Pathogens in Water

1
Pathogens in Water
Module 4 Pathogens in Water
Escherichia coliScanning electron micrograph

• History
• Introduction Overview
• Pathogenic Bacteria, Viruses and Protozoa
• Methods
• Regulations Standards

2
History

• HISTORY
• The determination of water-borne pathogens as
  the causative factor in an outbreak uses the
  classical detective work of epidemiological
  studies.
• In 1854, a cholera outbreak in London, England
  was shown to be linked to a pump that derived its
  water from a polluted section of the River
  Thames. People served by a pump that obtained its
  water upstream of London had a low incidence of
  cholera.
• In 1855-1856, Dudd and Shaw showed that there
  was an association between typhoid fever in a
  street in Bristol, England and the use of a
  particular pump for water. People on an alternate
  pump did no become infected. This was 30 years
  before the causative agent for typhoid was
  identified. The appreciation of water as a
  carrier of disease-producing organisms only came
  about in the mid-1880's with the establishment of
  the germ theory by Pasteur.
• At the beginning of this century, cities in the
  Ohio River watershed commonly had death rates due
  to typhoid on the order of 75 deaths per 100,000
  people. Upon the installation of drinking water
  treatment plants, the death rate fell to about
  15/100,000.

3
History

• The occurrence of pathogens in drinking water
  supplies is currently a serious problem in
  developing countries but is also of concern in
  developed countries.
• The number of water-borne disease outbreaks in
  the U.S. decreased steadily from 1920 - 1960 in
  direct relation to the increase of chlorination
  systems, but have increased since then.
• Whether this is a real trend, or a change in the
  ability to identify the disease source, is
  arguable.

4
Introduction
INTRODUCTION Although current public attention
is focused on chemical contamination of water
supplies, only 10 of identified health problems
(1971-1978) related to drinking water have been
attributed to such contamination. Pathogenic
micro-organisms have been responsible for 40,
while the cause of 50 of such problems has not
been identified. Since water supplies are used
by populations, the transmission of water-borne
pathogens may be expressed in the form of
epidemics or may be endemic in nature.
5
Introduction
The Figure shows a distinguishing feature of a
water-borne epidemic. But, sometimes the
identification of drinking water as a source of
infection is difficult due to long incubation
times. Propagation of the disease-causing
microorganisms can be by them being introduced to
water supplies via domestic effluent, then
infecting part of the population, who in turn
reintroduce infected waste to water supplies.
An examination of the micro-organisms in
domestic waste can be a good indicator of the
degree and variety of infection in the
population.
6
Pathogen Characteristics

• PATHOGEN CHARACTERISTICS (General)
• Most water-borne pathogens may be classified as
  viruses, bacteria, or protozoa (See Table on next
  slide).
• They typically cause intestinal diseases,
  leaving the host in the faecal material,
  contaminating the water supply, and then entering
  the recipient by ingestion.
• Their survival period in water varies widely and
  is influenced by many factors such as salinity,
  temperature, etc. It may be generalized that
  cellular viruses last longer than bacteria while
  protozoa can extend their survival time by
  encystation.

7
Disease Organisms
8
Viruses

• Viruses
• Although there are over 100 known water-borne
  human enteric viruses, infectious hepatitis A,
  poliovirus and viral gastroenteritis are of
  practical concern as water-borne viruses.
• All of them, with the exception of the
  infectious hepatitis agent, have been found in
  sewage and polluted rivers.
• Tests for the presence of viruses in water
  supplies are difficult and uncertain and so
  little is known of the survival time and
  concentration distribution of viruses in water
  sources.
• In general, enteric viruses survive less than
  three months in the environment but have been
  reported surviving up to five months in sewage.
• There is dispute over whether a minimum
  infectious dose is necessary as for bacteria,
  some researchers claiming that a single virus is
  sufficient for infection.
• Due to their small size and surface properties,
  viruses tend to be adsorbed onto surfaces.

9
Bacteria - details

• Bacteria
• Bacteria comprise the largest group of
  water-borne pathogens.
• A minimum infectious dose of several hundred to
  several thousand organisms is necessary to cause
  bacteriological infection.
• Pathogenic bacteria are usually poor competitors
  at low substrate levels found in natural waters
  and so tend to be eliminated by competition and
  predation.
• Low temperatures, sediment adsorption and anoxic
  conditions occasionally prolong their survival.
• The most common bacteriological diseases are
• Shigella sp., the cause of dysentery, is almost
  strictly a human affliction (minor in other
  primates). Most shigellosis and salmonellosis
  epidemics in developed countries are food-borne
  but a few are caused by drinking water.
  Transmission by drinking water is still the major
  route of infection in underdeveloped countries.
  Methodology for detection is not reliable.
  Die-off is rapid in sewage, although low salt
  concentrations and temperatures will extend
  survival times ( 25 days _at_ 13 degrees C, 4 days _at_
  37 degrees C).
• Salmonellosis incidence (food poisoning) is low
  and peaks seasonally in mid to late summer due to
  favourable conditions for food-borne salmonella.
  Salmonella sp. Are carried by humans (1-4 of
  population), farm animals (13-17 incidence) and
  wild animals. Most Salmonella sp. cause
  gastrointestinal diseases, while one, which is
  strictly a human pathogen, causes typhoid.

10
Bacteria - details
Enteropathogenic E. coli produce gastroenteritis
and urinary infections. Carrier rates vary but
may be 16 in mothers of newborne infants, 7 in
food handlers and 3 in children. Farm animals
are also carriers. Concentrations of E. coli in
effluents to natural waters reduce to 5 of
original levels in less than 5 days. Tularemia
is passed principally by ticks, rodents and
direct contact with sewage. Water contamination
occurs from rodents. The disease spreads via the
lymphatics and bloodstream. It grows
intracellularly and causes lesions in the lungs,
liver, spleen and brain. Leptospirosis, which
begins as a wound infection, is an occupational
disease among workers in close contact with
polluted water. Pigs, dogs, rodents and humans
are carriers and is excreted in urine of infected
animals. Cholera is a serious, highly
contagious disease causing dramatic and fatal
loss of water and electrolytes. Healthy carriers
may make up 1-9 or even 25 of the population
11
Protozoa
Protozoa Protozoans entering the host body by
ingestion are usually in cyst form. Two
protozoans of major concern as water-borne
pathogens are Giardia intestinalis and Entamoebia
histolytica. Giardiasis is the most prevalent
water-borne disease in the United States. One
to ten cysts of the flagellated Giardia is the
minimal infection dose and causes serious
diarrhea. Beavers have been implicated as a
source where no obvious human contamination was
found. Cysts of E. histolytica, which cause
amebic dysentery, survive for long periods of
time at low temperatures and damp conditions in
clean water, but only a few days in fecal
material. Cryptosporidium has also been
associated with gastrointestinal disease
outbreaks in a number of North American cities
(including Kitchener-Waterloo). Cyclospora
cayetanensis,  has been a recent problem
associated with imported fruit that has been in
contact with faeces.
WebLink
12
Standards

• STANDARDS
• The variety of pathogenic organisms in water
  supplies is large but their concentrations are
  low.
• Testing for each one in order to monitor water
  quality is an expensive and unsure proposition.
• Tests for some pathogens are unreliable and may
  require an unacceptably long incubation period
  for the quick response which may be required for
  public safety.
• Thus, the concept of an indicator organism is
  used to indicate the possible presence of
  disease-causing constituents.
• Such an indicator organism should behave as
  follows
• be applicable to all water - be present when
  pathogens are
• have no aftergrowth in water - be absent when
  pathogens are
• have constant characteristics - persist longer
  than pathogens
• be harmless to humans - correlate quantitatively
  with pathogens
• be present in greater numbers than pathogens
• be easily, accurately and quickly detected.

13
Standards

• STANDARDS
• The variety of pathogenic organisms in water
  supplies is large but their concentrations are
  low.
• Testing for each one in order to monitor water
  quality is an expensive and unsure proposition.
• Tests for some pathogens are unreliable and may
  require an unacceptably long incubation period
  for the quick response which may be required for
  public safety.
• Thus, the concept of an indicator organism is
  used to indicate the possible presence of
  disease-causing constituents.
• Such an indicator organism should behave as
  follows
• be applicable to all water - be present when
  pathogens are
• have no aftergrowth in water - be absent when
  pathogens are
• have constant characteristics - persist longer
  than pathogens
• be harmless to humans - correlate quantitatively
  with pathogens
• be present in greater numbers than pathogens
• be easily, accurately and quickly detected.

14
Indicator Organisms
Why are Escherichia coli and coliforms used as
indicator organisms ?
The coliform group nearly fulfills the criteria
listed above. Escherichia coli is considered a
reliable indicator of bacterial pathogens.
Protozoans and viruses, however, usually survive
longer than E. coli and may also survive
disinfection which is otherwise adequate for
bacteria. Filtration is usually successful in
extracting protozoans and viruses and should be
used in conjunction with disinfection procedures.
15
Guidelines

• The first comprehensive Canadian guidelines were
  published in 1968 and revised in 1978.
• In 1986 a federal-provincial advisory committee
  was established to revise and update the
  guidelines on a continuing basis.
• They are unenforceable at the moment, but laws
  may be introduced at the provincial, territorial
  or federal levels.
• The Ontario Ministry of the Environment used to
  sample and analyse municipal drinking water
  supplies.
• Municipalities usually conducted parallel
  monitoring now they are responsible for all
  monitoring.
• Situation changed when Ontario privatized the
  water monitoring testing
• See the Ministry of Environment
  Website for updates on procedures and regulations
  in Ontario (post-Walkerton)
• The Ontario Drinking Water Regulations
  are also posted
• More information is available on the
  Walkerton outbreak

WebLink
WebLink
WebLink
16
Canadian Water Quality Guidelines
Canadian Water Quality Guidelines (Adobe Acrobat
files)

• General Topics
• Derivation of Guidelines
• Summary of Guidelines for Canadian Drinking Water
  Quality
• Guide - Recreational Uses 1
• Guide - Recreational Uses 2
• Boil Water Advisories
• Drinking Water Treatment Units
• 9th National Conference on Drinking Water May 16,
  17 18 2000
• Non-Chemical factors
• Bacteria
• Protozoa
• Microbiological Quality
• Taste
• Colour
• Turbidity

17
Individual Compounds
All Individual Compounds Issues (Adobe Acrobat
Files) Aldicarb Aldrin Aluminum Ammonia
Arsenic Asbestos Atrazine Azinphos Bacteria
Barium Bendiocarb Benzo-a-pyrene Boron
Bromate Bromoxynil Cadmium Calcium Carbaryl
Carbofuran Carbon tetrachloride Chloramines
Chloride Chlorophenols Chlorpyriphos Chromium
Colour Copper Cyanazine Cyanide Diazinon
Dicamba Dichloroethane (1,2) Dichlorobenzene
Dichloromethane Diclofop-methyl Dimethoate
Dinoseb Diquat Diuron
Dichloroethylene (1,1) Fluoride Gasoline
Glyphosate Hardness Iron Lead Magnesium
Malathion Manganese Mercury Methoxychlor
Metolachlor Metribuzin Microbiological
Monochlorobenzene Nitrate NTA (nitriloacetic
acid) Odour Paraquat Parathion pH Phorate
Picloram Protozoa Radionuclides Radon
Selenium Silver Simazine Sodium Sulphate
Sulphide Taste Total Dissolved Solids
Temperature Terbufos Tetrachloroethylene
Toluene Trichloroethylene Trifluaralin
Trihalomethanes Turbidity Uranium Vinyl
Chloride Zinc
Canadian Water Quality Guidelines
18

• 2002 Ontario Drinking Water Quality Standards
• The standards for drinking water quality in
  Ontario are prescribed in O. Reg. 169/03 under
  the Safe Drinking Water Act, 2002.
• O. Reg. 169/03 prescribes standards for 158
  chemical/physical, microbiological and
  radiological parameters.
• Background and supporting information to the
  standards can be found in Technical Support
  Document for Ontario Drinking Water Standards,
  Objectives and Guidelines (formerly known as the
  Ontario Drinking Water Standards) available on
  the ministrys Web site at www.ene.gov.on.ca.
  Supporting documentation for the standards is
  also available from Health Canada at
• http//www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/doc
  _sup-appui/index_e.html
• The amended regulation has a revised and more
  stringent standard for trichloroethylene and
  eliminates standards for fecal coliforms,
  background colony counts and heterotrophic plate
  counts, parameters which are now considered
  outdated or which are no longer considered to be
  health-related by the scientific and technical
  community. Total coliforms and E. coli remain as
  the principal health indicators for the
  protection of drinking water.
• For more information, contact
• Public Information Centre
• Ministry of the Environment
• 135 St. Clair Avenue West
• Toronto ON M4V 1P5
• Tel [phone] or [phone]

19

• 2002 Ontario Drinking Water Quality Standards
• The standards for drinking water quality in
  Ontario are prescribed in O. Reg. 169/03 under
  the Safe Drinking Water Act, 2002.
• O. Reg. 169/03 prescribes standards for 158
  chemical/physical, microbiological and
  radiological parameters.
• Background and supporting information to the
  standards can be found in Technical Support
  Document for Ontario Drinking Water Standards,
  Objectives and Guidelines (formerly known as the
  Ontario Drinking Water Standards) available on
  the ministrys Web site at www.ene.gov.on.ca.
  Supporting documentation for the standards is
  also available from Health Canada at
• http//www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/doc
  _sup-appui/index_e.html
• The amended regulation has a revised and more
  stringent standard for trichloroethylene and
  eliminates standards for fecal coliforms,
  background colony counts and heterotrophic plate
  counts, parameters which are now considered
  outdated or which are no longer considered to be
  health-related by the scientific and technical
  community. Total coliforms and E. coli remain as
  the principal health indicators for the
  protection of drinking water.
• For more information, contact
• Public Information Centre
• Ministry of the Environment
• 135 St. Clair Avenue West
• Toronto ON M4V 1P5
• Tel [phone] or [phone]

20

• 2002 Ontario Drinking Water Quality Standards
• Technical Update
• For Municipal Residential Drinking Water Systems
  under O. Reg. 170/03
• Following public consultation, on June 5, 2006,
  O.Reg 169/03 was amended.The amendments revise or
  eliminate standards for parameters based on new
  health-related scientific information or which
  are now considered outdated by the scientific and
  technical community and by the Advisory Council
  on Drinking Water Quality and Testing Standards.
  This technical update describes Ontarios
  drinking water quality standards, for full
  details please see the regulation at
• http//www.e-laws.gov.on.ca/DBLaws/Regs/English/03
  0169_e.htm

21

• 2002 Ontario Drinking Water Quality Standards
• Technical Update
• MICROBIOLOGICAL STANDARDS
• Microbiological Parameter
• Standard (expressed as a maximum)
• 1. Escherichia coli (E. coli) Not detectable
• 2. Revoked O. Reg. 248/06, s. 1.
• 3. Total coliforms Not detectable
• 4. Revoked O. Reg. 248/06, s. 1.
• 5. Revoked O. Reg. 248/06, s. 1.
• O. Reg. 169/03, Sched. 1 O. Reg. 248/06, s. 1.

22

• 2002 Ontario Drinking Water Quality Standards
• Technical Update
• MICROBIOLOGICAL STANDARDS
• Microbiological Parameter
• Standard (expressed as a maximum)
• 1. Escherichia coli (E. coli) Not detectable
• 2. Revoked O. Reg. 248/06, s. 1.
• 3. Total coliforms Not detectable
• 4. Revoked O. Reg. 248/06, s. 1.
• 5. Revoked O. Reg. 248/06, s. 1.
• O. Reg. 169/03, Sched. 1 O. Reg. 248/06, s. 1.

23
Methods
Methodology

• One of the problems involved with the reliable
  detection of waterborne pathogens is that there
  is no one method suitable for all pathogens.
• The pathogens in the Enterobacteriaceae occur
  only sporadically in water and the environment is
  not conducive to growth and survival of these
  pathogens.
• This, together with many other technical
  reasons, makes the coliform test of dubious value
  in assessing the actual risk of ingesting water.
  In addition, detection of the actual pathogens
  themselves is technically difficult and often
  time consuming.
• Since the pathogens are usually present in very
  low concentrations in most water samples, the
  degree of concentration required to obtain usable
  numbers of the pathogenic organisms is often very
  high. This leads to other problems in water where
  other kinds of contaminants such as detritus,
  organic materials or other organisms are present.
  
• As examples of the problem, to detect Giardia
  cysts, over 400 litres of water is usually
  filtered.
• For Cryptosporidium this value may be 1200
  litres for viruses it may be as high as 2000 to
  5000 litres.

24
Methods

• Another reason for the difficulty are that
  recovery of organisms from the samples (even
  after concentration) is difficult due to possible
  damage to the organisms by the harsher
  environment in water compared to their normal
  habitat.
• This can, in the extreme cases, lead to
  organisms in water that cannot be cultured on
  normal media, but still retain their
  pathogenicity. These organisms are called
  "viable, but non-culturable

25
Viruses - Methods
Viruses Concentration of viruses is an important
facet of their detection and identification. The
most common method involves passing very large
volumes of water through filters that are
electronegative or electropositive in nature. The
virus particles are adsorbed to the surface of
the filter by electrostatic charges and are then
eluted by passing smaller volumes of a
protein-containing liquid (e.g. beef extract).
26
Viruses - Methods
Viruses Concentration of viruses is an important
facet of their detection and identification. The
most common method involves passing very large
volumes of water through filters that are
electronegative or electropositive in nature. The
virus particles are adsorbed to the surface of
the filter by electrostatic charges and are then
eluted by passing smaller volumes of a
protein-containing liquid (e.g. beef extract).
Detection is often through tissue culture of the
viruses in human or primate tissue samples. More
recently, immunofluorescence (antibodies linked
with fluorescent dyes such as FITC),
enzyme-linked immunosorbent assays (ELISA both
direct and indirect), nucleic acid probes, the
Polymerase Chain Reaction (PCR) and
radioimmunofocus assays (RIFA) have become more
common.
27
Bacteria - Methods

• Bacteria
• The most common indicator of potential pathogen
  contamination is still the coliform test in its
  various guises.
• The total coliform test has many forms the most
  common so far has been the membrane filter method
  in which a known volume of water is filtered
  through a 0.45 mM or 0.22 mM filter and the
  filter incubated on M-endo or LES-Endo agar. Red
  colonies with a metallic sheen are considered
  coliforms.
• A slower method, the Most Probable Number
  method, is also used and involves serial decimal
  dilution of the water sample followed by adding 5
  aliquots of the dilutions to 5 tubes of nutrient
  media. If the dilutions are chosen correctly,
  some of the serial decimal dilutions will have
  some tubes with growth and some without. A
  statistical procedure based on these sets gives
  the number of bacteria in the original sample.
  This test is sometimes referred to as "dilution
  to extinction" - an unfortunate phrase !

More recently, with changes in the drinking water
regulations of many countries with regard to
coliforms, no coliforms at all are allowed in
100-mL samples. This permits a simple
presence/absence test to be used where 100 mL of
sample is placed in nutrient medium at suitable
concentrations and incubated any growth means
the water has coliforms and so fails the
regulations.
28
Bacteria - Methods

• A remaining problem is the time taken to assay
  for coliform number or presence/absence up to 3
  days is required for some of the tests.
• The quickest tests (P/A and membrane filter)
  take 24 hours.
• Very often, quicker results reporting is
  essential or desirable.
• Rapid detection tests have been developed for
  these situations. They vary in their acceptance
  by the regulatory bodies, but at least one
  (Defined Substrate Systems) has achieved broad
  use and regulatory standing.
• In this test (various commercial versions are
  available), the water sample is filtered through
  0.45 mM membrane filters, removed and and
  incubated at 35C on a medium containing
  4-methylumbelliferone-beta-D-galactoside. If
  coliforms are present, they break the bond
  between the methylumbelliferone and the
  galactoside, releasing the fluorescent
  umbelliferone derivative. The degree of
  fluorescence is measured with a fluorimeter and
  shows the degree of contamination by coliforms.

29
Bacteria - Methods
A variant of this test (the Colilert method) uses
ortho-nitrophenyl-beta-D-galactosidase (ONPG) and
4-methyl umbelliferyl-beta-D-glucuronide (MUG)
for detecting total coliforms and Escherichia
coli in a single solution. The coliforms break
down ONPG with their beta-galactosidase enzymes
releasing the yellow coloured indicator portion
of the molecule. If E. coli is also present, the
enzyme glucuronidase hydrolyzes the MUG to
glucuronide and the indicator portion
4-methyl-umbelliferone that fluoresces under
ultraviolet light. This permits separate and
independent estimates of total coliform and E.
coli counts in the same sample. Levels as low as
1 CFU/100 mL have been reliably detected with
this method. This test can also be used with the
MPN or P/A tests to improve resolution and
sensitivity. Gene probe and PCR technology
(Polymerase Chain Reaction) General
Molecular Biology Methods - Donis-Keller
Lab.Laboratory Manual) could also be used to
produce more sensitive and specific test for
coliforms or E. coli
WebLink
30
Parasites - Protozoa
Waterborne parasites  Water can contain many
different protozoal parasites. The most
significant are Cryptosporidium spp. , Giardia
spp. and Entamoeba histolytica Giardia outbreaks
have become more common and in fact is the most
prominent cause of waterborne illness in the
U.S., accounting for 20 of all waterborne
disease cases. Cryptosporidium is less common
but there have been numerous outbreaks around the
world. The largest outbreaks occurred in
Milwaukee, Wisconsin affecting 400,000
individuals . A lake used as a water supply
became contaminated and the oocytes of
Cryptosporidium survived through water treatments
of coagulation, flocculation, rapid sand
filtration, sedimentation and chlorination. A
local outbreak occurred in the Region of Waterloo
after Grand River water was added to the normal
groundwater supply   Entamoeba histolytica
has three stages in the life cycle. The
trophozoite and precyst stages are not as
resistant as the cyst stage to disinfection. Even
the cyst stage is not able to tolerate
temperatures above 50 ºC, sunlight or extended
disinfection periods. The only source of the
organism is from humans and a few primates.
31
Protozoa - Giardia
Giardia is a flagellated protozoan with a life
cycle consisting of two stages the first is a
cyst that produces two flagellated trophozoites
that, when ingested, attach to the lining of the
intestines. These cysts can survive for long
periods (up to 2 months at 8C) and are much more
resistant to chlorine than most bacteria. The
only practical identification method is
microscopic examination of water after
concentration by filtration. No reliable culture
method for Giardia is available.
Giardia lamblia cyst stained with iodine
32
Protozoa - Cryptosporidium
Cryptosporidium is a protozoan parasite that is
coccoidal in form and develops within the gastric
or intestinal mucosal epithelial layer in
mammals. It has a complex life cycle consisting
of sexual and asexual stages and produces a very
stable and resistant oocyte structure which
"germinates" after ingestion to form 4 sporozites
that infect the lining of the intestine cells.
Many stages follow including trophozoites that
undergo asexual multiplication to form type I
meronts and then merozoites that can infect new
cells. The oocysts are formed from gametocytes
from Type II meronts that form merozoites that go
on to form microgameteocytes and
macrogametocytes. The oocytes are then excreted
and enter the water supply. The oocytes are even
more resistant to disinfection than Giardia. A
major source of Cryptosporidium is from cattle
and other animals. Most likely, manure pits and
storage areas overflowing into local rivers and
streams of the Grand River or cattle grazing on
river or stream bans were the cause of the
regional infection by Cryptosporidium. No culture
method is available for Cryptosporidium.
Cryptosporidium life cycle
33
Methods for Protozoa
Filtration method for detection of Giardia and
Cryptosporidium
34
Key Points
Key Points

• General properties of epidemiology of
  water-borne diseases
• Main causal agents of water-borne disease and
  their survival times in water
• How these factors (main causal agents and
  survival times) affect the
• relevance and importance of setting and using
  standards.
• The characteristics of "good" standards
• How present standards such as the coliform test
  match or do not match these characteristics
• Importance of sewage treatment and water
  treatment  on water-borne disease
• Main detection methods for viruses, bacteria and
  protozoa. Example of each. Problems associated
  with such detection methods.
• Relationship of detection methods to standards.

End of Module 4