COLIFORMS

1
COLIFORMS

• Indicator Bacteria
• and
• Water Quality Testing

2
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 lthttp//www.eoearth.org/article/Fecal_pol
lution_of_watergt
3
Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens
Name of micro-organisms Major diseases Major reservoirs and primary sources
Bacteria Bacteria Bacteria
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
4
Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens
Name of micro-organisms Major diseases Major reservoirs and primary sources
Enteric viruses Enteric viruses Enteric viruses
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 death Human feces
5
Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens Table 3. Examples of waterborne pathogens
Name of micro-organisms Major diseases Major reservoirs and primary sources
Protozoa Protozoa Protozoa
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
Helminths Helminths Helminths
Ascaris lumbricoides ascariosis Animal and human feces
Adapted from Ashbolt, 2004 Adapted from Ashbolt, 2004 Adapted from Ashbolt, 2004
6
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

7
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. 

8
Indicator Organisms

• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci
• Enterococci

9
Indicator Organisms

• Total Coliforms not all from fecal origin
• Fecal Coliforms
• E. coli
• Fecal Streptococci
• Enterococci

10
Indicator Organisms

• Total Coliforms not all from fecal origin
• Fecal Coliforms grow at higher temps
• E. coli
• Fecal Streptococci
• Enterococci

11
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

12
Indicator Organisms

• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci previously used to test for
  human origin
• Enterococci

13
Indicator Organisms

• Total Coliforms
• Fecal Coliforms
• E. coli
• Fecal Streptococci previously used to test for
  human origin
• Enterococci best indicator for salt water

14
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.

15
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,
  Gram-negative, 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.

16
Other Properties

• ß-galactosidase positive
• Enzyme that hydrolyzes lactose into glucose and
  galactose
• Oxidase negative
• Indicates the absence of Cytochrome-c in the
  electron transport chain.

17
Typical Coliform Genera

• Citrobacter
• Enterobacter
• Escherichia
• Hafnia
• Klebsiella
• Serratia

18
Typical Coliform Genera

• Citrobacter
• Enterobacter
• Escherichia
• Hafnia
• Klebsiella
• Serratia

Enterobacteriacea
19
Traditional Methods

• Multiple-Tube Fermentation
• very low-tech
• flexible
• depends on growth
• slow

20
Traditional Methods

• Membrane-Filtration
• suitable for highly dilute samples
• prone to interference
• eliminates injured cells

21
Enzymatic Methods

• Coliform specific enzymes
• ?-Galactosidase (lacZ) total coliforms
• Ability to breakdown lactose
• ?-D-Glucuronidase (uidA) fecal coliforms
• Ability to breakdown complex sugars

22
Enzymatic Methods

• Presence / Absence Tests
• like MTF but with enzymes

23
Enzymatic Methods

• Membrane Filtration
• more specific than traditional method so it does
  not require confirmation steps

24
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.

25
Molecular Methods

• Nucleic Acid based techniques
• Polymerase Chain Reaction (PCR)
• phylogenetic primers
• enzyme specific primers
• detection limit
• viability

26
Molecular Methods

• Nucleic Acid based techniques
• In Situ Hybridization
• radioactive vs. fluorescent
• very high-tech