Microbiology Awwa

2006 © American Water Works Association

AWWA was founded in 1881 during atime that saw pivotal developments in themicrobiological and public health sciences.Developments such as John Snow’s pioneer-ing work in tracing the source of a choleraepidemic to a contaminated water pump inLondon (1854), Louis Pasteur’s refutation ofspontaneous generation (1861) and develop-ment of pasteurization (1864), Joseph Lister’sfirst work on the use of antiseptics duringsurgery (1867), Ferdinand Cohn’s classifica-tion of bacteria (1875) and discovery of bac-terial spores (1877), and Robert Koch’s isola-tion of the anthrax bacterium (1876), set thestage for a revolution in microbiology duringthe 1880s. Koch’s laboratory in Germanyintroduced the use of pure culture techniquesfor handling bacteria, used agar-agar for thefirst time to produce a practical solid mediumfor culturing bacteria, identified the causativeagent of tuberculosis, and developed a series

of criteria (Koch’s postulates) for determiningthe cause of a disease. Koch later received theNobel Prize in 1905 for founding the scienceof bacteriology. In addition, Clostridiumtetani (the causative agent of tetanus) was dis-covered, the Gram stain was developed,Escherichia coli was identified as part of thenormal human intestinal flora, Pasteur andPaul Ehrlich began their pioneering work onimmunization, 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 beganduring the 1880s. Since these pioneeringdays, the microbiological and public healthsciences have progressed dramatically.Approximately 1,500 human pathogens arecurrently recognized (Cleaveland et al, 2001)and the modes of action and mechanisms oftransmission 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, andsome diseases have virtually been eradicated.Consequently, microbiology within the waterindustry has also advanced.

Infectious diseases exert a huge burden onlife expectancy, public health systems, andnational economies worldwide. The WorldHealth Organization (WHO) estimates thatglobally, 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, safedrinking water. Gastrointestinal diseasecaused by microbially contaminated drinkingwater is one of the leading causes of death inthe developing world, accounting for approx-imately 5 million deaths annually. Such fig-ures demonstrate the importance of efficientdrinking water disinfection. The benefits ofmicrobiologically safe water go beyond theabsence of disease within the community andaffect the productivity of industry as well asthe price of goods and services. Municipalwater systems designed to prevent water-borne infectious disease are probably one ofthe most effective investments of public fundsthat society can make. As such, despitepotential health effects of disinfection by-products, chlorination of drinking water iswidely regarded as one of the most significantpublic health advances in human history.Chlorine-based disinfectants remain animportant treatment for drinking waterbecause they provide a wide range of benefitsthat are not provided by any other single dis-infectant; they are the only disinfectants thatprovide a residual in the distribution system,which is a key part of the multibarrierapproach to preventing waterborne disease.

Nevertheless, despite the advances madein understanding the ecology, pathogenicity,occurrence, and epidemiology of humanpathogens, improvements made in drinkingwater treatment practices, the development ofalternative disinfection technologies, and abetter understanding of watershed manage-ment practices, the water industry mustremain vigilant with respect to the microbiol-ogy of drinking water. Waterborne diseaseoutbreaks continue to occur, even in affluentnations operating modern treatment facilities(Hrudey & Hrudey, 2004). As our under-standing of microbes improves, changes inthe way they are handled and treated may benecessary. 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 aswaterborne threats to public health; thecausative agent is not identified in approxi-mately 30–50% of waterborne outbreaks.Also, the past five years have seen the threat


Changesin Recognized

Species ofCryptosporidium

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. parvum were previously referred to as

C. parvum genotypes 1 and 2, respectively.

1912C. muris

C. parvum

1995C. baileyi

C. meleagridis

C. muris

C. nasorum

C. parvum

C. serpentis

1997C. baileyi

C. felis

C. meleagridis

C. muris

C. nasorum

C. parvum

C. serpentis

C. wrairi

2006C. 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. wrairiSpe

cies


of intentional drinking water contamination,with organisms that may not normally befound in drinking water, emerging as a signifi-cant concern. Consequently, there is a strongincentive for continued assessment of micro-bial risk, development of improved analyticalmethods, and proactive microbial research bythe water industry.

Although the primary focus of this reviewis on those microorganisms that cause dis-ease, the water industry is interested in fourgeneral groups of microorganisms:

• Frank and opportunistic pathogens thatcause disease such as Cryptosporidium spp.,enteropathogenic E. coli, and enteric viruses.

• Microorganisms such as coliforms, E.coli, enterococci, and bacteriophages(although not necessarily harmful themselvesthey indicate that water quality has beencompromised).

• Nuisance organisms that can cause aes-thetic problems or degrade water quality.These include nitrifying bacteria and taste-and odor-producing cyanobacteria and arethe subject of AWWA Manual M7, ProblemOrganisms in Water (AWWA, 2004).

• Beneficial microbes such as those thatform the basis of biologically active filters ororganisms that may be used to reduce wasteproducts generated by water treatment (e.g.,bioremediation of waste salts produced dur-ing desalination).

Pathogens in drinking waterEnvironmental waters that are used as

sources of drinking water may be contami-nated with a wide range of pathogenicmicroorganisms (Table 1) intermixed with adominant background of naturally occurringnonpathogenic microbial populations.Although water treatment facilities that areoperated correctly remove the vast majority ofmicrobial 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 yearsof the 20th century, waterborne diseases suchas cholera (caused by Vibrio cholerae) andtyphoid (Salmonella typhi) were still majorpublic 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 thedeveloped world (McGuire, 2006).

However, as these diseases disappeared,new ones emerged. These include the para-sitic protozoa Cryptosporidium and Giardia,pathogenic E. coli, a variety of enteric viruses,and toxin-producing cyanobacteria. Figure 1shows the changing nature of waterborne dis-ease outbreaks from the 1930s to 2000. Inthe 1930s amebiasis (caused by the intestinalparasite Entamoeba histolytica) and typhoidwere the predominantly recognized causes ofwaterborne disease, along with a handful ofhepatitis A cases. In the period from 1940 to1950, more organisms became recognized asagents of waterborne disease. Giardia wasrecognized as an agent of waterborne diseasein Japan in 1946, but it was not until the1960s that the first waterborne cases werereported in the United States. Once thewaterborne route for giardiasis was under-stood and surveillance instituted, a 100-foldincrease of cases was reported in the periodfrom 1976 to 1980. The Milwaukee, Wis.,cryptosporidiosis outbreak in 1993 addedanother >100-fold increase in the number ofcases of waterborne disease, but other emerg-ing pathogens began to appear as waterbornedisease agents in the 1990s. Cyclosporacayetanensis, a coccidian parasite similar toCryptosporidium, was documented as thecause of an outbreak in a Chicago, Ill., hospi-tal and E. coli O157:H7, previously known asan agent of foodborne disease outbreaks,caused several large waterborne disease out-breaks, with some deaths directly attributedto contaminated drinking water.

In 2005, there were just six cases ofcholera and 261 cases of typhoid fever in theUnited States, compared with 7,212 cases ofcryptosporidiosis, 2,368 cases of E. coliO175:H7 infection, 17,256 cases of giardia-sis, 9,877 cases of hepatitis, and 1,952 casesof legionellosis; however, only a portion ofthese reported cases were waterborne (CDC,2005). Although cholera and typhoid remainmajor scourges in the developing world, at thedawn of the 21st century in the United States,they have been replaced by microbes that donot cause widespread mortality but do repre-

Gia

rdia


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 Cryptosporidium spp. 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

Hea

lth E

ffects

166 MARCH 2006 | JOURNAL AWWA • 98:3 | ROCHELLE & CLANCY 2006 © American Water Works Association


sent waterborne threats to public health andcan, in some instances, be fatal. All water-borne outbreaks are the result of some form ofdrinking water contamination, and althoughmany can be attributed to specific failures intreatment plant operations or poor managerialdecisions, some outbreaks occur even in theabsence of operational violations. The largestand thus most famous waterborne disease out-break in 1993 in Milwaukee was caused by aprotozoan parasite, Cryptosporidium, andaffected approximately 403,000 residents andcaused 100 deaths (Mackenzie et al, 1994).Probably no other organism better highlightsthe technological advances in drinking watermicrobiology over the past 20 years. Theinterest spurred by the Milwaukee incidentled to the development of improved oocystrecovery and purification methods, manypolymerase chain reaction- (PCR-) basedmethods, including real-time PCR and geno-typing procedures, microarray assays, and cellculture–based infectivity assays.

Cryptosporidium muris was first identifiedin 1907 and C. parvum five years later

(Tyzzer, 1907; 1912), but it was not recog-nized as a human pathogen until 1976. Sincethen the taxonomy of the genus has been amoving target (sidebar on page 164),although the past 10 years have seen signifi-cant improvements in our understanding ofthe parasite’s speciation. The first knownwaterborne cryptosporidiosis outbreakoccurred in 1984 in Texas (Hrudey &Hrudey, 2004) and at least nine other out-breaks were reported before the Milwaukeeincident, two of which had an estimated dis-ease incidence of more than 10,000 individu-als. Many outbreaks of cryptosporidiosis havenow been associated with drinking water orrecreational use of water worldwide, and out-breaks continue despite the lessons of thepast 20 years. The most recent outbreak in2005 affected more than 200 people in NorthWales in the United Kingdom (CDR, 2005).Occurrence studies in the United States andCanada report finding oocysts in 4.5–100%of raw water samples (Rose et al, 1997; Walliset al, 1996; LeChevallier & Norton, 1995),and water samples that were from source


1935

1950

1965

1980

2000 Amebiasis

Typhoid

Tularemia

Paratyphoid

Hepatitis A

Leptospirosis

Shigellosis

Giardiasis

Salmonellosis

Cyclospora

E. coli 0157

Cryptosporidiosis

0.0001

0.001

0.01

0.1

1

10

100

1,000

10,000

100,000

Year

Ave

rag

e A

nn

ual

Occ

urr

ence

FIGURE 1 The changing nature of waterborne disease

Wat

erbor

ne d

iseas

e


waters receiving domestic and agriculturalwaste had oocyst concentrations as high as5,800/L (Madore et al, 1987). The US Envi-ronmental Protection Agency’s (USEPA’s)Information Collection Rule (ICR) survey of5,838 untreated source water samplesthroughout the United States reported anaverage occurrence of 6.8%, with a mean con-centration of 0.067 oocysts/L (Messner &Wolpert, 2003). Oocysts have also beendetected in up to 40% of treated drinkingwater samples (Rose et al, 1997). An AwwaResearch Foundation study of 100 surfacewater plants showed that 76 were positive forCryptosporidium in source water samples,whereas 15 plants had Cryptosporidium pre-sent in the filter effluents (McTigue et al,1998). In another study of Cryptosporidiumoccurrence, approximately 10% of 518source water samples collected over a one-year period from six watersheds were posi-tive; the mean oocyst concentration was0.015/L (LeChevallier et al, 2002).

The disparity in the reports of very highnumbers of Cryptosporidium oocysts in bothraw and treated water in early studies com-pared with much lower concentrations inmore recent studies may be a reflection ofimproved analytical methods. In the late1990s, the USEPA developed method 1622,a significant improvement to the ICR method(USEPA, 2001). The filtration step wasimproved to permit total capture of oocystsand excellent recovery through elution; theuse of immunomagnetic separation wasincorporated to significantly improve separa-tion of oocysts from sample debris; and addi-tional microscopic confirmation criteria(internal morphology) reduced the number offalse-positives reported (Clancy, 2000;Clancy et al, 1999).

Because the presence of C. parvum waswell documented in cattle, it was generallyassumed that cows were responsible formuch of the Cryptosporidium contaminationdetected in surface waters. However, as aresult of the application of a variety of molec-ular methods in the 1990s, it was recognizedthat the oocysts identified microscopically asC. parvum could be categorized as two sepa-rate types: genotype 1 or anthroponoticoocysts were isolated almost exclusively from

Water Quality-relatedMicrobes Whose Entire

Genomes Have BeenSequenced

Acanthamoeba castellani

Human adenoviruses (types A–F)

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.htmlMicr

obes


humans; genotype 2 or zoonotic isolates weredetected in a wide range of animals, includ-ing humans. Based on genetic, physiological,and host animal differences, and the apparentlack of recombination between the two geno-types, these two groups have been classifiedas separate species. The zoonotic genotyperetained the name C. parvum, and theanthroponotic genotype was named C.hominis (Morgan-Ryan et al, 2002). There isconsiderable strain level variation in thegenomic sequences within each species. C.parvum and C. hominis oocysts (or organ-isms morphologically indistinguishable fromC. parvum and C. hominis) have beenreported in at least 152 species of mammals,including humans (Fayer et al, 2000), and

these two species are responsible for mostcases of human cryptosporidiosis. However,other species or genetically distinct isolates ofCryptosporidium that have also been isolatedfrom human infections are C. canis, C. felis,C. meleagridis, C. suis, and C. muris (Gatei etal, 2002; Morgan-Ryan et al, 2002; Fayer etal, 2001; Pedraza-Diaz et al, 2001; Xiao et al,2001; Morgan et al, 2000; Pieniazek et al,1999). C. meleagridis was originallydescribed in 1955 (Slavin, 1955) but is nowrecognized as an emerging human pathogenin the United Kingdom, where it is responsi-ble for 1% of all human cryptosporidiosiscases (Caccio et al, 2005). Also, there appearto be many animal host-specific strains ofCryptosporidium that are detected in water

Mic

robi

olog

yDate 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 Cryptosporidium sp. 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 1712006 © American Water Works Association

Significant Events in the Historyof Drinking Water Microbiology

1854 John Snow traced cholera outbreak to water pump in London’s 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

Cryptosporidium first 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 Cryptosporidium in 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., Cryptosporidium waterborne 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 world’s 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 isnot yet known. Genotyping data from 22waterborne cryptosporidiosis outbreaksdemonstrated that 67% were caused by C.hominis and 33% by C. parvum (McLauchlinet al, 2000; Sulaiman et al, 1998). Humanfecal contamination is therefore responsiblefor many of these outbreaks.

For an organism that was not recognized asa human pathogen until 30 years ago, there hasbeen a tremendous amount of research aimedat improving detection methods, evaluatingdisinfectants and antimicrobial agents, refiningviability and infectivity assays, and increasingour understanding of the organism’s biology,epidemiology, and occurrence in the environ-ment. An AWWA symposium dedicatedentirely to waterborne cryptosporidiosis(Fricker et al, 1997) laid the groundwork formuch of the research that has been completedduring the past 10 years. Some of the detec-tion methods that have been developed,including those that were investigated butabandoned, include flow cytometry, spec-troscopy, immunoassays, continuous cen-trifugation, vortex flow filtration, ultrafiltra-tion, probe hybridization, in-situhybridization, nucleic acid sequence basedamplification, PCR, reverse transcriptase

PCR, real-time-PCR, andmicroarrays. Development of C.parvum in cell culture was firstreported in 1984 (Current &Haynes, 1984), and in the1990s many cell lines wereshown to support infection.Cell culture–based infectivityassays were developed specifi-cally for drinking water applica-tions (Rochelle et al, 1997;Slifko et al, 1997; Di Giovanniet al, 1999), and the equiva-lency of cell culture with a stan-dard mouse infectivity assay hasbeen demonstrated (Rochelle etal, 2002). A study utilizing cellculture to assess infectivityreported that 27% of surfacewater treatment plants werereleasing infectious oocysts intheir finished water, and over-

all, 1.4% of treated drinking water samplescontained infectious oocysts (Aboytes et al,2004). This finding raises doubts concerningthe ability of conventional treatment plants tomeet the USEPA’s risk goals. Cryptosporid-ium isolates can be speciated and genotypedby PCR targeting at least five genomic targets(18S rDNA, -tubulin, hsp70, COWP, actingenes) and differentiation at the subgenotypelevel is achieved using micro- and minisatel-lite repeat sequences. In addition, thegenomes of both C. hominis and C. parvumhave been sequenced in their entirety (Abra-hamsen et al, 2004; Xu et al, 2004), allowingin-depth genetic comparisons between thetwo species. Because of its widespread occur-rence, low infectious dose, and resistance toconventional chlorine disinfection (Korich etal, 1990), the organism has been targeted byrecently promulgated drinking water regula-tions (USEPA, 2006).

Although not the subject of such an intenseresearch effort in recent years, our under-standing of Giardia spp. has also progressed.Giardia was originally described by Antonievon Leeuwenhoek in 1675; it was then redis-covered by William Lamble in 1859 andnamed after him—Giardia 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 wasfirst recognized as a waterborne pathogen inJapan in 1946, and the first documentedwaterborne outbreak of giardiasis in theUnited States was in Aspen, Colo., during the1965–66 ski season, affecting 120 skiers (Lin,1985). Giardia was consistently the mostcommonly identified pathogen in waterbornedisease outbreaks in the United Statesbetween 1971 and 1996, with 115 outbreaksand 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 arenow known to comprise at least seven geneti-cally distinct assemblages (which may even-tually be designated as new species), onlytwo of which (A and B) appear to infecthumans (Caccio et al, 2005). Despite themany waterborne outbreaks of giardiasis, therole of animals and person-to-person diseasetransmission, and the relative risk of acquir-ing infection through drinking water are yetto be resolved.

Whereas Pasteur (1822–95) and Koch(1843–1910) are seen as the founders ofmedical bacteriology, Sergei Winogradsky(1856–1953) and Martinus Beijerinck(1851–1931) are recognized as the origina-tors of environmental microbiology. Beijer-inck discovered sulfate-reducing bacteria anddeveloped the first enrichment cultures.Enrichment cultures are used today to isolatebacteria with specific physiological propertiesand to improve detection sensitivities for avariety of microbial pathogens. Beijerinckwas awarded the Leeuwenhoek Medal in1905. Other medal awardees whose work isstill influencing the study and development ofmicrobiology within the water industry arePasteur (1895), Felix d’Herelle (1925, co-dis-coverer of bacteriophages), Winogradsky(1935), Roger Stanier (1981; who estab-lished that blue/green algae are bacteria [thecyanobacteria]), and Carl Woese (1992) whoredefined the tree of life based on phyloge-netic analysis of 16S ribosomal RNAsequences. Most important, Beijerinck dis-

covered viruses, a name he coined in 1898,following on from Dmitri Iwanowski’s worksix years earlier. Poliomyelitis was the firsthuman disease shown to be caused by a virusin 1909. During the 1930s to 1950s, patho-genic viruses were cultured in chick embryosand other animal systems. The first in-vitrocell culture systems were developed in 1949,and a viral plaque assay was developed in1952 allowing accurate quantitation of animalviruses. The 1960s onward saw continueddevelopment of improved detection methodsfor viruses, including radio-immunoassays,immunofluorescence, Western blots, andenzyme-linked immunosorbent assays. Awaterborne outbreak in 1968 was attributedto enteric viruses but the 1971 edition ofStandard Methods for the Examination ofWater and Wastewater stated that “No rou-tine examination of water or wastewater forenteric viruses is practical or necessarilymeaningful at the present time.” However, in1979 the WHO Scientific Group on HumanViruses in Water, Wastewater, and Soil con-cluded that contamination of water by viruseswas a significant threat to public health, evenin the developed world.

A large waterborne outbreak caused bycoxsackie virus B3 and hepatitis A virusaffected approximately 7,900 people in Texasin 1980 (Hrudey & Hrudey, 2004), and therehave been many viral waterborne outbreakssince. It is speculated that many of theapproximately 30–50% of outbreaks forwhich no causative agent is identified may becaused by viruses. Since a 1965 symposiumon transmission of viruses by water, therehave been concerted efforts to develop andimprove viral detection methods for watermatrices. The 15th edition of Standard Meth-ods (1981) included a tentative method fordetecting enteric viruses in up to 1,000 L offinished water based on a two-stage filteradsorption–elution procedure. In 1984, theUSEPA published a Manual of Methods forVirology (USEPA, 1984) that included the“best methods” available at the time. Theexpress intent of the manual was to make itpossible for any competent water bacteriol-ogy laboratory to concentrate and recoverviruses from water. This manual has now S

tand

ard

Meth

ods


been revised at least seven times as better andnewer methods have been developed. Themethod used for the ICR (USEPA, 1996)involved recovery of viruses by capture onpositively charged filters, organic floccula-tion, and analysis by a total culturable virusassay on Buffalo Green Monkey kidney(BGMK) cells. Viral detection methods arenow routinely used in many laboratories andimprovements have led to quantitative assaysthat are used for enumeration and disinfec-tion studies (photo on page 184), faster sam-ple turnaround, and assays that support awider range of viruses. Molecular detectionmethods were first applied in the 1980s withPCR and its many variants used for directdetection in environmental samples anddetection of infection in cell cultures. Theexponential increase in the amount of nucleicacid sequence data has allowed for greaterspecificity in probe and primer design andthe genomes of some potentially waterborneviruses have been sequenced in their entirety(sidebar on page 170). However, furtheradvances in virological methods are still nec-essary to assess the public health significanceof waterborne viruses. For example, an in-vitro cell culture method is not available forhuman caliciviruses and a more reliable quan-titative plaque assay is required for aden-ovirus types 40 and 41, but both of thesesissues are currently being investigated.

The Safe Drinking Water Act requires theUSEPA to publish a list of contaminants thatmay require regulation in the future but arenot currently subject to any proposed or pro-mulgated regulations. This ContaminantCandidate List (CCL) contains nine groupsof microbes that are known or anticipated tooccur in water, but for which the analyticaldetection methods are inadequate and insuf-ficient information is available on the healtheffects, occurrence, and treatment efficacy toallow a regulatory decision (sidebar on page179). For some organisms, information islacking for all of these criteria. In otherinstances, there is ample health effects infor-mation but occurrence data are inadequate.For example, the health effects of humanpathogenic species of microsporidia, Entero-cytozoon bieneusi, and Encephalitozoon spp.,

are well documented (Wittner et al, 1999).Microsporidia were first recognized ashuman pathogens in the 1970s and wereoriginally classified as amitochondrial proto-zoa, but phylogenetic analyses based on sev-eral genes indicated that they are moreclosely related to fungi (Katinka et al, 2001;Weiss & Vossbrinck, 1999). Encephalitozooncuniculi has 11 chromosomes, and with 2million bases, has one of the smallest knowneukaryotic genomes (Katinka et al, 2001).The routes of transmission to humans arenot clearly understood, but many wild anddomestic animals can carry microsporidia soit is possible that surface waters can becomecontaminated and consequently may serve asa route of transmission to humans. Prototypemethods have been developed for detectingmicrosporidia spores in environmentalwaters and recent work has demonstratedthat microsporidia are sensitive to chlorineand ultraviolet (UV) disinfection at levelstypically used for drinking water treatment(Huffman et al, 2002; Johnson et al, 2003).A restrospective epidemiological study of acluster of microsporidiosis cases indicatedan association with the municipal water dis-tribution system but no evidence of contami-nation was found (Cotte et al, 1999), andthere is some doubt as to whether this inci-dent truly represented a waterborne out-break (Hunter, 2000). Human-pathogenicmicrosporidia have been detected in surfacewater and groundwater, tertiary-treatedsewage effluent, and food crop irrigationwater (Dowd et al, 2003; Thurston-Enriquezet al, 2002; Dowd et al, 1998; Sparfell et al,1997). However, methods are still inade-quate to allow a definitive assessment of theirsignificance to waterborne disease and thepotential for zoonotic transmission has notyet been fully investigated.

Adenoviruses are nonenveloped, icosohe-dral, double-stranded DNA viruses (80–110nm in diameter) that are widespread innature, infecting birds and many mammals;human adenoviruses are classified into sixspecies (A–F; Horwitz, 2001). The virusescause a variety of clinical manifestations, andthe case fatality rate is as much as 50% amongthe immunocompromised. There have been a


few studies of adenovirus occurrence insource waters. Chapron et al (2000) analyzedsamples collected during the ICR usingBGMK cells as well as nested PCR and foundthat 38% of the samples contained infectiveviruses and that 48% of the samples werepositive for adenovirus DNA. Human aden-ovirus DNA has been detected in surfacewaters, seawater, and treated drinking waterusing PCR-based methods (Fong et al, 2005;van Heerden et al, 2005; Pina et al, 1998),but detection of pathogens by PCR-basedmethods alone does not necessarily correlatewith the presence of infectious organisms.For example, a real-time PCR assay detectedadenovirus DNA in 16% of samples, butnone of them contained infectious aden-oviruses when assayed on two cell lines (Choi& Jiang, 2005). Infectious adenoviruses weredetected in urban rivers affected by domesticand industrial wastewaters (Lee et al, 2004).A few outbreaks of adenovirus infection havebeen 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 waterbornepathogens. Consequently, although they arelisted on the CCL with the caution that thereis insufficient scientific information on aden-oviruses, they have become the regulatorydriver for setting UV dose requirements forvirus inactivation credit (USEPA, 2006).

Microbial indicators and otherorganisms

Drinking water treatment plants tradition-ally monitor fecal coliform and other indica-tor organisms to provide an approximatemeasure of potential fecal contamination andevaluate the efficacy of removal or inactiva-tion of pathogenic microorganisms. The firstedition 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. Theeighth edition of Standard Methods (which

Microorganisms on the CCLThe 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

CCL—Contaminant Candidate List

*Microsporidia were originally classified as protozoa but are now recognized as being more closely related to fungi. Mic

roor

gani

sms


became known as Standard Methods for theExamination of Water and Wastewater withthe publication of the 11th edition in 1960),published in 1936, described pour plates forthe bacteriological examination of water andlactose fermentation tubes and Endomedium to confirm the presence of “coli-aerogenes” bacteria (today’s coliform group).In the intervening 70 years, great advanceshave been made in the development of meth-ods for detecting specific microorganisms,including those such as viruses and protozoathat cannot be readily cultured on agarplates, 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. Pourplates, mEndo medium, most probable num-ber tables, and gas production upon lactosefermentation are all still used in modernwater quality laboratories. Advances such asthe introduction into Standard Methods of a

membrane filter method for fecal coliforms(1971), development of API identificationstrips for enteric bacteria in the 1970s, andthe development in the 1980s of fluorescentmedia containing 4-methyl-umbelliferyl-D-glucuronide (MUG) for detecting coliformsin drinking water have streamlined the pro-cedures and decreased analysis time.

The Total Coliform Rule (TCR; USEPA,1989) requires all public water systems tomonitor for coliform bacteria in their distrib-ution systems, specifies a followup monitor-ing schedule whenever positive samples aredetected, and requires public notification ofpositive samples exceeding the maximumcontaminant level. However, there are someconcerns over the value of the total coliformtest for public health protection. Progressivechanges to the analytical methods aimed atstreamlining the procedure and decreasingthe time to obtain results have reduced itsspecificity such that a positive result does notnecessarily imply treatment failure or conta-mination of the distribution system. A totalcoliform–positive may simply representgrowth of nonpathogenic environmental bac-teria in distribution system water or pipebiofilms. Many researchers, public healthofficials, and AWWA have suggested that E.coli should be adopted as the sole microbialindicator for compliance purposes, and theavailability of relatively simple rapid culture-based and molecular tests for detecting andidentifying E. coli make this a feasible alterna-tive. The TCR is currently undergoingreview by the USEPA, with the revised ruledue to be published in 2006.

Microbial source tracking (MST) repre-sents a further potential development of E.coli as an indicator of contamination. MST isbased on the assumption that different animalspecies have developed different intestinalmicrobial flora and that these differences canbe discerned with an appropriate tool andmay be used to identify sources of fecal cont-amination in water. There are many proposedMST methods, but many of them involvephenotypic methods (e.g., antibiotic resis-tance analysis) or genotypic analysis such asrepetitive-PCR, ribotyping, and pulse-fieldgel electrophoresis of E. coli or enterococci

Although today’s

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

oli


isolates recovered from potential source ani-mals and the body of water under investiga-tion. However, most MST methods areresearch-level tools that are not yet ready forroutine implementation (Griffith et al, 2003;Stoeckel et al, 2004). Despite early claims ofsuccess in the use of E. coli to determinesources of contamination in water,intraspecies heterogeneity, growth and per-sistence of these bacteria in the environment,and a relatively cosmopolitan distributionamong various animal host species, suggestthat it may not be the most appropriate targetorganism 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, anddiarrheal diseases. There are currently 170serotypes of diarrheagenic E. coli strains thatare classified as enterotoxigenic, enteropatho-genic, enteroaggregative, enteroinvasive, andenterohemorrhagic, each characterized bydiffering pathogenic features (Nataro &Kaper, 1998). The most prominent patho-genic strain is enterohemorrhagic E. coliO157:H7 that produces Shigella-like toxinsand is therefore also referred to as shiga toxinE. coli along with approximately 60 otherserotypes. The 13th edition of StandardMethods (1971) considered waterborne infec-tions by pathogenic E. coli to be “quiteimprobable,” but this prediction held true foronly four years. The first waterborne out-break attributed to pathogenic E. coli(O6:H16) occurred in 1975 and sickened2,200 people (Rosenberg et al, 1977). Thenin 1989 a waterborne outbreak of E. coliO157:H7 in Cabool, Mo., sickened 243 peo-ple and killed four (Hrudey & Hrudey,2004). Although this bacterium is unlikely tobe a common contaminant in surface watersthat are not directly affected by raw fecalmaterial and it is effectively inactivated by allof 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 shouldhave been a wakeup call for the water indus-try. Two other E. coli O157:H7 waterbornedisease outbreaks occurred in the 1990s; one

in Alpine, Wyo., with 114 cases in 1998, fol-lowed by an outbreak at a small county fair inupstate New York, resulting in 781 cases andtwo deaths. In spite of these warnings, theexperience was repeated in 2000 on a largescale in Walkerton, Ont., Canada, with 2,300cases and seven deaths attributed to E. coliO157:H7 contamination of drinking water(O’Conner, 2002). The political, economic,and personal ramifications of the Walkertonoutbreak still reverberate today. There havebeen at least 30 waterborne outbreaks causedby E. coli O157:H7, and the many methodsthat are now available for detecting and iden-tifying pathogenic strains of E. coli (particu-larly O157:H7) attest to their importanceand public health effects. These includeselective culture media, immunomagneticpurification kits, fluorescent antibodies, latexagglutination tests, enzyme linkedimmunosorbent assays, and molecular assaystargeting the shiga toxin and antigen biosyn-thesis genes, among others. A particular con-cern from a water industry perspective is thatsome diarrheagenic strains, including manyenteroinvasive are typically lactose-negative(Nataro & Kaper, 1998). In addition, moststrains of E. coli O157:H7 do not produce afunctional �-glucuronidase (Strockbine et al,1998) and will therefore not be detected byMUG-based methods. A recent study evalu-ated four antibody-based methods and aPCR assay for detecting E. coli O157:H7 inwater (Bukhari et al, 2005). The authorsreported comparable performance for one ofthe immunological tests and the PCR-basedmethod with a reproducible detection sensi-tivity of 20 cfu/200 mL water.

A further example of how methodologicaldevelopments have improved the waterindustry’s understanding of and ability tocontrol bacterial processes that relate towater quality is provided by nitrifying bacte-ria. Winogradsky conducted the earliestwork on nitrifying bacteria in 1891, demon-strating that oxidation of ammonia is a two-step process, and identified several genera ofimportant nitrifying bacteria: Nitrosomonasspp. (the predominant ammonia oxidizer),Nitrosococcus spp., Nitrobacter spp. (the pre-dominant nitrite oxidizer), and Nitrospira


spp. Apart from playing a significant role inthe biogeochemical nitrogen cycle, nitrifyingbacteria are also important in the mainte-nance of finished water quality. Ammonia inwater, including that introduced through theuse of chloramine disinfection, can lead tobiological instability in drinking water distri-bution systems by promoting the growth ofnitrifying bacteria. The resulting cell massand the products of ammonia oxidationdeplete the residual disinfectant and maysustain the growth of nitrite-oxidizing andheterotrophic bacteria. The reduction inchloramine residual and development of amicrobial community in the distribution sys-tem generally leads to an overall deteriora-tion in water quality, including the formationof nitrite or nitrate. Nitrification in chlorami-nated drinking water was first addressed inthe late 1980s with investigations of the sea-sonal occurrence and distribution of ammo-nia-oxidizing bacteria as well as theirresponse to disinfection (Wolfe et al, 1990).Techniques that have been applied to thestudy of nitrifying bacteria include PCR, typ-ically targeting the 16S rRNA or ammoniamonooxygenase genes, and comparativenucleotide sequence analysis (Baribeau et al,2000), real-time PCR, fluorescent in-situhybridization, confocal microscopy, anddenaturing gradient gel electrophoresis toinvestigate community structure (Kowalchuk& Stephen, 2001). Through the applicationof such tools over the past 15 years, theoccurrence, 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 relativelywell understood (Wolfe & Lieu, 2002).Recent research has focused on using a com-bination of molecular detection tools andmeasurement of various physical and chemi-cal water parameters in distribution systemsas an early warning of potential nitrificationconditions (Regan et al, 2002).

Detection methodsThe 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 physiologicalfunctions such as growth on particular sub-strates or production of specific compounds.The Society of American Bacteriologists,later to become the American Society ofMicrobiology, applied this technique to pre-pare a report on the classification of bacteriathat evolved in 1923 into Bergey’s Manual ofDeterminative Bacteriology. This classifica-tion scheme became the standard for manyyears. However, as more diverse bacteriawere isolated from a wider range of habitats,it became apparent that classification basedon growth and morphological characteristicswas unrealistic and often generated inconsis-tent identifications. Agar was first used as asolidifying agent in bacterial culture media in1881 and for the next 100 years, growingbacteria on a variety of media was the stan-dard procedure for studying waterbornemicrobes. Media were initially nonspecificbut selective media that either preferentiallysupport the growth of particular bacteria orallow differentiation between species basedon indicator compounds are now available forcoliforms, E. coli, E. coli O157:H7,pseudomonads, enterococci, Legionella spp.,mycobacteria, Campylobacter spp., Yersiniaspp., Burkholderia cepacia, Clostridium spp.,and Salmonella spp., among others. How-ever, protozoa, viruses, and many bacterialpathogens cannot be cultured on agar plates.Therefore, although the agar plate is still themainstay of coliform compliance monitoringin the water industry, many alternative meth-ods have been developed for working withthe ever-broadening array of microorganismsthat confront the microbial water quality lab-oratory. Cell culture–based methods forviruses and protozoa have become routine—ifnot standardized—in many laboratories, anti-body-based separation and detection meth-ods are available for a broad array ofpathogens, and an almost bewildering arrayof detection platforms have been developed(Figure 2; photo on page 186). Rapidity ofdetection has become one of the major dri-vers in the development of analytical meth-ods. Over the past 15 years molecular biol-ogy–based techniques, and in particularPCR, have revolutionized the detection of

Bac

teria



pathogenic bacteria, viruses, and protozoa inboth clinical and environmental samples.Molecular methods enable rapid detection ofpathogens in water by providing levels of sen-sitivity and specificity difficult to achieve withtraditional culture-based assays, which oftentake days to perform.

The double-helix structure of DNA wasidentified in 1953 by James Watson, FrancisCrick, Maurice Wilkins, and RosalindFranklin. DNA hybridization was first used tocompare species in 1961, and nucleic acidreassociation was developed in 1969 to clas-sify enterobacteria. DNA sequencing wasinvented in 1977, automated sequencing wasdeveloped in 1986, and the first completebacterial genome was sequenced in 1995

(Haemophilus influenzae). Then in 2005, anentire microbial genome (Mycoplasma geni-talium) was sequenced in less than a day on asingle instrument (Margulies et al, 2005).Pathogens of interest to the water industry forwhich entire genome sequences are availableare shown in the sidebar on page 170. ThisDNA timeline illustrates the technique-drivenadvances in modern microbiology over thepast 40 years that have led to the current situ-ation in which data processing and analysis isthe limiting factor, rather than data genera-tion. Molecular methods have revolutionizedour understanding of the composition, phy-logeny, physiology, and function of microbialcommunities in the environment. Publishedapplications of molecular techniques to

Water sample(100 mL – 1,000 L)

Elution and centrifugation

Nucleic acid extraction

Direct extractionof nucleic acidswith or without

membranedissolution

Reverse transcriptionfor RNA targets

Nonselective or selective cultural

enrichment

Immunomagneticpurfication

of target pathogen

Purification to remove inhibitors

Probe capture

DNA sequencedatabases

Primer/probedesign

Empirical specificitytesting with non-target

organisms andassessment of sensitivity

Gel electrophoresis

Hybridization withconfirmatory probes

Restriction digestion and/orDNA sequencing for

confirmation and identification

Microarray hybridization

Simultaneous probing,real-time detection, and

quantification with QPCR

Amplification withuniversal primers

Microarray hybridizationtargeting multiple pathogens

(with speciation and strain typing)

Concentrate by filtration:Membranes, capsules, cartridges, hollow fiber, electropositive, electronegative

QPCR—quantitative polymerase chain reaction

Selection of sample concentration method, the level of sample enhancement or purification (red), andparticular 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 detectionof pathogens in water, fecal source tracking ofeither indicator microorganisms or specificpathogens, and detection methods for in-vitroinfectivity and disinfection assays (Table 3).Although not formally standardized orapproved for monitoring purposes within thewater industry, nucleic acid–based techniqueshave been widely used for detecting and ana-lyzing microorganisms in water.

Molecular methods were first applied tothe detection of potential waterbornepathogens in the 1980s, but they have notbeen adopted on a routine basis by the waterindustry, as they have been in clinical, foren-sic, and food industry laboratories. There areseveral reasons for the lack of responsivenessin the water industry. There is a lack of stan-dardization of molecular methods, and rigor-ous quality assurance and control measureshave only recently been applied to thesemethods. There are also relatively fewresearchers and still fewer utilities using mole-cular tools to address water-related microbialissues. Then there is the unique challengepresented by attempting to detect very lowconcentrations of target organisms in rela-

tively large volumes of water (typically1–1,000 L). In addition, water is a very com-plex matrix; even finished drinking water cancontain a plethora of microorganisms. This isa significant difference between water andclinical applications. For example, when apathogen is identified in a clinical sample, theanalyst can be nearly 100% certain it is thecorrect identification, and, if the individual isexperiencing symptoms of the suspected dis-ease, it is highly likely that the patient has thatdisease. In a water sample, there are hun-dreds of organisms that may be present, pro-viding a larger challenge in determiningwhether there are pathogens in the mix. Thepossibility of false-positive identifications indrinking 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 forCryptosporidium using PCR. They cautionedabout the use of molecular methods in envi-ronmental samples where organisms with aclose phylogenetic relationship may co-exist.

There are many approaches for applyingmolecular methods to the study of microbialwater 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 ofpathogens and other microorganisms inwater. The replacement of radioisotopicmethods with nonradioactive alternatives inthe 1990s made the methods available to awider range of laboratories. However, a cer-

tain amount of evaluation and optimization isstill necessary to ensure that results obtainedusing these techniques are reliable and con-sistent, and it is important that molecularmethods should not be considered as analternative to conventional microbiologicaltechniques. 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

Cryptosporidium spp. Surface water Cell culture/PCR LeChevallier et al, 2003

Cryptosporidium spp. 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

ELISA—enzyme-linked immunosorbent assay, FISH—fluorescent in-situ hybridization, NASBA—nucleic acid sequence based

amplification, PCR—polymerase chain reaction, RT-PCR—reverse transcriptase PCR, qPCR—quantitative 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. Incorporationof appropriate controls into molecular assaysis critical, as is understanding the significanceof molecular “positives” in the absence ofculture-based methods.

Microscopy has always been, and willremain, fundamental to the study of microor-ganisms, simply because there is no substi-tute for being able to actually see the objectof investigation (photo on page 180). One ofthe earliest rudimentary microscopes wasused to identify Giardia in 1675, andalthough the basic principle did not change,significant advances in the science and engi-neering of optics were necessary to maketheir use routine. The electron microscopewas invented in 1931, phase contrastmicroscopy was developed in 1934, andNomarski differential interference contrastmicroscopy was patented in 1953. The prin-ciples of fluorescence microscopy were ini-tially realized in the early 1900s, but thedevelopment of epifluorescence illumination

in the mid 1970s brought thetechnology into the main-stream. Although the modernwater quality laboratorymicroscope would be recog-nized by a microbiologistfrom 125 years ago, thediversity of illumination,observation, image capture,image analysis, and automa-tion functions would nodoubt make them envious.Many fluorescent antibodiesand fluorogenic compoundsare now available for observ-ing both intact microbes andtheir internal constituents.Despite the many technologi-cal developments in pathogendetection methods, therecently promulgated LongTerm 2 Enhanced SurfaceWater Treatment Rule(USEPA, 2006) includes fluo-rescence microscopy as theonly approved method fordetecting Cryptosporidium

oocysts. Nevertheless, there is a relatively highlevel of subjectivity in the microscopic identi-fication of Giardia cysts and Cryptosporidiumoocysts in environmental samples, even byqualified analysts. In some circumstances,such identification can best be described asqualified guesswork (Clancy, 2000).

The crystal ballIn parallel with the broader scientific

community, the microbial sciences withinthe water industry have progressed steadilyover the past 125 years, with occasionaltechnological leaps and recognition of newhuman pathogens opening up entirely newareas of investigation. The future is likely tofollow the same pattern. Emerging and re-emerging pathogens will necessitate contin-ued development of analytical methods.Fundamental studies of pathogen healtheffects and epidemiology, and surveys ofpathogen occurrence will be needed todetermine the true role of drinking water inhuman 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 alternativesources, new pathogens may emerge asthreats to public health and various previ-ously unrecognized water quality problemsmay come to the fore. For example, as desali-nation of seawater and brackish waterbecomes more widespread, utilities mayneed to develop methods to reduce and han-dle the waste salts that are produced by theprocess; sulfate-reducing bacteria may pro-vide a mechanism for conversion of reverseosmosis–generated sulfate waste to a com-mercially viable product rather than wasterequiring expensive and regulated disposal(Lee et al, 2005). In addition, recent yearshave seen the industry consider the likeli-hood and consequences of terrorist or othercriminal contamination of water supplies.The potentially lethal nature of likely conta-minating agents coupled with the rapid andlarge-scale distribution of the agents througha public water supply necessitates the devel-opment of rapid detection technologies.This has led to water microbiologistsbecoming familiar with technologies andequipment that were probably not contem-plated before 2001 (photo on page 174).Field portable equipment is available forrapid detection of selected pathogens butcaution needs to be exercised in their appli-cation to routine monitoring. The possibilityof false-positive detections can result intremendous disruption and expense for allinvolved—utilities, government officials,businesses, and individuals. This hasoccurred with continuous on-line air moni-toring at government buildings on severaloccasions, where false-positive identificationsof anthrax and nerve gas have been reported(US Government Subcommittee, 2005). Anadded problem of frequent false alarms is the“boy-who-cried-wolf syndrome,” where thepublic becomes desensitized to warnings andwill not take them seriously. Early detectionof microbiological contaminants by on-line,real-time monitoring devices is possible, andthese 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 thatdata are reliable.

Microbiology will play a much larger rolein the water utility laboratory of the future.This will be independent of regulation andwill be driven by the utility’s desire for opti-mized water quality for consumers. Currentlyit is primarily large utilities that haveadvanced microbiological capabilities (detec-tion of parasites, viruses, taste- and odor-causing bacteria and algae), but moremedium-sized utilities are recognizing thebenefits 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 understandmicrobial problems and develop the bestresponse, requires professional microbiolo-gists. Understanding the complex role thatmicrobes play in water supplies and manag-ing these supplies to consistently producehigh-quality water will continue to challengethe industry for the foreseeable future.

Paul Rochelle is a principal microbiologist inthe Water Quality Laboratory at the Metropoli-tan Water District of Southern California wherehe manages the Microbiology Development Team.He has undergraduate degrees in biology andmicrobiology from Sheffield and ManchesterPolytechnics in the United Kingdom and a micro-biology PhD from the University of Wales Insti-tute of Science and Technology. He has more than20 years of experience in the application ofmicrobiological and molecular biology techniquesto the detection and study of microorganisms inenvironmental samples and has published widelyon issues relating to microbial water quality.Jennifer Clancy is a microbiologist and presidentof Clancy Environmental Consultants Inc. in St.Albans, Vt. She has a BS degree from CornellUniversity, an MS degree from the University ofVermont, and a PhD degree from McGill Univer-sity, all in microbiology, as well as an MS degreein Environmental Law from Vermont LawSchool. She has more than 30 years experience inmedical and environmental microbiology. Shehas been involved in microbiological methoddevelopment and validation for the AmericanSociety for Testing and Materials and the USEnvironmental Protection Agency.


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tion of Viruses in Groundwater by PCR. Appl. &Envir. Microbiol., 65:444.

Abd El Galil, K.H. et al, 2004. Combined Immuno-magnetic Separation-Molecular Beacon-ReverseTranscription-PCR Assay for Detection ofHepatitis A Virus From Environmental Samples.Appl. & Envir. Microbiol., 70:4371.

Aboytes, R. et al, 2004. Detection of Infectious Cryp-tosporidium in Filtered Drinking Water. Jour.AWWA, 96:9:88.

Abrahamsen, M.S. et al, 2004. Complete GenomeSequence of the Apicomplexan, Cryptosporid-ium parvum. Science, 304:441.

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Baribeau, H. et al, 2000. Microbial Population Charac-terization of Suspended and Fixed Biomass inDrinking Water Reservoirs. Proc. 2000 AWWAWQTC. AWWA, Denver.

Bernhard, A.K. & Field, K.G., 2000. Identification ofNon-point Sources of Fecal Pollution in CoastalWaters by Using Host-specific 16S RibosomalDNA Genetic Markers From Fecal Anaerobes.Appl. & Envir. Microbiol., 66:1587.

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