters are 35
cfu/100mL for a 30 day mean and 104 – 501 cfu/100mL
for a single sample, while levels in fresh water should
be less than 33 cfu/100mL for a 30 day mean and 61 –
151 cfu/100 mL as a single sample reading.
 Clostridium perfringens
• Clostridium perfringens is an effective indicator of
faecal contamination.
• Unlike E. coli, C. perfringens can withstand processing
treatments and unfavorable growth conditions.
• Clostridium perfringens is found in undercooked or
improperly sterilized canned foods (germination of
endospores) and in water (surface water).
• The natural contamination source is human and animal
faeces transmitted into food products primarily by
water. C. perfringens produces an extensive range of
invasins and exotoxins.
• The enterotoxins cause the undesirable, predominantly
meat-associated, food poisoning, as well as wound and
surgical infections that lead to gas gangrene.
• C. perfringens plays a subsidiary role in water
examination
• Clostridia are spore builders and are resistant to
heating, chlorination and other stress factors. In
contrast to vegetative cells like coliforms (E. coli,
enterococci), which are less resistant, C.
perfringens has the advantage of a robust
capability to survive
• Therefore, while faecal contamination is
detected primarily by coliforms as an indicator,
which could disappear after a processing step, C.
perfringens remains present. The organism is not
a hazard in water; rather, it is problematic when
the water comes in contact with food.
 Yeasts and molds
• Fungi are widely distributed in nature and can occur as
unicellular yeast or filamentous and, multicellular
molds.
• Yeasts and molds are able to contaminate foods and
are responsible for quick spoilage of the infested food
stuff.
• acid/alkaline requirements for yeast and mold growth
in a wide range food products is quite broad, ranging
from pH 2 to above pH 9.
• produce toxic or allergenic substances molds are
especially predestinated to be a potential health risk.
• spread by dusts and aerosols, surfaces in the
production environment will be consistently
contaminated
• Yeasts are facultative anaerobe, mono-cellular
fungi (Ascosporidae), fermenting sugar substrate
to CO2 and H2O.
• Under anaerobic conditions yeasts ferment sugar
to alcohol and CO2. Those qualities are
commercially used in the brewing, wine and
baking industry.
• Saccharomyces cerevisiae is the most common
strain used for industrial purposes.
• In terms of food spoilage genera of Candida play
a major role. This germ is located on the human
and animal mucosa (nose, throat).
• The term „mold“ is commonly used for the visible
part of the fungi present on the surface of
contaminated food.
• Under the surface the fungi forms mycelium which
cannot be recognized with the naked eye. multiply
through vegetative reproduction
• Specific molds as well as yeast are used for
industrial purposes (e.g. cheese production).
• Harmful genera of molds exist as well and are able
to produce toxins (mycotoxins).
• Almost all molds have an allergenic potential related
to their spore form capabilities
• Mold cannot undergo the process of
photosynthesis to derive nutrition, therefore,
they require organic hosts and specific organic
matter for such purposes. This need for
nutrition is why you often see mold growing
on decaying food.
 Bacteriophage as a indicator organism
• Bacteriophages that infect coliform bacteria are
known as coliphages.
• Phages are valuable prototypes for enteric viruses
because they share many underlying properties and
features notably composition, morphology, structure,
size and site of replication.
• Inaddition, their resistances against environmental
factors make coliphages more applicable than faecal
bacteria for indicating faecal contamination of water
• The survival and incidence of bacterial viruses
(phages) in water environments resemble those of
human viruses more closely than most other
bacterial indicators commonly used.
• Phage detection in environmental water samples
involves concentration of the sample, decontamination
of the concentrate, and carrying out of phage (plaque)
assay by the double- or single-layer agar methods
• somatic coliphages are detectable by simple,
inexpensive and rapid techniques, and the phages
occur in large numbers in any water environment
exposed to human oranimal excreta.
• Phages have proven to be valuable tools in research on
viruses and havebeen projected as microbial indicators
of water quality, as they share many fundamenta
lproperties with human enteric viruses which pose a
health risk, if present in water contaminated with
human faeces
• Of all human enteric viruses, those commonly
detected in the environment that havebeen
proposed as indicators of wastewater
contamination in aquatic environment
includeEnteroviruses(EV) HepatitisA virus
(HAV),Rotaviruses(RV),Adenoviruses(AdV) and
HumanPolyomaviruses
• Membrane Filtration Test
–Quantify bacteria numbers by filtering water, growing
bacteria, and counting
•all organisms that produce a dark colony (generally
purplish green) with a metallic sheen within 24 hours
at 35oC of incubation on M-Endo medium. (The green
metallic sheen may vary in size, covering the entire
colony or only appearing in a central area or in the
periphery)
•more sensitive and precise, but too sensitive for sewage
system,will cause overwhelming result
•cheaper and faster to operate; used for testing water
that are supposedly not contaminated, e.g., tap water,
raw water →can identify a specific indicator.
Known volume of water is filtered through a filter
(0.45 μm) that is capable of trapping all bacteria
•Filter transferred to Petri dish containing growth
media
•Individual bacterial cells will grow on the filter into
visible colonies in 24 hours
•m-ColiBlue24 broth
•Due to the metabolism of the bacteria on the
media: –Blue colonies indicate E. coli–Red
colonies indicate other Total coliformbacteria–E.
coli turn bluefrom the action of β-
glucuronidaseenzyme on 5-bromo-4-chloro-3-
indolyl-Beta-B-glucuronide
• MPN TEST