Microbiology Awwa

2006 American Water Works Association
AWWA was founded in 1881 during a

time that saw pivotal developments in the
microbiological and public health sciences.
Developments such as John Snows pioneer-
ing work in tracing the source of a cholera
epidemic to a contaminated water pump in
London (1854), Louis Pasteurs refutation of
spontaneous generation (1861) and develop-
ment of pasteurization (1864), Joseph Listers
first work on the use of antiseptics during
surgery (1867), Ferdinand Cohns classifica-
tion of bacteria (1875) and discovery of bac-
terial spores (1877), and Robert Kochs isola-
tion of the anthrax bacterium (1876), set the
stage for a revolution in microbiology during
the 1880s. Kochs laboratory in Germany
introduced the use of pure culture techniques
for handling bacteria, used agar-agar for the
first time to produce a practical solid medium
for culturing bacteria, identified the causative
agent of tuberculosis, and developed a series
of criteria (Kochs postulates) for determining
the cause of a disease. Koch later received the
Nobel Prize in 1905 for founding the science
of bacteriology. In addition, Clostridium
tetani (the causative agent of tetanus) was dis-
covered, the Gram stain was developed,
Escherichia coli was identified as part of the
normal human intestinal flora, Pasteur and
Paul Ehrlich began their pioneering work on
immunization, the petri dish was developed,
the causative agent of brucellosis was identi-
fied, a diphtheria antitoxin was developed,
and the first work on nitrifying bacteria began
during the 1880s. Since these pioneering
days, the microbiological and public health
sciences have progressed dramatically.
Approximately 1,500 human pathogens are
currently recognized (Cleaveland et al, 2001)
and the modes of action and mechanisms of
transmission of many are fully understood.
Many infections are easily controlled by the
The evolution
of microbiology in the
drinking water industry
ROCHELLE & CLANCY | 98: 3 JOURNAL AWWA | MARCH 2006 163
Paul Rochelle and
Jennifer Clancey
164 MARCH 2006 | JOURNAL AWWA 98: 3 | ROCHELLE & CLANCY
judicious use of antibiotics and vaccines, and
some diseases have virtually been eradicated.
Consequently, microbiology within the water
industry has also advanced.
Infectious diseases exert a huge burden on
life expectancy, public health systems, and
national economies worldwide. The World
Health Organization (WHO) estimates that
globally, 2.6 billion people do not have ade-
quate sanitation and that more than 1.4 bil-
lion people do not have access to clean, safe
drinking water. Gastrointestinal disease
caused by microbially contaminated drinking
water is one of the leading causes of death in
the developing world, accounting for approx-
imately 5 million deaths annually. Such fig-
ures demonstrate the importance of efficient
drinking water disinfection. The benefits of
microbiologically safe water go beyond the
absence of disease within the community and
affect the productivity of industry as well as
the price of goods and services. Municipal
water systems designed to prevent water-
borne infectious disease are probably one of
the most effective investments of public funds
that society can make. As such, despite
potential health effects of disinfection by-
products, chlorination of drinking water is
widely regarded as one of the most significant
public health advances in human history.
Chlorine-based disinfectants remain an
important treatment for drinking water
because they provide a wide range of benefits
that are not provided by any other single dis-
infectant; they are the only disinfectants that
provide a residual in the distribution system,
which is a key part of the multibarrier
approach to preventing waterborne disease.
Nevertheless, despite the advances made
in understanding the ecology, pathogenicity,
occurrence, and epidemiology of human
pathogens, improvements made in drinking
water treatment practices, the development of
alternative disinfection technologies, and a
better understanding of watershed manage-
ment practices, the water industry must
remain vigilant with respect to the microbiol-
ogy of drinking water. Waterborne disease
outbreaks continue to occur, even in affluent
nations operating modern treatment facilities
(Hrudey & Hrudey, 2004). As our under-
standing of microbes improves, changes in
the way they are handled and treated may be
necessary. In addition, water utilities cur-
rently monitor for only a few select microor-
ganisms and pathogens not previously con-
sidered by the industry may emerge as
waterborne threats to public health; the
causative agent is not identified in approxi-
mately 3050% of waterborne outbreaks.
Also, the past five years have seen the threat
Changes
in Recognized
Species of
Cryptosporidium
By 1980, there were at least 21 named species of Cryptosporidi-
um(e.g., C. bovis, C. crotali, C.garngami, C. agni, C. rhesi, C.
cuniculus), but many of these have since been invalidated
because of wrongly identified parasites, failure to recognize pre-
viously named species, or insufficient scientific support.
*C. hominis and C. parvumwere previously referred to as
C. parvumgenotypes 1 and 2, respectively.
1912
C. muris
C. parvum
1995
C. baileyi
C. meleagridis
C. muris
C. nasorum
C. parvum
C. serpentis
1997
C. baileyi
C. felis
C. meleagridis
C. muris
C. nasorum
C. parvum
C. serpentis
C. wrairi
2006
C. andersoni
C. baileyi
C. canis
C. felis
C. galli
C. hominis*
C. meleagridis
C. muris
C. nasorum
C. parvum*
C. saurophilum
C. serpentis
C. suis
C. wrairi
S
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s
of intentional drinking water contamination,
with organisms that may not normally be
found in drinking water, emerging as a signifi-
cant concern. Consequently, there is a strong
incentive for continued assessment of micro-
bial risk, development of improved analytical
methods, and proactive microbial research by
the water industry.
Although the primary focus of this review
is on those microorganisms that cause dis-
ease, the water industry is interested in four
general groups of microorganisms:
Frank and opportunistic pathogens that
cause disease such as Cryptosporidiumspp.,
enteropathogenic E. coli, and enteric viruses.
Microorganisms such as coliforms, E.
coli, enterococci, and bacteriophages
(although not necessarily harmful themselves
they indicate that water quality has been
compromised).
Nuisance organisms that can cause aes-
thetic problems or degrade water quality.
These include nitrifying bacteria and taste-
and odor-producing cyanobacteria and are
the subject of AWWA Manual M7, Problem
Organisms in Water (AWWA, 2004).
Beneficial microbes such as those that
form the basis of biologically active filters or
organisms that may be used to reduce waste
products generated by water treatment (e.g.,
bioremediation of waste salts produced dur-
ing desalination).
Pathogens in drinking water
Environmental waters that are used as
sources of drinking water may be contami-
nated with a wide range of pathogenic
microorganisms (Table 1) intermixed with a
dominant background of naturally occurring
nonpathogenic microbial populations.
Although water treatment facilities that are
operated correctly remove the vast majority of
microbial contaminants, pathogens can some-
times break through to the distribution sys-
tem, as evidenced by the number of water-
borne disease outbreaks (Hrudey & Hrudey,
2004), some of which affected many thou-
sands of people (Table 2). In the early years
of the 20th century, waterborne diseases such
as cholera (caused by Vibrio cholerae) and
typhoid (Salmonella typhi) were still major
public health issues in the United States but
widespread installation of filtration and disin-
fection treatment plants and improved sanita-
tion largely eradicated these diseases in the
developed world (McGuire, 2006).
However, as these diseases disappeared,
new ones emerged. These include the para-
sitic protozoa Cryptosporidiumand Giardia,
pathogenic E. coli, a variety of enteric viruses,
and toxin-producing cyanobacteria. Figure 1
shows the changing nature of waterborne dis-
ease outbreaks from the 1930s to 2000. In
the 1930s amebiasis (caused by the intestinal
parasite Entamoeba histolytica) and typhoid
were the predominantly recognized causes of
waterborne disease, along with a handful of
hepatitis A cases. In the period from 1940 to
1950, more organisms became recognized as
agents of waterborne disease. Giardia was
recognized as an agent of waterborne disease
in Japan in 1946, but it was not until the
1960s that the first waterborne cases were
reported in the United States. Once the
waterborne route for giardiasis was under-
stood and surveillance instituted, a 100-fold
increase of cases was reported in the period
from 1976 to 1980. The Milwaukee, Wis.,
cryptosporidiosis outbreak in 1993 added
another >100-fold increase in the number of
cases of waterborne disease, but other emerg-
ing pathogens began to appear as waterborne
disease agents in the 1990s. Cyclospora
cayetanensis, a coccidian parasite similar to
Cryptosporidium, was documented as the
cause of an outbreak in a Chicago, Ill., hospi-
tal and E. coli O157:H7, previously known as
an agent of foodborne disease outbreaks,
caused several large waterborne disease out-
breaks, with some deaths directly attributed
to contaminated drinking water.
In 2005, there were just six cases of
cholera and 261 cases of typhoid fever in the
United States, compared with 7,212 cases of
cryptosporidiosis, 2,368 cases of E. coli
O175:H7 infection, 17,256 cases of giardia-
sis, 9,877 cases of hepatitis, and 1,952 cases
of legionellosis; however, only a portion of
these reported cases were waterborne (CDC,
2005). Although cholera and typhoid remain
major scourges in the developing world, at the
dawn of the 21st century in the United States,
they have been replaced by microbes that do
not cause widespread mortality but do repre-
G
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Pathogen Health Effects
Bacteria Aeromonas hydrophila Gastroenteritis
Campylobacter spp. Acute gastroenteritis
Cyanobacteria (toxin producers) Gastrointestinal disease,
liver and nerve toxicity
Pathogenic Escherichia coli Bloody diarrhea, hemorrhagic
colitis, and hemolytic uremic
syndrome
Helicobacter pylori Chronic gastritis, peptic and
duodenal ulcers, and gastric
cancer
Legionella pneumophila Severe lung inflammation and
influenza-like symptoms
Mycobacterium avium complex Pulmonary disease
Salmonella typhimurium Gastroenteritis, nausea, acute
watery diarrhea
Shigella spp. Severe diarrhea
Vibrio cholera Severe vomiting and diarrhea,
fatal dehydration
Yersinia enterocolitica Gastrointestinal infections
Protozoa Cryptosporidiumspp. Self-limiting to severe diarrhea
Encephalitozoon spp.* Diarrhea
Enterocytozoon bieneusi* Diarrhea and widely disseminated
infections
Giardia duodenalis Self-limiting to severe diarrhea
Toxoplasma gondii Flu-like symptoms, blindness in
babies of infected mothers
Viruses Adenoviruses Respiratory infections,
conjunctivitis, and gastroenteritis
Caliciviruses Diarrhea and vomiting
Coxsackieviruses Diarrhea and vomiting, heart
inflammation
Echoviruses Aseptic meningitis and heart
inflammation
Hepatitis viruses Gastroenteritis
Rotavirus Gastroenteritis
*Although originally classified as protozoa, the microsporidia group of parasites is now considered to be more
closely related to fungi.
TABLE 1 Health effects associated with potential waterborne pathogens
H
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sent waterborne threats to public health and
can, in some instances, be fatal. All water-
borne outbreaks are the result of some form of
drinking water contamination, and although
many can be attributed to specific failures in
treatment plant operations or poor managerial
decisions, some outbreaks occur even in the
absence of operational violations. The largest
and thus most famous waterborne disease out-
break in 1993 in Milwaukee was caused by a
protozoan parasite, Cryptosporidium, and
affected approximately 403,000 residents and
caused 100 deaths (Mackenzie et al, 1994).
Probably no other organism better highlights
the technological advances in drinking water
microbiology over the past 20 years. The
interest spurred by the Milwaukee incident
led to the development of improved oocyst
recovery and purification methods, many
polymerase chain reaction- (PCR-) based
methods, including real-time PCR and geno-
typing procedures, microarray assays, and cell
culturebased infectivity assays.
Cryptosporidiummuris was first identified
in 1907 and C. parvumfive years later
(Tyzzer, 1907; 1912), but it was not recog-
nized as a human pathogen until 1976. Since
then the taxonomy of the genus has been a
moving target (sidebar on page 164),
although the past 10 years have seen signifi-
cant improvements in our understanding of
the parasites speciation. The first known
waterborne cryptosporidiosis outbreak
occurred in 1984 in Texas (Hrudey &
Hrudey, 2004) and at least nine other out-
breaks were reported before the Milwaukee
incident, two of which had an estimated dis-
ease incidence of more than 10,000 individu-
als. Many outbreaks of cryptosporidiosis have
now been associated with drinking water or
recreational use of water worldwide, and out-
breaks continue despite the lessons of the
past 20 years. The most recent outbreak in
2005 affected more than 200 people in North
Wales in the United Kingdom (CDR, 2005).
Occurrence studies in the United States and
Canada report finding oocysts in 4.5100%
of raw water samples (Rose et al, 1997; Wallis
et al, 1996; LeChevallier & Norton, 1995),
and water samples that were from source
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FIGURE 1 The changing nature of waterborne disease
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waters receiving domestic and agricultural
waste had oocyst concentrations as high as
5,800/L (Madore et al, 1987). The US Envi-
ronmental Protection Agencys (USEPAs)
Information Collection Rule (ICR) survey of
5,838 untreated source water samples
throughout the United States reported an
average occurrence of 6.8%, with a mean con-
centration of 0.067 oocysts/L (Messner &
Wolpert, 2003). Oocysts have also been
detected in up to 40% of treated drinking
water samples (Rose et al, 1997). An Awwa
Research Foundation study of 100 surface
water plants showed that 76 were positive for
Cryptosporidiumin source water samples,
whereas 15 plants had Cryptosporidiumpre-
sent in the filter effluents (McTigue et al,
1998). In another study of Cryptosporidium
occurrence, approximately 10% of 518
source water samples collected over a one-
year period from six watersheds were posi-
tive; the mean oocyst concentration was
0.015/L (LeChevallier et al, 2002).
The disparity in the reports of very high
numbers of Cryptosporidiumoocysts in both
raw and treated water in early studies com-
pared with much lower concentrations in
more recent studies may be a reflection of
improved analytical methods. In the late
1990s, the USEPA developed method 1622,
a significant improvement to the ICR method
(USEPA, 2001). The filtration step was
improved to permit total capture of oocysts
and excellent recovery through elution; the
use of immunomagnetic separation was
incorporated to significantly improve separa-
tion of oocysts from sample debris; and addi-
tional microscopic confirmation criteria
(internal morphology) reduced the number of
false-positives reported (Clancy, 2000;
Clancy et al, 1999).
Because the presence of C. parvum was
well documented in cattle, it was generally
assumed that cows were responsible for
much of the Cryptosporidiumcontamination
detected in surface waters. However, as a
result of the application of a variety of molec-
ular methods in the 1990s, it was recognized
that the oocysts identified microscopically as
C. parvumcould be categorized as two sepa-
rate types: genotype 1 or anthroponotic
oocysts were isolated almost exclusively from
Water Quality-related
Microbes Whose Entire
Genomes Have Been
Sequenced
Acanthamoeba castellani
Human adenoviruses (types AF)
Anabaena variabilis
Astrovirus
Bifidobacterium longum
Burkholderia spp.
Campylobacter jejuni
Cryptosporidium hominis
Cryptosporidium parvum
Desulfovibrio desulfuricans
Encephalitozoon cuniculi
Entamoeba histolytica
Enterococcus faecalis
Escherichia coli
Giardia duodenalis
Hepatitis A, C, and E viruses
Helicobacter pylori
Legionella pneumophila
Mycobacterium avium
Nitrobacter spp.
Nitrosomonas spp.
Norwalk virus
Pseudomonas spp.
Salmonella spp.
Shigella spp.
Synechococcus spp.
Toxoplasma gondii
Vibrio spp.
See www.ncbi.nlm.nih.gov/Genomes/ and http://genome.jgi-
psf.org/mic_home.html
M
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humans; genotype 2 or zoonotic isolates were
detected in a wide range of animals, includ-
ing humans. Based on genetic, physiological,
and host animal differences, and the apparent
lack of recombination between the two geno-
types, these two groups have been classified
as separate species. The zoonotic genotype
retained the name C. parvum, and the
anthroponotic genotype was named C.
hominis (Morgan-Ryan et al, 2002). There is
considerable strain level variation in the
genomic sequences within each species. C.
parvumand C. hominis oocysts (or organ-
isms morphologically indistinguishable from
C. parvumand C. hominis) have been
reported in at least 152 species of mammals,
including humans (Fayer et al, 2000), and
these two species are responsible for most
cases of human cryptosporidiosis. However,
other species or genetically distinct isolates of
Cryptosporidiumthat have also been isolated
from human infections are C. canis, C. felis,
C. meleagridis, C. suis, and C. muris (Gatei et
al, 2002; Morgan-Ryan et al, 2002; Fayer et
al, 2001; Pedraza-Diaz et al, 2001; Xiao et al,
2001; Morgan et al, 2000; Pieniazek et al,
1999). C. meleagridis was originally
described in 1955 (Slavin, 1955) but is now
recognized as an emerging human pathogen
in the United Kingdom, where it is responsi-
ble for 1% of all human cryptosporidiosis
cases (Caccio et al, 2005). Also, there appear
to be many animal host-specific strains of
Cryptosporidiumthat are detected in water
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Date Location Agent Outbreak Size
1854 London, England, UK Vibrio cholerae 578 deaths
1978 Colorado, USA Giardia duodenalis 5,000 cases
1978 Vermont, USA Campylobacter jejuni 3,000 cases
1981 Eagle-Vail, Colo., USA Rotavirus 80 cases
1985 Pittsfield, Mass., USA Giardia duodenalis 3,800 cases
1987 Carrollton, Ga., USA Cryptosporidiumsp. 13,000 cases
1989 Cabool, Mo., USA Escherichia coli O157:H7 243 cases, 4 deaths
1993 Gideon, Mo., USA Salmonella sp. 600 cases, 7 deaths
1993 Milwaukee, Wis., USA Cryptosporidium hominis 403,000 cases, 100 deaths
1995 Florida, USA Giardia duodenalis 1,449 cases
1995 Victoria, Canada Toxoplasma gondii 100 cases
1996 Ogose, Japan Cryptosporidium sp. 9,000 cases
1996 Florida, USA Norwalk-like virus 594 cases
1998 Wyoming, USA Escherichia coli O157:H7 157 cases
1999 New York, USA Escherichia coli O157:H7 781 cases, 2 deaths
2000 Walkerton, Ont., Canada Escherichia coli O157:H7 2,300 cases, 7 deaths
2001 North Battleford, Canada Cryptosporidium sp. 7,100 cases
2002 Connecticut, USA Norovirus 142 cases
2002 Transtrand, Sweden Norwalk-like virus 500 cases
2005 North Wales, UK Cryptosporidium hominis ~220 cases
TABLE 2 Selected waterborne disease outbreaks
ROCHELLE & CLANCY | 98: 3 JOURNAL AWWA | MARCH 2006 171 2006 American Water Works Association
Significant Events in the History
of Drinking Water Microbiology
1854 John Snow traced cholera outbreak to water pump in Londons Broad Street
1881 AWWA founded
Robert Koch introduces bacterial pure culture techniques
Walther and Angelina Hesse develop agar-based bacterial growth medium
1882 Robert Koch identifies causative agent of tuberculosis
1884 Robert Koch isolates Vibrio cholerae from Elbe River
Gram stain introduced
Escherichia coli identified as normal inhabitant of human gut
1893 First use of ozone as a drinking water disinfectant in Holland
1897 Initial standardization of bacteriological methods by the American Public Health
Association
1905 First edition of Standard Methods of Water Analysis published
1906 First use of ozone as a drinking water disinfectant in France
1907 Charles Louis awarded Nobel Prize for demonstrating that protozoa cause infectious diseases
Cryptosporidiumfirst identified by E.E. Tyzzer
1908 Jersey City, N.J., is the first US community to begin chlorination of drinking water
1914 First drinking water bacteriological standard established (2 coliforms per 100 mL,)
1916 First ultraviolet installation in United States for drinking water disinfection
1940 First installation of ozone in the United States for taste and odor control
1953 Watson, Crick, and Franklin determine the double helix structure of DNA
1954 Polio vaccine developed
Enders, Weller, and Robbins receive Nobel Prize for growing poliovirus in cell cultures
1970 US Environmental Protection Agency (USEPA) established
1971 Membrane filter method for fecal coliforms introduced into Standard Methods for the
Examination of Water and Wastewater
1973 First cloning of DNA
1974 Authorization of the Safe Drinking Water Act in the United States
1975 First documented waterborne outbreaks attributed to enterotoxigenic E. coli
1976 First cases of human cryptosporidiosis reported
Tentative Standard Method introduced for recovery of enteric viruses from water
First documented waterborne outbreaks of Giardia in humans attributed to
contamination by beavers
1978 First documented waterborne outbreaks attributed to Campylobacter
1983 Polymerase chain reaction (PCR) invented
First report of 2-methylisoborneol (MIB) producing unicellular cyanobacteria
1984 First waterborne outbreak of cryptosporidiosis
1985 Initial development of method for detecting Cryptosporidiumin water
First presentation at Water Quality Treatment Conference of molecular methods for
detecting waterborne pathogens (dot blot hybridization)
Rotavirus and enterovirus isolated from treated drinking water from plants meeting all
required standards
1986 First use of immunomagnetic separation (IMS) for recovery of waterborne
pathogens
1988 Development of Colilert for detecting coliforms in drinking water
1989 Promulgation of the Surface Water Treatment Rule
Publication of Total Coliform Rule
Cabool, Mo., E. coli O157:H7 waterborne outbreak with 243 cases and 4 deaths
First report of IMS for detection of waterborne protozoa at AWWA Water Quality
Technology Conference
1990 First application of PCR for detecting waterborne pathogens
1993 Milwaukee, Wis., Cryptosporidiumwaterborne outbreak affected 403,000 people;
100 deaths
1995 Partnership for Safe Water initiated by the USEPA
1995 First complete bacterial genome is sequenced (Haemophilus influenzae)
First and only waterborne outbreak attributed to Toxoplasma gondii in a developed
country (Canada)
1996 Large waterborne cryptosporidiosis outbreaks in Canada (14,000 cases) and Japan
(9,000 cases)
Reauthorization of Safe Drinking Water Act
Adenoviruses shown to be resistant to ultraviolet disinfection
1997 First complete eukaryotic genome sequenced (yeast)
1998 USEPA publishes first Contaminant Candidate List
1999 First AWWA International Symposium on Waterborne Pathogens
2000 Walkerton E. coli O157:H7 waterborne outbreak with 2,300 cases and 7 deaths
2001 First use of microarrays targeting waterborne pathogens
2005 Candidate Contaminant List 2 published
Contract awarded for construction of the worlds largest ultraviolet drinking water
facility (2.2 bgd) in New York
2006 Promulgation of the Long Term 2 Enhanced Surface Water Treatment Rule
Third AWWA International Symposium on Waterborne Pathogens
and whose significance to human health is
not yet known. Genotyping data from 22
waterborne cryptosporidiosis outbreaks
demonstrated that 67% were caused by C.
hominis and 33% by C. parvum(McLauchlin
et al, 2000; Sulaiman et al, 1998). Human
fecal contamination is therefore responsible
for many of these outbreaks.
For an organism that was not recognized as
a human pathogen until 30 years ago, there has
been a tremendous amount of research aimed
at improving detection methods, evaluating
disinfectants and antimicrobial agents, refining
viability and infectivity assays, and increasing
our understanding of the organisms biology,
epidemiology, and occurrence in the environ-
ment. An AWWA symposium dedicated
entirely to waterborne cryptosporidiosis
(Fricker et al, 1997) laid the groundwork for
much of the research that has been completed
during the past 10 years. Some of the detec-
tion methods that have been developed,
including those that were investigated but
abandoned, include flow cytometry, spec-
troscopy, immunoassays, continuous cen-
trifugation, vortex flow filtration, ultrafiltra-
tion, probe hybridization, in-situ
hybridization, nucleic acid sequence based
amplification, PCR, reverse transcriptase
PCR, real-time-PCR, and
microarrays. Development of C.
parvumin cell culture was first
reported in 1984 (Current &
Haynes, 1984), and in the
1990s many cell lines were
shown to support infection.
Cell culturebased infectivity
assays were developed specifi-
cally for drinking water applica-
tions (Rochelle et al, 1997;
Slifko et al, 1997; Di Giovanni
et al, 1999), and the equiva-
lency of cell culture with a stan-
dard mouse infectivity assay has
been demonstrated (Rochelle et
al, 2002). A study utilizing cell
culture to assess infectivity
reported that 27% of surface
water treatment plants were
releasing infectious oocysts in
their finished water, and over-
all, 1.4% of treated drinking water samples
contained infectious oocysts (Aboytes et al,
2004). This finding raises doubts concerning
the ability of conventional treatment plants to
meet the USEPAs risk goals. Cryptosporid-
iumisolates can be speciated and genotyped
by PCR targeting at least five genomic targets
(18S rDNA, -tubulin, hsp70, COWP, actin
genes) and differentiation at the subgenotype
level is achieved using micro- and minisatel-
lite repeat sequences. In addition, the
genomes of both C. hominis and C. parvum
have been sequenced in their entirety (Abra-
hamsen et al, 2004; Xu et al, 2004), allowing
in-depth genetic comparisons between the
two species. Because of its widespread occur-
rence, low infectious dose, and resistance to
conventional chlorine disinfection (Korich et
al, 1990), the organism has been targeted by
recently promulgated drinking water regula-
tions (USEPA, 2006).
Although not the subject of such an intense
research effort in recent years, our under-
standing of Giardia spp. has also progressed.
Giardia was originally described by Antonie
von Leeuwenhoek in 1675; it was then redis-
covered by William Lamble in 1859 and
named after himGiardia lamblia (synony-
mous with G. duodenalis and G. intestinalis).
The integrity
of a Class III
biohazard
containment
glovebox is
evaluated
something not
considered
necessary by the
water industry just
a few years ago.
Until human volunteers established the infec-
tivity of G. lamblia in the 1950s, it was com-
monly thought to be a nonpathogenic inhabi-
tant of the gut (Rendtorff, 1954). Giardia was
first recognized as a waterborne pathogen in
Japan in 1946, and the first documented
waterborne outbreak of giardiasis in the
United States was in Aspen, Colo., during the
196566 ski season, affecting 120 skiers (Lin,
1985). Giardia was consistently the most
commonly identified pathogen in waterborne
disease outbreaks in the United States
between 1971 and 1996, with 115 outbreaks
and 28,000 cases (Craun & Calderon, 1999).
As with Cryptosporidium spp., the taxon-
omy of Giardia spp. is undergoing revision.
Cysts previously identified as G. lamblia are
now known to comprise at least seven geneti-
cally distinct assemblages (which may even-
tually be designated as new species), only
two of which (A and B) appear to infect
humans (Caccio et al, 2005). Despite the
many waterborne outbreaks of giardiasis, the
role of animals and person-to-person disease
transmission, and the relative risk of acquir-
ing infection through drinking water are yet
to be resolved.
Whereas Pasteur (182295) and Koch
(18431910) are seen as the founders of
medical bacteriology, Sergei Winogradsky
(18561953) and Martinus Beijerinck
(18511931) are recognized as the origina-
tors of environmental microbiology. Beijer-
inck discovered sulfate-reducing bacteria and
developed the first enrichment cultures.
Enrichment cultures are used today to isolate
bacteria with specific physiological properties
and to improve detection sensitivities for a
variety of microbial pathogens. Beijerinck
was awarded the Leeuwenhoek Medal in
1905. Other medal awardees whose work is
still influencing the study and development of
microbiology within the water industry are
Pasteur (1895), Felix dHerelle (1925, co-dis-
coverer of bacteriophages), Winogradsky
(1935), Roger Stanier (1981; who estab-
lished that blue/green algae are bacteria [the
cyanobacteria]), and Carl Woese (1992) who
redefined the tree of life based on phyloge-
netic analysis of 16S ribosomal RNA
sequences. Most important, Beijerinck dis-
covered viruses, a name he coined in 1898,
following on from Dmitri Iwanowskis work
six years earlier. Poliomyelitis was the first
human disease shown to be caused by a virus
in 1909. During the 1930s to 1950s, patho-
genic viruses were cultured in chick embryos
and other animal systems. The first in-vitro
cell culture systems were developed in 1949,
and a viral plaque assay was developed in
1952 allowing accurate quantitation of animal
viruses. The 1960s onward saw continued
development of improved detection methods
for viruses, including radio-immunoassays,
immunofluorescence, Western blots, and
enzyme-linked immunosorbent assays. A
waterborne outbreak in 1968 was attributed
to enteric viruses but the 1971 edition of
Standard Methods for the Examination of
Water and Wastewater stated that No rou-
tine examination of water or wastewater for
enteric viruses is practical or necessarily
meaningful at the present time. However, in
1979 the WHO Scientific Group on Human
Viruses in Water, Wastewater, and Soil con-
cluded that contamination of water by viruses
was a significant threat to public health, even
in the developed world.
A large waterborne outbreak caused by
coxsackie virus B3 and hepatitis A virus
affected approximately 7,900 people in Texas
in 1980 (Hrudey & Hrudey, 2004), and there
have been many viral waterborne outbreaks
since. It is speculated that many of the
approximately 3050% of outbreaks for
which no causative agent is identified may be
caused by viruses. Since a 1965 symposium
on transmission of viruses by water, there
have been concerted efforts to develop and
improve viral detection methods for water
matrices. The 15th edition of Standard Meth-
ods (1981) included a tentative method for
detecting enteric viruses in up to 1,000 L of
finished water based on a two-stage filter
adsorptionelution procedure. In 1984, the
USEPA published a Manual of Methods for
Virology (USEPA, 1984) that included the
best methods available at the time. The
express intent of the manual was to make it
possible for any competent water bacteriol-
ogy laboratory to concentrate and recover
viruses from water. This manual has now
S
t
a
n
d
a
r
d

M
e
t
h
o
d
s
been revised at least seven times as better and
newer methods have been developed. The
method used for the ICR (USEPA, 1996)
involved recovery of viruses by capture on
positively charged filters, organic floccula-
tion, and analysis by a total culturable virus
assay on Buffalo Green Monkey kidney
(BGMK) cells. Viral detection methods are
now routinely used in many laboratories and
improvements have led to quantitative assays
that are used for enumeration and disinfec-
tion studies (photo on page 184), faster sam-
ple turnaround, and assays that support a
wider range of viruses. Molecular detection
methods were first applied in the 1980s with
PCR and its many variants used for direct
detection in environmental samples and
detection of infection in cell cultures. The
exponential increase in the amount of nucleic
acid sequence data has allowed for greater
specificity in probe and primer design and
the genomes of some potentially waterborne
viruses have been sequenced in their entirety
(sidebar on page 170). However, further
advances in virological methods are still nec-
essary to assess the public health significance
of waterborne viruses. For example, an in-
vitro cell culture method is not available for
human caliciviruses and a more reliable quan-
titative plaque assay is required for aden-
ovirus types 40 and 41, but both of theses
issues are currently being investigated.
The Safe Drinking Water Act requires the
USEPA to publish a list of contaminants that
may require regulation in the future but are
not currently subject to any proposed or pro-
mulgated regulations. This Contaminant
Candidate List (CCL) contains nine groups
of microbes that are known or anticipated to
occur in water, but for which the analytical
detection methods are inadequate and insuf-
ficient information is available on the health
effects, occurrence, and treatment efficacy to
allow a regulatory decision (sidebar on page
179). For some organisms, information is
lacking for all of these criteria. In other
instances, there is ample health effects infor-
mation but occurrence data are inadequate.
For example, the health effects of human
pathogenic species of microsporidia, Entero-
cytozoon bieneusi, and Encephalitozoon spp.,
are well documented (Wittner et al, 1999).
Microsporidia were first recognized as
human pathogens in the 1970s and were
originally classified as amitochondrial proto-
zoa, but phylogenetic analyses based on sev-
eral genes indicated that they are more
closely related to fungi (Katinka et al, 2001;
Weiss & Vossbrinck, 1999). Encephalitozoon
cuniculi has 11 chromosomes, and with 2
million bases, has one of the smallest known
eukaryotic genomes (Katinka et al, 2001).
The routes of transmission to humans are
not clearly understood, but many wild and
domestic animals can carry microsporidia so
it is possible that surface waters can become
contaminated and consequently may serve as
a route of transmission to humans. Prototype
methods have been developed for detecting
microsporidia spores in environmental
waters and recent work has demonstrated
that microsporidia are sensitive to chlorine
and ultraviolet (UV) disinfection at levels
typically used for drinking water treatment
(Huffman et al, 2002; Johnson et al, 2003).
A restrospective epidemiological study of a
cluster of microsporidiosis cases indicated
an association with the municipal water dis-
tribution system but no evidence of contami-
nation was found (Cotte et al, 1999), and
there is some doubt as to whether this inci-
dent truly represented a waterborne out-
break (Hunter, 2000). Human-pathogenic
microsporidia have been detected in surface
water and groundwater, tertiary-treated
sewage effluent, and food crop irrigation
water (Dowd et al, 2003; Thurston-Enriquez
et al, 2002; Dowd et al, 1998; Sparfell et al,
1997). However, methods are still inade-
quate to allow a definitive assessment of their
significance to waterborne disease and the
potential for zoonotic transmission has not
yet been fully investigated.
Adenoviruses are nonenveloped, icosohe-
dral, double-stranded DNA viruses (80110
nm in diameter) that are widespread in
nature, infecting birds and many mammals;
human adenoviruses are classified into six
species (AF; Horwitz, 2001). The viruses
cause a variety of clinical manifestations, and
the case fatality rate is as much as 50% among
the immunocompromised. There have been a
few studies of adenovirus occurrence in
source waters. Chapron et al (2000) analyzed
samples collected during the ICR using
BGMK cells as well as nested PCR and found
that 38% of the samples contained infective
viruses and that 48% of the samples were
positive for adenovirus DNA. Human aden-
ovirus DNA has been detected in surface
waters, seawater, and treated drinking water
using PCR-based methods (Fong et al, 2005;
van Heerden et al, 2005; Pina et al, 1998),
but detection of pathogens by PCR-based
methods alone does not necessarily correlate
with the presence of infectious organisms.
For example, a real-time PCR assay detected
adenovirus DNA in 16% of samples, but
none of them contained infectious aden-
oviruses when assayed on two cell lines (Choi
& Jiang, 2005). Infectious adenoviruses were
detected in urban rivers affected by domestic
and industrial wastewaters (Lee et al, 2004).
A few outbreaks of adenovirus infection have
been associated with recreational water
(Craun et al, 2003; Kukkula et al, 1997; Mar-
tone et al, 1980), but the role of water in
transmission of adenoviruses is unclear.
Many studies conducted in the past 10 years,
however, have demonstrated that aden-
oviruses are far more resistant to UV disinfec-
tion than other potential waterborne
pathogens. Consequently, although they are
listed on the CCL with the caution that there
is insufficient scientific information on aden-
oviruses, they have become the regulatory
driver for setting UV dose requirements for
virus inactivation credit (USEPA, 2006).
Microbial indicators and other
organisms
Drinking water treatment plants tradition-
ally monitor fecal coliform and other indica-
tor organisms to provide an approximate
measure of potential fecal contamination and
evaluate the efficacy of removal or inactiva-
tion of pathogenic microorganisms. The first
edition of Standard Methods of Water Analy-
sis was published in 1905 and the first drink-
ing water bacteriological standard of 2 col-
iforms/100 mL was established in 1914. The
eighth edition of Standard Methods (which
Microorganisms on the CCL
The Safe Drinking Water Act requires the US Environmental Protection Agency to publish a list of
contaminants (every five years) that may require regulation in the future but are not currently subject
to any proposed or promulgated regulations. Contaminants are placed on the list because they are
known or anticipated to occur in water but there is currently insufficient information on the health
effects, occurrence, treatment efficacy, and analytical methods to allow a regulatory decision. Microor-
ganisms currently included on the CCL are:
Viruses Adenoviruses
Caliciviruses
Coxsackieviruses
Echoviruses
Bacteria Aeromonas hydrophila
Cyanobacteria (toxin-producers)
Helicobacter pylori
Mycobacterium avium-intracellulare complex
Protozoa/Fungi Microsporidia (Encephalitozoon spp. and Enterocytozoon spp.)*
Source: USEPA, 2005
CCLContaminant Candidate List
*Microsporidia were originally classified as protozoa but are now recognized as being more closely related to fungi.
M
i
c
r
o
o
r
g
a
n
i
s
m
s
became known as Standard Methods for the
Examination of Water and Wastewater with
the publication of the 11th edition in 1960),
published in 1936, described pour plates for
the bacteriological examination of water and
lactose fermentation tubes and Endo
medium to confirm the presence of coli-
aerogenes bacteria (todays coliform group).
In the intervening 70 years, great advances
have been made in the development of meth-
ods for detecting specific microorganisms,
including those such as viruses and protozoa
that cannot be readily cultured on agar
plates, but the overall approach to the rou-
tine and regulated microbiological examina-
tion of drinking water remains the same.
Detection of coliform bacteria, the fecal sub-
set of coliforms, and E. coli is the corner-
stone of microbial water quality testing. Pour
plates, mEndo medium, most probable num-
ber tables, and gas production upon lactose
fermentation are all still used in modern
water quality laboratories. Advances such as
the introduction into Standard Methods of a
membrane filter method for fecal coliforms
(1971), development of API identification
strips for enteric bacteria in the 1970s, and
the development in the 1980s of fluorescent
media containing 4-methyl-umbelliferyl-D-
glucuronide (MUG) for detecting coliforms
in drinking water have streamlined the pro-
cedures and decreased analysis time.
The Total Coliform Rule (TCR; USEPA,
1989) requires all public water systems to
monitor for coliform bacteria in their distrib-
ution systems, specifies a followup monitor-
ing schedule whenever positive samples are
detected, and requires public notification of
positive samples exceeding the maximum
contaminant level. However, there are some
concerns over the value of the total coliform
test for public health protection. Progressive
changes to the analytical methods aimed at
streamlining the procedure and decreasing
the time to obtain results have reduced its
specificity such that a positive result does not
necessarily imply treatment failure or conta-
mination of the distribution system. A total
coliformpositive may simply represent
growth of nonpathogenic environmental bac-
teria in distribution system water or pipe
biofilms. Many researchers, public health
officials, and AWWA have suggested that E.
coli should be adopted as the sole microbial
indicator for compliance purposes, and the
availability of relatively simple rapid culture-
based and molecular tests for detecting and
identifying E. coli make this a feasible alterna-
tive. The TCR is currently undergoing
review by the USEPA, with the revised rule
due to be published in 2006.
Microbial source tracking (MST) repre-
sents a further potential development of E.
coli as an indicator of contamination. MST is
based on the assumption that different animal
species have developed different intestinal
microbial flora and that these differences can
be discerned with an appropriate tool and
may be used to identify sources of fecal cont-
amination in water. There are many proposed
MST methods, but many of them involve
phenotypic methods (e.g., antibiotic resis-
tance analysis) or genotypic analysis such as
repetitive-PCR, ribotyping, and pulse-field
gel electrophoresis of E. coli or enterococci
Although todays
microscopes would
be recognized
by microbiologists
of previous eras,
imaging capabilities
have improved
greatly. The
microscopist in the
photo above is
viewing a Giardia
cyst at 1,000
magnification using
Nomarski
differential
interference
contrast optics
overlaid with a
fluorescence image
of DAPI-stained
nuclei.
E
.

c
o
l
i
isolates recovered from potential source ani-
mals and the body of water under investiga-
tion. However, most MST methods are
research-level tools that are not yet ready for
routine implementation (Griffith et al, 2003;
Stoeckel et al, 2004). Despite early claims of
success in the use of E. coli to determine
sources of contamination in water,
intraspecies heterogeneity, growth and per-
sistence of these bacteria in the environment,
and a relatively cosmopolitan distribution
among various animal host species, suggest
that it may not be the most appropriate target
organism for MST.
Although it is generally used as an indica-
tor of fecal and therefore pathogen contami-
nation, E. coli itself can be pathogenic, caus-
ing urinary tract infections, sepsis, and
diarrheal diseases. There are currently 170
serotypes of diarrheagenic E. coli strains that
are classified as enterotoxigenic, enteropatho-
genic, enteroaggregative, enteroinvasive, and
enterohemorrhagic, each characterized by
differing pathogenic features (Nataro &
Kaper, 1998). The most prominent patho-
genic strain is enterohemorrhagic E. coli
O157:H7 that produces Shigella-like toxins
and is therefore also referred to as shiga toxin
E. coli along with approximately 60 other
serotypes. The 13th edition of Standard
Methods (1971) considered waterborne infec-
tions by pathogenic E. coli to be quite
improbable, but this prediction held true for
only four years. The first waterborne out-
break attributed to pathogenic E. coli
(O6:H16) occurred in 1975 and sickened
2,200 people (Rosenberg et al, 1977). Then
in 1989 a waterborne outbreak of E. coli
O157:H7 in Cabool, Mo., sickened 243 peo-
ple and killed four (Hrudey & Hrudey,
2004). Although this bacterium is unlikely to
be a common contaminant in surface waters
that are not directly affected by raw fecal
material and it is effectively inactivated by all
of the drinking water disinfectants in com-
mon use, its presence (coupled with subopti-
mal treatment practices) can have severe con-
sequences, and the Cabool outbreak should
have been a wakeup call for the water indus-
try. Two other E. coli O157:H7 waterborne
disease outbreaks occurred in the 1990s; one
in Alpine, Wyo., with 114 cases in 1998, fol-
lowed by an outbreak at a small county fair in
upstate New York, resulting in 781 cases and
two deaths. In spite of these warnings, the
experience was repeated in 2000 on a large
scale in Walkerton, Ont., Canada, with 2,300
cases and seven deaths attributed to E. coli
O157:H7 contamination of drinking water
(OConner, 2002). The political, economic,
and personal ramifications of the Walkerton
outbreak still reverberate today. There have
been at least 30 waterborne outbreaks caused
by E. coli O157:H7, and the many methods
that are now available for detecting and iden-
tifying pathogenic strains of E. coli (particu-
larly O157:H7) attest to their importance
and public health effects. These include
selective culture media, immunomagnetic
purification kits, fluorescent antibodies, latex
agglutination tests, enzyme linked
immunosorbent assays, and molecular assays
targeting the shiga toxin and antigen biosyn-
thesis genes, among others. A particular con-
cern from a water industry perspective is that
some diarrheagenic strains, including many
enteroinvasive are typically lactose-negative
(Nataro & Kaper, 1998). In addition, most
strains of E. coli O157:H7 do not produce a
functional -glucuronidase (Strockbine et al,
1998) and will therefore not be detected by
MUG-based methods. A recent study evalu-
ated four antibody-based methods and a
PCR assay for detecting E. coli O157:H7 in
water (Bukhari et al, 2005). The authors
reported comparable performance for one of
the immunological tests and the PCR-based
method with a reproducible detection sensi-
tivity of 20 cfu/200 mL water.
A further example of how methodological
developments have improved the water
industrys understanding of and ability to
control bacterial processes that relate to
water quality is provided by nitrifying bacte-
ria. Winogradsky conducted the earliest
work on nitrifying bacteria in 1891, demon-
strating that oxidation of ammonia is a two-
step process, and identified several genera of
important nitrifying bacteria: Nitrosomonas
spp. (the predominant ammonia oxidizer),
Nitrosococcus spp., Nitrobacter spp. (the pre-
dominant nitrite oxidizer), and Nitrospira
spp. Apart from playing a significant role in
the biogeochemical nitrogen cycle, nitrifying
bacteria are also important in the mainte-
nance of finished water quality. Ammonia in
water, including that introduced through the
use of chloramine disinfection, can lead to
biological instability in drinking water distri-
bution systems by promoting the growth of
nitrifying bacteria. The resulting cell mass
and the products of ammonia oxidation
deplete the residual disinfectant and may
sustain the growth of nitrite-oxidizing and
heterotrophic bacteria. The reduction in
chloramine residual and development of a
microbial community in the distribution sys-
tem generally leads to an overall deteriora-
tion in water quality, including the formation
of nitrite or nitrate. Nitrification in chlorami-
nated drinking water was first addressed in
the late 1980s with investigations of the sea-
sonal occurrence and distribution of ammo-
nia-oxidizing bacteria as well as their
response to disinfection (Wolfe et al, 1990).
Techniques that have been applied to the
study of nitrifying bacteria include PCR, typ-
ically targeting the 16S rRNA or ammonia
monooxygenase genes, and comparative
nucleotide sequence analysis (Baribeau et al,
2000), real-time PCR, fluorescent in-situ
hybridization, confocal microscopy, and
denaturing gradient gel electrophoresis to
investigate community structure (Kowalchuk
& Stephen, 2001). Through the application
of such tools over the past 15 years, the
occurrence, ecology, activity, and interac-
tions of the various bacteria involved in nitri-
fication of drinking water, as well as the fac-
tors that lead to nitrification, are relatively
well understood (Wolfe & Lieu, 2002).
Recent research has focused on using a com-
bination of molecular detection tools and
measurement of various physical and chemi-
cal water parameters in distribution systems
as an early warning of potential nitrification
conditions (Regan et al, 2002).
Detection methods
The pioneering work of Winogradsky and
Beijerinck led to great enthusiasm for identi-
fying and classifying bacteria in the environ-
ment. In 1909, Sigurd Orla-Jensen proposed
classifying bacteria based on physiological
functions such as growth on particular sub-
strates or production of specific compounds.
The Society of American Bacteriologists,
later to become the American Society of
Microbiology, applied this technique to pre-
pare a report on the classification of bacteria
that evolved in 1923 into Bergeys Manual of
Determinative Bacteriology. This classifica-
tion scheme became the standard for many
years. However, as more diverse bacteria
were isolated from a wider range of habitats,
it became apparent that classification based
on growth and morphological characteristics
was unrealistic and often generated inconsis-
tent identifications. Agar was first used as a
solidifying agent in bacterial culture media in
1881 and for the next 100 years, growing
bacteria on a variety of media was the stan-
dard procedure for studying waterborne
microbes. Media were initially nonspecific
but selective media that either preferentially
support the growth of particular bacteria or
allow differentiation between species based
on indicator compounds are now available for
coliforms, E. coli, E. coli O157:H7,
pseudomonads, enterococci, Legionella spp.,
mycobacteria, Campylobacter spp., Yersinia
spp., Burkholderia cepacia, Clostridiumspp.,
and Salmonella spp., among others. How-
ever, protozoa, viruses, and many bacterial
pathogens cannot be cultured on agar plates.
Therefore, although the agar plate is still the
mainstay of coliform compliance monitoring
in the water industry, many alternative meth-
ods have been developed for working with
the ever-broadening array of microorganisms
that confront the microbial water quality lab-
oratory. Cell culturebased methods for
viruses and protozoa have become routineif
not standardizedin many laboratories, anti-
body-based separation and detection meth-
ods are available for a broad array of
pathogens, and an almost bewildering array
of detection platforms have been developed
(Figure 2; photo on page 186). Rapidity of
detection has become one of the major dri-
vers in the development of analytical meth-
ods. Over the past 15 years molecular biol-
ogybased techniques, and in particular
PCR, have revolutionized the detection of
B
a
c
t
e
r
i
a
pathogenic bacteria, viruses, and protozoa in
both clinical and environmental samples.
Molecular methods enable rapid detection of
pathogens in water by providing levels of sen-
sitivity and specificity difficult to achieve with
traditional culture-based assays, which often
take days to perform.
The double-helix structure of DNA was
identified in 1953 by James Watson, Francis
Crick, Maurice Wilkins, and Rosalind
Franklin. DNA hybridization was first used to
compare species in 1961, and nucleic acid
reassociation was developed in 1969 to clas-
sify enterobacteria. DNA sequencing was
invented in 1977, automated sequencing was
developed in 1986, and the first complete
bacterial genome was sequenced in 1995
(Haemophilus influenzae). Then in 2005, an
entire microbial genome (Mycoplasma geni-
talium) was sequenced in less than a day on a
single instrument (Margulies et al, 2005).
Pathogens of interest to the water industry for
which entire genome sequences are available
are shown in the sidebar on page 170. This
DNA timeline illustrates the technique-driven
advances in modern microbiology over the
past 40 years that have led to the current situ-
ation in which data processing and analysis is
the limiting factor, rather than data genera-
tion. Molecular methods have revolutionized
our understanding of the composition, phy-
logeny, physiology, and function of microbial
communities in the environment. Published
applications of molecular techniques to
Water sample
(100 mL 1,000 L)
Elution and centrifugation
Nucleic acid extraction
Direct extraction
of nucleic acids
with or without
membrane
dissolution
Reverse transcription
for RNA targets
Nonselective
or selective cultural
enrichment
Immunomagnetic
purfication
of target pathogen
Purification
to remove inhibitors
Probe capture
DNA sequence
databases
Primer/probe
design
Empirical specificity
testing with non-target
organisms and
assessment of sensitivity
Gel electrophoresis
Hybridization with
confirmatory probes
Restriction digestion and/or
DNA sequencing for
confirmation and identification
Microarray hybridization
Simultaneous probing,
real-time detection, and
quantification with QPCR
Amplification with
universal primers
Microarray hybridization
targeting multiple pathogens
(with speciation and strain typing)
Concentrate by filtration:
Membranes, capsules, cartridges, hollow fiber, electropositive, electronegative
QPCRquantitative polymerase chain reaction
Selection of sample concentration method, the level of sample enhancement or purification (red), and
particular detection assay (blue) depend on the required sensitivity and specificity of the assay in
addition to sample throughput, technical capacity of the laboratory, time, and cost constraints.
Pathogen-specific amplification
FIGURE 2 A general approach for the application of molecular methods to detect
pathogens in water
drinking water issues include direct detection
of pathogens in water, fecal source tracking of
either indicator microorganisms or specific
pathogens, and detection methods for in-vitro
infectivity and disinfection assays (Table 3).
Although not formally standardized or
approved for monitoring purposes within the
water industry, nucleic acidbased techniques
have been widely used for detecting and ana-
lyzing microorganisms in water.
Molecular methods were first applied to
the detection of potential waterborne
pathogens in the 1980s, but they have not
been adopted on a routine basis by the water
industry, as they have been in clinical, foren-
sic, and food industry laboratories. There are
several reasons for the lack of responsiveness
in the water industry. There is a lack of stan-
dardization of molecular methods, and rigor-
ous quality assurance and control measures
have only recently been applied to these
methods. There are also relatively few
researchers and still fewer utilities using mole-
cular tools to address water-related microbial
issues. Then there is the unique challenge
presented by attempting to detect very low
concentrations of target organisms in rela-
tively large volumes of water (typically
11,000 L). In addition, water is a very com-
plex matrix; even finished drinking water can
contain a plethora of microorganisms. This is
a significant difference between water and
clinical applications. For example, when a
pathogen is identified in a clinical sample, the
analyst can be nearly 100% certain it is the
correct identification, and, if the individual is
experiencing symptoms of the suspected dis-
ease, it is highly likely that the patient has that
disease. In a water sample, there are hun-
dreds of organisms that may be present, pro-
viding a larger challenge in determining
whether there are pathogens in the mix. The
possibility of false-positive identifications in
drinking water was demonstrated by Stur-
baum et al (2002). Examining natural waters,
these researchers noted that a sample contain-
ing a harmless dinoflagellate was positive for
Cryptosporidiumusing PCR. They cautioned
about the use of molecular methods in envi-
ronmental samples where organisms with a
close phylogenetic relationship may co-exist.
There are many approaches for applying
molecular methods to the study of microbial
water quality (Figure 2), and they have pro-
The quantitative viral
plaque assay is used
to assess infectivity
and the efficacy
of disinfectants.
vided much-needed insight into the occur-
rence, survival, viability, and inactivation of
pathogens and other microorganisms in
water. The replacement of radioisotopic
methods with nonradioactive alternatives in
the 1990s made the methods available to a
wider range of laboratories. However, a cer-
tain amount of evaluation and optimization is
still necessary to ensure that results obtained
using these techniques are reliable and con-
sistent, and it is important that molecular
methods should not be considered as an
alternative to conventional microbiological
techniques. Rather, they provide an addi-
Organism Matrix Assay method Reference
Adenoviruses River water and urban sewage PCR Pina et al, 1998
Adenoviruses River water Cell culture/RT-PCR Lee et al, 2004
Caliciviruses Source and treated drinking water RT-PCR Huang et al, 2000
Hepatitis A virus Spiked wastewater NASBA Jean et al, 2001
Hepatitis A virus Spiked groundwater RT-PCR/molecular beacon Abd El Galil et al, 2004
Hepatitis A virus Groundwater RT-PCR Abbaszadegan et al, 1999
Noroviruses River water RT-PCR Lodder & de Roda Husman, 2005
Reoviruses Surface water Cell culture/RT-PCR Spinner & DiGiovanni, 2001
Rotavirus Groundwater RT-PCR Abbaszadegan et al, 1999
Bacteroidetes Coastal water PCR and qPCR Bernhard & Field, 2000
Campylobacter spp. Surface water PCR-ELISA Sails et al, 2002
Campylobacter spp. River water and sewage PCR and FISH Moreno et al, 2003
Cyanobacteria Surface water qPCR Foulds et al, 2002
Escherichia coli O157:H7 Artificial wetlands qPCR Ibekwe et al, 2002
Legionella pneumophila Hospital water systems qPCR Wellinghausen et al, 2001
Mycobacterium avium Spiked drinking water NASBA/molecular beacon Rodriguez-Lazaro et al, 2004
Nitrifying bacteria Chloraminated drinking water PCR and TRFLP Regan et al, 2002
Salmonella spp. Surface water Enrichment-PCR Yanko et al, 2004
Cryptosporidiumspp. Surface water Cell culture/PCR LeChevallier et al, 2003
Cryptosporidiumspp. Stormwater PCR and fingerprinting Xiao et al, 2000
Cyclospora cayetanensis Spiked surface water concentrate PCR-RFLP Shields & Olson, 2003
Giardia lamblia Wastewater PCR Mayer & Palmer, 1996
Naegleria fowleri Drinking water Nested-PCR/sequencing Marciano-Cabral et al, 2003
Toxoplasma gondii Surface water PCR Villena et al, 2004
Microsporidia Surface water and groundwater PCR/sequencing Dowd et al, 1998
ELISAenzyme-linked immunosorbent assay, FISHfluorescent in-situ hybridization, NASBAnucleic acid sequence based
amplification, PCRpolymerase chain reaction, RT-PCRreverse transcriptase PCR, qPCRquantitative PCR
TABLE 3 Examples of molecular assays for detection of pathogens and indicators in water
tional suite of tools that can be used to com-
plement traditional methods. Incorporation
of appropriate controls into molecular assays
is critical, as is understanding the significance
of molecular positives in the absence of
culture-based methods.
Microscopy has always been, and will
remain, fundamental to the study of microor-
ganisms, simply because there is no substi-
tute for being able to actually see the object
of investigation (photo on page 180). One of
the earliest rudimentary microscopes was
used to identify Giardia in 1675, and
although the basic principle did not change,
significant advances in the science and engi-
neering of optics were necessary to make
their use routine. The electron microscope
was invented in 1931, phase contrast
microscopy was developed in 1934, and
Nomarski differential interference contrast
microscopy was patented in 1953. The prin-
ciples of fluorescence microscopy were ini-
tially realized in the early 1900s, but the
development of epifluorescence illumination
in the mid 1970s brought the
technology into the main-
stream. Although the modern
water quality laboratory
microscope would be recog-
nized by a microbiologist
from 125 years ago, the
diversity of illumination,
observation, image capture,
image analysis, and automa-
tion functions would no
doubt make them envious.
Many fluorescent antibodies
and fluorogenic compounds
are now available for observ-
ing both intact microbes and
their internal constituents.
Despite the many technologi-
cal developments in pathogen
detection methods, the
recently promulgated Long
Term 2 Enhanced Surface
Water Treatment Rule
(USEPA, 2006) includes fluo-
rescence microscopy as the
only approved method for
detecting Cryptosporidium
oocysts. Nevertheless, there is a relatively high
level of subjectivity in the microscopic identi-
fication of Giardia cysts and Cryptosporidium
oocysts in environmental samples, even by
qualified analysts. In some circumstances,
such identification can best be described as
qualified guesswork (Clancy, 2000).
The crystal ball
In parallel with the broader scientific
community, the microbial sciences within
the water industry have progressed steadily
over the past 125 years, with occasional
technological leaps and recognition of new
human pathogens opening up entirely new
areas of investigation. The future is likely to
follow the same pattern. Emerging and re-
emerging pathogens will necessitate contin-
ued development of analytical methods.
Fundamental studies of pathogen health
effects and epidemiology, and surveys of
pathogen occurrence will be needed to
determine the true role of drinking water in
human disease. As populations continue to
Many new pathogen
detection tools are
undergoing evalua-
tion in larger micro-
bial water quality
and academic labo-
ratories.
grow and water utilities look for alternative
sources, new pathogens may emerge as
threats to public health and various previ-
ously unrecognized water quality problems
may come to the fore. For example, as desali-
nation of seawater and brackish water
becomes more widespread, utilities may
need to develop methods to reduce and han-
dle the waste salts that are produced by the
process; sulfate-reducing bacteria may pro-
vide a mechanism for conversion of reverse
osmosisgenerated sulfate waste to a com-
mercially viable product rather than waste
requiring expensive and regulated disposal
(Lee et al, 2005). In addition, recent years
have seen the industry consider the likeli-
hood and consequences of terrorist or other
criminal contamination of water supplies.
The potentially lethal nature of likely conta-
minating agents coupled with the rapid and
large-scale distribution of the agents through
a public water supply necessitates the devel-
opment of rapid detection technologies.
This has led to water microbiologists
becoming familiar with technologies and
equipment that were probably not contem-
plated before 2001 (photo on page 174).
Field portable equipment is available for
rapid detection of selected pathogens but
caution needs to be exercised in their appli-
cation to routine monitoring. The possibility
of false-positive detections can result in
tremendous disruption and expense for all
involvedutilities, government officials,
businesses, and individuals. This has
occurred with continuous on-line air moni-
toring at government buildings on several
occasions, where false-positive identifications
of anthrax and nerve gas have been reported
(US Government Subcommittee, 2005). An
added problem of frequent false alarms is the
boy-who-cried-wolf syndrome, where the
public becomes desensitized to warnings and
will not take them seriously. Early detection
of microbiological contaminants by on-line,
real-time monitoring devices is possible, and
these will become important tools for moni-
toring water supplies for natural or intro-
duced contaminants once the issues encoun-
tered with false-positives are resolved so that
data are reliable.
Microbiology will play a much larger role
in the water utility laboratory of the future.
This will be independent of regulation and
will be driven by the utilitys desire for opti-
mized water quality for consumers. Currently
it is primarily large utilities that have
advanced microbiological capabilities (detec-
tion of parasites, viruses, taste- and odor-
causing bacteria and algae), but more
medium-sized utilities are recognizing the
benefits of having these capabilities in-house.
To be able to provide enhanced routine mon-
itoring, and have the ability to conduct spe-
cific research projects tailored to understand
microbial problems and develop the best
response, requires professional microbiolo-
gists. Understanding the complex role that
microbes play in water supplies and manag-
ing these supplies to consistently produce
high-quality water will continue to challenge
the industry for the foreseeable future.
Paul Rochelle is a principal microbiologist in
the Water Quality Laboratory at the Metropoli-
tan Water District of Southern California where
he manages the Microbiology Development Team.
He has undergraduate degrees in biology and
microbiology from Sheffield and Manchester
Polytechnics in the United Kingdom and a micro-
biology PhD from the University of Wales Insti-
tute of Science and Technology. He has more than
20 years of experience in the application of
microbiological and molecular biology techniques
to the detection and study of microorganisms in
environmental samples and has published widely
on issues relating to microbial water quality.
Jennifer Clancy is a microbiologist and president
of Clancy Environmental Consultants Inc. in St.
Albans, Vt. She has a BS degree from Cornell
University, an MS degree from the University of
Vermont, and a PhD degree from McGill Univer-
sity, all in microbiology, as well as an MS degree
in Environmental Law from Vermont Law
School. She has more than 30 years experience in
medical and environmental microbiology. She
has been involved in microbiological method
development and validation for the American
Society for Testing and Materials and the US
Environmental Protection Agency.
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