COLIFORMS
Indicator Bacteria
and
Water Quality Testing
FECAL POLLUTION
• Exposure to fecally contaminated water does not always translate into
infection. However, the higher the fecal bacterial levels in water, the
higher the chances of pathogens to be present in significant numbers too.
• Among the diseases associated with poor microbial water quality, those
causing dehydrating diarrhea are of critical importance as they could lead
to death within 48 hours after the initial symptoms. These extreme cases
are more predominant in countries where overcrowding and poor sanitary
conditions are the norm.
• Examples of fecal waterborne diseases are gastroenteritis, typhoid and
paratyphoid fevers, salmonellosis, cholera, meningitis, hepatitis,
encephalitis, amoebic meningoencephalitis, cryptosporidiosis, giardiasis,
dysentery, and amoebic dysentery (Table 3
From
Jorge W. Santo Domingo, Nicholas J. Ashbolt (Lead Author);Avanish K. Panikkar (Topic Editor) . "Fecal pollution of water". In:
Encyclopedia of Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition, National Council for
Science and the Environment). [First published in the Encyclopedia of Earth March 18, 2010; Last revised Date March 18, 2010;
Retrieved October 10, 2010 <http://www.eoearth.org/article/Fecal_pollution_of_water>
Table 3. Examples of waterborne pathogens
Major diseases
Major reservoirs and primary
sources
Salmonella typhi
Typhoid fever
Human feces
Salmonella paratyphi
Paratyphoid fever
Human feces
Other Salmonella
Salmonellosis
Human and animal feces
Shigella spp.
Bacillary dysentery
Human feces
Vibrio cholera
Cholera
Human feces and freshwater
zooplankton
Enteropathogenic E. coli
Gastroenteritis
Human feces
Yersinia enterocolitica
Gastroenteritis
Human and animal feces
Campylobacter jejuni
Gastroenteritis
Human and animal feces
Leptospira spp.
Leptospirosis
Animal and human urine
Various mycobacteria
Pulmonary illness
Soil and water
Name of micro-organisms
Bacteria
Table 3. Examples of waterborne pathogens
Major diseases
Major reservoirs and primary
sources
Polio viruses
Poliomyelities
Human feces
Coxsackie viruses A
Aseptic meningitis
Human feces
Coxsackie viruses B
Aseptic meningitis
Human feces
Echo viruses
Aseptic meningitis
Human feces
Norovirus
Gastroenteritis
Human feces to fomites and water
Rotaviruses
Gastroenteritis
Human feces
Adenoviruses
Upper respiratory and
gastrointestinal illness
Human feces
Hepatitis A virus
Infectious hepatitis
Human feces
Hepatitis E virus
Infectious hepatitis; miscarriage and
Human feces
death
Name of micro-organisms
Enteric viruses
Table 3. Examples of waterborne pathogens
Major diseases
Major reservoirs and primary
sources
Acanthamocba castellani
Amoebic meningoencephalitis
Human feces
Balantidium coli
Balantidosis (dysentery)
Human and animal feces
Cryptosporidium homonis, C.
parvum
Cryptosporidiosis (gastroenteritis)
Water, human and other mammal
feces
Entamoeba histolytica
Amoebic dysentery
Human and animal feces
Giardia lamblia
Giardiasis (gastroenteritis)
Water and animal feces
Naegleria fowleri
Primary amoebic
meningoencephalitis
Warm water
ascariosis
Animal and human feces
Name of micro-organisms
Protozoa
Helminths
Ascaris lumbricoides
Adapted from Ashbolt, 2004
Indicator Organisms
• organisms that behave in approximately the
same manner as the pathogens of concern,
but are present in higher numbers or are
otherwise easier to detect
Indicator Organisms
• A good indicator microorganism has qualities which make it …
– Easy to test for in the lab.
– Present in greater numbers than the pathogens they indicate.
– Safer to work with in the lab than pathogens since the indicators cause
no or only mild illnesses.
• Indicators must not only be easy to work with in the lab, they must
also predict the presence of pathogens.
• Coliform bacteria are good indicators because they only reproduce
in the intestines of animals, so they will not be present in water
unless the water has been contaminated with sewage.
• Coliform bacteria are also able to survive outside animals'
intestines, so they will be present in water for several days after the
water has been contaminated.
Indicator Organisms
• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci
• Enterococci
Indicator Organisms
• Total Coliforms – not all from fecal origin
• Fecal Coliforms
• E. coli
• Fecal Streptococci
• Enterococci
Indicator Organisms
• Total Coliforms – not all from fecal origin
• Fecal Coliforms – grow at higher temps
• E. coli
• Fecal Streptococci
• Enterococci
Indicator Organisms
• Total Coliforms – not all from fecal origin
• Fecal Coliforms – grow at higher temps
• E. coli – best indicator of health risk
• Fecal Streptococci
• Enterococci
Indicator Organisms
• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci – previously used to test for
human origin
• Enterococci
Indicator Organisms
• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci – previously used to test for
human origin
• Enterococci – best indicator for salt water
Escherichia coli
• E. coli normally colonizes an infant's
gastrointestinal tract within 40 hours of birth,
arriving with food or water or with the individuals
handling the child.
• In the bowel, it adheres to the mucus of the large
intestine. It is the primary facultative anaerobe of
the human gastrointestinal tract.
• As long as these bacteria do not acquire genetic
elements encoding for virulence factors, they
remain benign commensals.
Definitions of (Total) Coliforms
• Multiple-Tube Fermentation
– All aerobic and facultative anaerobic, Gram-negative,
non-spore-forming, rod shaped bacteria that ferment
lactose with gas and acid formation within 48 hr. at
35C.
• Membrane Filtration
– All aerobic and many facultative anaerobic, Gramnegative, non-spore-forming, rod-shaped bacteria
that develop a red colony with a metallic sheen within
24 hr. at 35C on an Endo-type medium containing
lactose.
Other Properties
• β-galactosidase positive
– Enzyme that hydrolyzes lactose into glucose and
galactose
• Oxidase negative
– Indicates the absence of Cytochrome-c in the
electron transport chain.
Typical Coliform Genera
•
•
•
•
•
•
Citrobacter
Enterobacter
Escherichia
Hafnia
Klebsiella
Serratia
Typical Coliform Genera
•
•
•
•
•
•
Citrobacter
Enterobacter
Escherichia
Hafnia
Klebsiella
Serratia
Enterobacteriacea
Traditional Methods
• Multiple-Tube Fermentation
– very low-tech
– flexible
– depends on growth
– slow
Traditional Methods
• Membrane-Filtration
– suitable for highly dilute samples
– prone to interference
– eliminates injured cells
Enzymatic Methods
• Coliform specific enzymes
– -Galactosidase (lacZ) –total coliforms
• Ability to breakdown lactose
– -D-Glucuronidase (uidA) –fecal coliforms
• Ability to breakdown complex sugars
Enzymatic Methods
• Presence / Absence Tests
– like MTF but with enzymes
Enzymatic Methods
• Membrane Filtration
– more specific than traditional method so it does
not require confirmation steps
Enzymatic Methods
• Direct Fluorimetry
– Similar to presence/absence test but fluorescence
is monitored over time to determine relative
numbers of bacteria
• Solid-Phase Cytometry
– Filtered cells are induced and exposed to
fluorescent substrate.
– Scanning fluorimeter detects individual cells and
micro-colonies.
Molecular Methods
• Nucleic Acid based techniques
– Polymerase Chain Reaction (PCR)
•
•
•
•
phylogenetic primers
enzyme specific primers
detection limit
viability
Molecular Methods
• Nucleic Acid based techniques
– In Situ Hybridization
• radioactive vs. fluorescent
• very high-tech