Chapter 23

Indicator Microorganisms
Charles P. Gerba

23.1 The Concept of Indicator 23.3 Fecal Coliforms and 23.9 Standards and Criteria for
Organisms Escherichia coli Indicators
23.2 Total Coliforms 23.4 Fecal Streptococci 23.10 Microbial Source Tracking
23.2.1 The Most Probable 23.5 Clostridium perfringens Questions and Problems
Number (MPN) Test 23.6 Heterotrophic Plate Count References and Recommended
23.2.2 The Membrane Filter (MF) 23.7 Bacteriophage Readings
Test 23.8 Other Potential Indicator
23.2.3 The Presence–Absence Organisms
(P–A) Test

23.1 THE CONCEPT OF INDICATOR may also be present and that the water is potentially unsafe
ORGANISMS to drink.
In 1914 the U.S. Public Health Service adopted the
The routine examination of environmental samples for the coliform group as an indicator of fecal contamination of
presence of intestinal pathogens is often a tedious, difficult, drinking water. Many countries have adopted coliforms
and time-consuming task. Thus, it has been customary to and other groups of bacteria as official standards for drink-
tackle such examinations by looking first for certain indi- ing water, recreational bathing waters, wastewater dis-
cator microorganisms whose presence indicates that patho- charges, and various foods. Indicator microorganisms have
genic microorganisms may also be present. Developed at also been used to assess the efficacy of food processing
the turn of the twentieth century for assessing fecal con- and water and wastewater treatment processes. As an ideal
tamination, the indicator concept depends on the fact that assessor of fecal contamination, it has been suggested that
certain nonpathogenic bacteria occur in the feces of all they meet the criteria listed in Table 23.1. Unfortunately, no
warm-blooded animals. These bacteria can easily be iso-
lated and quantified by simple bacteriological methods.
Detection of these bacteria in water means that fecal con- TABLE 23.1 Criteria for an Ideal Indicator Organism
tamination has occurred and suggests that enteric patho- ● The organism should be useful for all types of water
gens may also be present. ● The organism should be present whenever enteric
For example, coliform bacteria, which normally occur pathogens are present
in the intestines of all warm-blooded animals, are excreted ● The organism should have a reasonably longer survival time
in great numbers in feces. In polluted water, coliform than the hardiest enteric pathogen
bacteria are found in densities roughly proportional to ● The organism should not grow in water
the degree of fecal pollution. Because coliform bacteria ● The testing method should be easy to perform
are generally hardier than disease-causing bacteria, their ● The density of the indicator organism should have some
absence from water is an indication that the water is bac- direct relationship to the degree of fecal pollution
teriologically safe for human consumption. Conversely, the ● The organism should be a member of the intestinal
microflora of warm-blooded animals
presence of the coliform group of bacteria is indicative that
other kinds of microorganisms capable of causing disease

Environmental Microbiology
Copyright © 2000, 2009 by Academic Press. Inc. All rights of reproduction in any form reserved. 485
486 PART | VI Water- and Foodborne Pathogens

TABLE 23.2 Estimated Levels of Indicator TABLE 23.4 Definitions and Examples of Indicator
Organisms in Raw Sewage Microorganisms

Group Definition and examples

Organism CFU per 100 ml
Process indicator A group of organisms that demonstrate
Coliforms 107–109
the efficacy of a process, such as total
Fecal coliforms 106–107
heterotrophic bacteria or total coliforms
Fecal streptococci 105–106
for chlorine disinfection
Enterococci 104–105
Clostridium perfringens 104 Fecal indicator A group of organisms that indicate the
Staphylococcus (coagulase positive) 103 presence of fecal contamination, such
Pseudomonas aeruginosa 105 as the fecal coliforms or Escherichia coli
Acid-fast bacteria 102
Coliphages 102–103 Index and model A group or species indicative of
Bacteroides 107–1010 organisms pathogen presence and behavior,
respectively, such as E. coli as index for
Salmonella and male-specific coliphages
as models for human enteric viruses

Modified from Ashbolt et al., 2001.

TABLE 23.3 Microbial Flora of Animal Feces

Animal Average density per gram

group
Fecal Fecal Clostridium
coliforms streptococci perfringens
Farm animals
Cow 230,000 1,300,000 200
Pig 3,300,000 84,000,000 3,980
Sheep 16,000,000 38,000,000 199,000
Horse 12,600 6,300,000 1
Duck 33,000,000 54,000,000 —
Chicken 1,300,000 3,400,000 250
Turkey 290,000 2,800,000 —
Animal pets
Cat 7,900,000 27,000,000 25,100,000
FIGURE 23.1 Relationships between indicators in three Enterobacteriaceae.
Dog 23,000,000 — —
Wild animals
Mouse 330,000 7,700,000 1
Rabbit 20 47,000 1 As such, the use of indicators is better defined by their
Chipmunk 148,000 6,000,000 —
intended purpose (Table 23.4). Thus, process indicators
are used to assess the efficacy of a treatment process (e.g.,
Human 13,000,000 3,000,000 1,580 drinking water treatment), while fecal indicators indicate
Modified from Geldreich, 1978. the presence of fecal contamination. An index (or model)
organism represents the presence and behavior of a patho-
gen in a given environment.

23.2 TOTAL COLIFORMS

one indicator meets all these criteria. Thus, various groups
of microorganisms have been suggested and used as indi- The coliform group, which includes Escherichia, Citrobacter,
cator organisms. Concentrations of indicator bacteria found Enterobacter, and Klebsiella species, is relatively easy
in wastewater and feces are shown in Tables 23.2 and 23.3. to detect (Fig. 23.1). Specifically, this group includes all
Indicators have traditionally been used to suggest the aerobic and facultatively anaerobic, gram-negative, non-
presence of enteric pathogens; however, today we recognize spore-forming, rod-shaped bacteria that produce gas upon
that there is rarely a direct correlation between bacterial lactose fermentation in prescribed culture media within
indicators and human pathogens (Ashbolt et al., 2001). 48 h at 35°C.
Chapter | 23 Indicator Microorganisms 487

23.2.1 The Most Probable Number

TABLE 23.5 Deficiencies with the Use of Coliform
Bacteria as Indicators of Water Quality
(MPN) Test
The MPN test allows detection of the presence of coli-
● Regrowth in aquatic environments
forms in a sample and estimation of their numbers (see also
● Regrowth in distribution systems
Section 10.1.3). This test consists of three steps: a presump-
● Suppression by high background bacterial growth
tive test, a confirmed test, and a completed test. In the pre-
● Not indicative of a health threat
sumptive test (Fig. 23.2A), lauryl sulfate–tryptose–lactose
● No relationship with enteric protozoan and viral
concentrations
broth is placed in a set of test tubes with different dilutions
of the water to be tested. Usually, three to five test tubes
Modified from Gleeson and Gray, 1997. are prepared per dilution. These test tubes are incubated at
35°C for 24 to 48 h, then examined for the presence of coli-
forms, which is indicated by gas and acid production. Once
the positive tubes have been identified and recorded, it is
The coliform group has been used as the standard for possible to estimate the total number of coliforms in the
assessing fecal contamination of recreational and drinking original sample by using an MPN table that gives numbers
waters since early in the twentieth century. Through expe- of coliforms per 100 ml. In the confirming test (Fig. 23.2B),
rience it has been learned that absence of this organism in the presence of coliforms is verified by inoculating selec-
100 ml of drinking water ensures the prevention of bacte- tive bacteriological agars such as Levine’s eosin–methy-
rial waterborne disease outbreaks. However, it has been lene blue (EMB) agar or Endo agar with a small amount
learned that a number of deficiencies in the use of this of culture from the positive tubes. Lactose-fermenting
indicator exist (Table 23.5). bacteria are indicated on the medium by the production of
All members of the coliform group have been observed colonies with a green sheen or colonies with a dark center.
to regrow in natural surface and drinking water distribution In some cases a completed test (not shown in Fig. 23.2) is
systems (Gleeson and Gray, 1997). The die-off rate of coli- performed in which colonies from the agar are inoculated
form bacteria depends on the amount and type of organic back into lauryl sulfate–tryptose–lactose broth to demon-
matter in the water and its temperature. If the water con- strate the production of acid and gas.
tains significant concentrations of organic matter and is
at an elevated temperature, the bacteria may increase in
numbers. This phenomenon has been observed in eutro- 23.2.2 The Membrane Filter (MF) Test
phic tropical waters, waters receiving pulp and paper mill
effluents, wastewater, aquatic sediments, and organically The MF test also allows scientists to determine the number
enriched soil (i.e., sewage sludge amended) after periods of of coliforms in a sample, but it is easier to perform than the
heavy rainfall. Of greatest concern is the growth or recov- MPN test because it requires fewer test tubes and less labor
ery of injured coliform bacteria in a distribution system (Fig. 23.3) (see also Section 10.2.1.3). In this technique,
because this may give a false indication of fecal contami- a measured amount of water (usually 100 ml for drink-
nation. Coliforms may colonize and grow in the biofilm ing water) is passed through a membrane filter (pore size
found on the distribution system pipes, even in the pres- 0.45 m) that traps bacteria on its surface. This membrane
ence of free chlorine. Escherichia coli is 2400 times more is then placed on a thin absorbent pad that has been satu-
resistant to free chlorine when attached to a surface than as rated with a specific medium designed to permit growth
free cells in water (LeChevallier et al., 1988). and differentiation of the organisms being sought. For
Because large numbers of heterotrophic bacteria in example, if total coliform organisms are sought, a modified
the water may mask the growth of coliform bacteria on Endo medium is used.
selective media used for their isolation, true numbers of For coliform bacteria, the filter is incubated at 35°C
coliforms may be underestimated. This often becomes a for 18–24 h. The success of the method depends on using
problem when aerobic heterotrophic bacterial numbers effective differential or selective media that can facilitate
exceed 500/ml. Finally, the longer survival and greater identification of the bacterial colonies growing on the
resistance to disinfectants of pathogenic enteric viruses membrane filter surface (see Fig. 23.3). To determine the
and protozoan parasites limit the use of coliform bacte- number of coliform bacteria in a water sample, the colo-
ria as an indicator for these organisms. Still, the coliform nies having a green sheen are enumerated.
group of bacteria has proved its merit in assessing the bac-
terial quality of water. Three methods are commonly used
to identify coliforms in water. These are the most probable
23.2.3 The Presence–Absence (P–A) Test
number (MPN), the membrane filter (MF), and the pres- Presence–absence tests (P–A tests) are not quantitative
ence–absence (P–A) tests. tests; instead, they answer the simple question of whether
488 PART | VI Water- and Foodborne Pathogens

(A) Presumptive test

(B) Confirmed test

FIGURE 23.2 Procedure for performing an MPN test for coliforms on water samples: (A) presumptive test and (B) confirmed test.
DSLB, double strength sulfate broth; SSLB, single strength lauryl sulfate broth.

the target organism is present in a sample or not. A single test or an MPN assay. The Colilert system (Fig. 23.4) is
tube of lauryl sulfate–tryptose–lactose broth as used in the one such assay: It is based on the fact that total coliform
MPN test, but without dilutions, would be used in a P–A bacteria produce the enzyme -galactosidase, which hydro-
test. Enzymatic assays have been developed that allow lyzes the substrate o-nitrophenyl--d-galactopyranoside
the simultaneous detection of total coliform bacteria and (ONPG) to yellow nitrophenol. E. coli can be detected at
E. coli in drinking water. The assay can be a simple P–A the same time by incorporation of a fluorogenic substrate,
Chapter | 23 Indicator Microorganisms 489

(A) (B)

(C) (D) (E)

(G)
(F)

FIGURE 23.3 Membrane filtration for determining the coliform count in a water sample using vacuum filtration.

4-methylumbelliferoyl glucuronide (MUG) (Fig. 23.5), tubes that contain powdered ingredients consisting of salts
which produces a fluorescent end product after interaction or specific enzyme substrates that serve as the only carbon
with the enzyme -glucuronidase found in E. coli but not source for the organisms (Fig. 23.4A). After 24 h of incuba-
in other coliforms. The end product is detected with a long- tion, samples positive for total coliforms turn yellow (Fig.
wave ultraviolet (UV) lamp. The Colilert test is performed 23.4B), whereas E. coli–positive samples fluoresce under
by adding the sample to a single bottle (P–A test) or MPN long-wave UV illumination in the dark (Fig. 23.4C).
490 PART | VI Water- and Foodborne Pathogens

(A) (B) (C)

FIGURE 23.4 Detection of indicator bacteria with Colilert. (A) Addition of salts and enzyme substrates to water
sample; (B) yellow color indicating the presence of coliform bacteria; (C) fluorescence under long wave ultraviolet
light indicating the presence of E. coli.

CH3 Fecal streptococci

COOH

Enterococci
Enterococcus faecalis

Group D streptococci
H O O O O
H Enterococcus faecium
OH H
HO H Group Q streptococci
H OH

FIGURE 23.5 The structure of 4-methylumbelliferyl--d-glucuronide

Viridans

Streptococci bovis
(MUG).
Streptococci equinus
Streptococci mitis
23.3 FECAL COLIFORMS AND Streptococci salivarius
ESCHERICHIA COLI
FIGURE 23.6 Definition of the terms “enterococci,” “group D strepto-
Although the total coliform group has served as the main cocci,” and “fecal streptococci” based on Streptococcus species belonging
indicator of water pollution for many years, many of the to each group.
organisms in this group are not limited to fecal sources.
Thus, methods have been developed to restrict the enumera- the same limitations in use as the coliform bacteria (i.e.,
tion to coliforms that are more clearly of fecal origin—that regrowth and less resistant to water treatment than viruses
is, the fecal coliforms (Fig. 23.1). These organisms, which and protozoa).
include the genera Escherichia and Klebsiella, are differ- Fecal coliforms may be detected by methods similar to
entiated in the laboratory by their ability to ferment lactose those used for coliform bacteria. For the MPN method EC
with the production of acid and gas at 44.5°C within 24 h. In broth is used, and for the membrane filter method m-FC
general, then, this test indicates fecal coliforms; it does not, agar is used for water analysis. A medium known as m-T7
however distinguish between human and animal contami- agar has been proposed for use in the recovery of injured
nation. The frequent occurrence of coliform and fecal coli- fecal coliforms from water (LeChevallier et al., 1983) and
form bacteria in unpolluted tropical waters, and their ability results in greater recovery from water. The Colilert test has
to survive for considerable periods of time outside the intes- the advantage of detecting coliforms and E. coli, the prin-
tine in these waters, have suggested that these organisms cipal fecal coliform, simultaneously within 24 h.
occur naturally in tropical waters (Toranzos, 1991) and that
new indicators for these waters need to be developed.
Some have suggested the use of E. coli as an indica- 23.4 FECAL STREPTOCOCCI
tor, because it can easily be distinguished from other mem-
bers of the fecal coliform group (e.g., absence of urease The fecal streptococci are a group of gram-positive Lance-
and presence of -glucuronidase) and is more likely to field group D streptococci (Fig. 23.6). The fecal strepto-
indicate fecal pollution. Fecal coliforms also have some of cocci belong to the genera Enterococcus and Streptococcus
Chapter | 23 Indicator Microorganisms 491

The enterococci have been suggested as useful indica-

TABLE 23.6 The FC/FS Ratio tors of risk of gastroenteritis for recreational bathers and
standards have been recommended (Cabelli, 1989). They
FC/FS ratio Source of pollution have been suggested as useful indicators of the presence of
4.0 Strong evidence that pollution is of human enteric viruses in the environment.
origin
2.0–4.0 Good evidence of the predominance of
human wastes in mixed pollution 23.5 CLOSTRIDIUM PERFRINGENS
0.7–2.0 Good evidence of the predominance of
domestic animal wastes in mixed pollution Clostridium perfringens is a sulfite-reducing anaerobic
spore former; it is gram positive, rod shaped, and exclu-
0.7 Strong evidence that pollution is of animal
origin sively of fecal origin. The spores are very heat resistant
(75°C for 15 minutes), persist for long periods in the envi-
ronment, and are very resistant to disinfectants. The hardy
spores of this organism limit its usefulness as an indicator.
However, it has been suggested that it could be an indica-
tor of past pollution, a tracer of less hardy indicators, and
(Gleeson and Gray, 1997). The genus Enterococcus includes an indicator of removal of protozoan parasites or viruses
all streptococci that share certain biochemical properties during drinking water and wastewater treatment (Payment
and have a wide range of tolerance of adverse growth con- and Franco, 1993).
ditions. They are differentiated from other streptococci by Other anaerobic bacteria such as Bifidobacterium
their ability to grow in 6.5% sodium chloride, pH 9.6, and and Bacteroides have been suggested as potential indica-
45°C and include Ent. avium, Ent. faecium, Ent. durans, tors. Because some Bifidobacterium are primarily associ-
Ent. faecalis, and Ent. gallinarium. In the water industry ated with humans, they could potentially help distinguish
the genus is often given as Streptococcus for this group. between human and animal contamination. However, bet-
Of the genus Streptococcus, only S. bovis and S. equinus ter and more standard methods are needed for detection of
are considered to be true fecal streptococci. These two all of the anaerobic bacteria in the environment before they
species of Streptococcus are predominately found in ani- can be adequately monitored in a routine fashion.
mals; Ent. faecalis and Ent. faecium are more specific to
the human gut. It has been suggested that a fecal coliform/
fecal streptococci (FC/FS) ratio of 4 or more indicates a 23.6 HETEROTROPHIC PLATE COUNT
contamination of human origin, whereas a ratio below 0.7
is indicative of animal pollution (Geldreich and Kenner, An assessment of the numbers of aerobic and faculta-
1969) (Table 23.6). However, the validity of the FC/FS tively anaerobic bacteria in water that derive their carbon
ratio has been questioned. Further, this ratio is valid only and energy from organic compounds is conducted via the
for recent (24 h) fecal pollution. heterotrophic plate count or HPC. This group includes
Both the membrane filtration method and MPN method gram-negative bacteria belonging to the following genera:
may also be used for the isolation of fecal streptococci. Pseudomonas, Aeromonas, Klebsiella, Flavobacterium,
The membrane filter method uses fecal Streptococcus agar Enterobacter, Citrobacter, Serratia, Acinetobacter, Proteus,
with incubation at 37°C for 24 h. All red, maroon, and pink Alcaligenes, Enterobacter, and Moraxella. The heterotro-
colonies (due to reduction of 2,4,5-triphenyltetrazolium phic plate counts of microorganisms found in untreated
chloride to formazan, a red dye) are counted as presump- drinking water and chlorinated distribution water are
tive fecal streptococci. Confirmation of fecal streptococci shown in Table 23.7 (LeChevallier et al., 1980). These
is by subculture on bile aesculin agar and incubation for bacteria are commonly isolated from surface waters and
18 h at 44°C. Fecal streptococci form discrete colonies sur- groundwater, and are widespread in soil and vegetation
rounded by a brown or black halo due to aesculin hydroly- (including many vegetables eaten raw). Some members of
sis, and Ent. faecalis are considered to be more specific to this group are opportunistic pathogens (e.g., Aeromonas,
the human gut. Fecal streptococci are considered to have Pseudomonas), but no conclusive evidence is available to
certain advantages over the coliform and fecal coliform demonstrate their transmission by drinking water. In drink-
bacteria as indicators: ing water, the number of HPC bacteria may vary from less
than 1 to more than 104 CFU/ml, and they are influenced
● They rarely multiply in water mainly by temperature, presence of residual chlorine, and
● They are more resistant to environmental stress and level of assimilable organic matter. In reality, these counts
chlorination than coliforms themselves have no or little health significance. However,
● They generally persist longer in the environment there has been concern because the HPC can grow to
(Gleeson and Gray, 1997) large numbers in bottled water and charcoal filters on
492 PART | VI Water- and Foodborne Pathogens

Although the HPC is not a direct indicator of fecal con-

TABLE 23.7 Identification of HPC Bacteria in tamination, it does indicate variation in water quality and
Untreated Drinking Water and in the Chlorinated potential for pathogen survival and regrowth. These bac-
Distribution System teria may also interfere with coliform and fecal coliform
detection when present in high numbers. It has been rec-
Organism Abundance (% of total number ommended that the HPC should not exceed 500 per ml in
of organisms identified) tap water (LeChevallier et al., 1980).
Distribution Untreated Heterotrophic plate counts are normally done by the
water drinking water spread plate method using yeast extract agar incubated at
35°C for 48 h. A low-nutrient medium, R2A (Reasoner and
Actinomyces spp. 10.7 0
Geldreich, 1985), has seen widespread use and is recom-
Arthrobacter spp. 2.3 1.3 mended for disinfectant-damaged bacteria. This medium
Bacillus spp. 4.9 0.6 is recommended for use with an incubation period of 5–7
Corynebacterium spp. 8.9 1.9
days at 28°C. HPC numbers can vary greatly depending on
the incubation temperature, growth medium, and length of
Micrococcus luteus 3.5 3.2 incubation.
Staphylococcus aureus 0.6 0
S. epidermidis 5.2 5.1
Acinetobacter spp. 5.5 10.8
23.7 BACTERIOPHAGE
Alcaligenes spp. 3.7 0.6 Because of their constant presence in sewage and polluted
Flavobacterium 2.0 0 waters, the use of bacteriophage (or bacterial viruses) as
meningosepticum appropriate indicators of fecal pollution has been proposed.
Moraxella spp. 0.3 0.6
These organisms have also been suggested as indicators of
viral pollution. This is because the structure, morphology,
Pseudomonas alcaligenes 6.9 2.5 and size as well as the behavior in the aquatic environment
P. cepacia 1.2 0 of many bacteriophage closely resemble those of enteric
P. fluorescens 0.6 0 viruses. For these reasons, they have also been used exten-
sively to evaluate virus resistance to disinfectants, to evalu-
P. mallei 1.4 0
ate virus fate during water and wastewater treatment, and
P. maltophilia 1.2 5.7 as surface and groundwater tracers.
Pseudomonas spp. 2.9 0 The use of bacteriophage as indicators of fecal pollution
is based on the assumption that their presence in water sam-
Aeromonas spp. 9.5 15.9
ples denotes the presence of bacteria capable of supporting
Citrobacter freundii 1.7 5.1 the replication of the phage. Two groups of phage in partic-
Enterobacter agglomerans 1.2 11.5 ular have been studied: the somatic coliphage, which infect
E. coli host strains through cell wall receptors, and the
Escherichia coli 0.3 0
F-specific RNA coliphage, which infect strains of E. coli and
Yersinia enterocolitica 0.9 6.4 related bacteria through the F  or sex pili. A significant
Hafnia alvei 0 5.7 advantage of using coliphage is that they can be detected
by simple and inexpensive techniques that yield results in
Enterobacter aerogenes 0 0.6
8–18 h. Both a plating method (the agar overlay method)
Enterobacter cloacae 0 0.6 and the MPN method can be used to detect coliphage (Fig.
Klebsiella pneumoniae 0 0 23.7) in volumes ranging from 1 to 100 ml. The F-specific
Serratia liquefaciens 0 0.6
coliphage (male-specific phage) have received the great-
est amount of attention because they are similar in size and
Unidentified 18.7 17.8 shape to many of the pathogenic human enteric viruses.
Modified from LeChevallier et al., 1980. Reprinted with permission from the American Coliphage f2, X174, MS2, and PRD-1 are the ones most
Society for Microbiology Journals Department. commonly used as tracers and for evaluation of disinfec-
tants. Because F-specific phage are infrequently detected
in human fecal matter and show no direct relationship to
household taps. In response to this concern, studies have the fecal pollution level, they cannot be considered indi-
been performed to evaluate the impact of HPC on illness. cators of fecal pollution (Havelaar et al., 1990). However,
These studies have not demonstrated a conclusive impact their presence in high numbers in wastewaters and their
on illness in persons who consume water with high HPC. relatively high resistance to chlorination contribute to their
Chapter | 23 Indicator Microorganisms 493

(A) Preparation of the Top Agar

(B) Plating and Detection

FIGURE 23.7 Technique for performing a bacteriophage assay.

consideration as an index of wastewater contamination and in certain applications (e.g., recreational waters). These
as potential indicators of enteric viruses. include Pseudomonas spp., yeasts, acid-fast mycobacteria
Bacteriophage of Bacteroides fragilis have also been (Mycobacterium fortuitum and M. phlei), Aeromonas, and
suggested as potential indicators of human viruses in Staphylococcus.
the environment (Tartera and Jofre, 1987). Bacteroides Within the genus Pseudomonas, the species of signifi-
spp. are strict anaerobes and are a major component of cant public health concern is P. aeruginosa, a gram-negative,
human feces, so bacteriophage active against these organ- nonsporulating, rod-shaped bacterium. The most common
isms have the potential to be suitable indicators of viral diseases associated with this organism are eye, ear, nose,
contamination. and throat infections. It is also the most common opportu-
Bacteriophage that infect B. fragilis appear to be exclu- nistic pathogen causing life-threatening infections in burn
sively human in origin (Tartera and Jofre, 1987) and appear patients and immunocompromised individuals. A char-
to be present only in environmental samples contaminated acteristic of the pseudomonad is that it can produce the
with human fecal pollution. This may help to differenti- blue-green pigment pyocyanin or the fluorescent pigment
ate human from animal contamination. They are absent fluorescein or both. Numerous cases of folliculitis, derma-
from natural habitats, which is a considerable advantage titis, and ear (swimmer’s ear) and urinary tract infections
over coliphages, which are found in habitats other than are due to P. aeruginosa associated with swimming in con-
the human gut. They are unable to multiply in the environ- taminated water or poorly maintained swimming pools and
ment (Tartera et al., 1989), and their decay rate in the envi- hot tubs. Because of this association and its consistent pres-
ronment appears similar to that of human enteric viruses. ence in high numbers in sewage, P. aeruginosa has been
However, their host is an anaerobic bacterium that involves suggested as a potential indicator for water in swimming
a complicated and tedious methodology, which limits their pools, hot tubs, and other recreational waters (Cabelli,
suitability as a routine indicator organism. 1978). However, as this organism is known to be ubiqui-
tous in nature and can multiply under natural conditions
(it can even grow in distilled water), it is believed to be of
23.8 OTHER POTENTIAL INDICATOR little value for fecal contamination studies.
ORGANISMS Coliforms have been used for many years to assess the
safety of swimming pool water, yet contamination is often
A number of other organisms have also been considered to not of fecal origin, with infections associated primarily
have potential as alternative indicator organisms or for use with the respiratory tract, skin, and eyes. For this reason
494 PART | VI Water- and Foodborne Pathogens

Staphylococcus aureus and Candida albicans, a gram-

positive bacterium and a yeast, respectively, have been TABLE 23.8 U.S. Federal and State Standards for
proposed as better indicators of this type of infection asso- Microorganisms
ciated with swimming. Recreational waters may serve as a
vehicle for skin infections caused by S. aureus, and some Authority Standards
observers have recommended that this organism be used as U.S. EPA
an additional indicator of the sanitary quality of recreational Safe Drinking Water Act 0 coliforms/100 ml
waters, because its presence is associated with human activ-
Clean Water Act
ity in recreational waters (Charoenca and Fujioka, 1993).
The genus Aeromonas includes straight gram-negative Wastewater discharges 200 fecal coliforms/100 ml
rods, facultatively anaerobic, that are included in the fam- Sewage sludge 1000 fecal coliforms/4 g
ily Vibrionaceae. Only Aeromonas hydrophila has received 3 Salmonella/4 g
attention as an organism of potential sanitary significance. 1 enteric virus/4 g
Aeromonas occur in uncontaminated waters as well as in 1 helminth oval/4 g
sewage and sewage-contaminated waters. The organism California
can be pathogenic for humans, other warm-blooded ani- Wastewater reclamation for 2.2 MPN/100 ml coliforms
mals, and cold-blooded animals including fish. Foodborne irrigation
outbreaks associated with A. hydrophila have been docu-
Food and Drug Administration
mented and it is considered an opportunistic pathogen in
humans. Because of its association with nutrient-rich con- Shellfish growing areasa 14 MPN/100 ml fecal coliforms
ditions, it has been suggested as an indicator of the nutrient a
FDA, 2005.
status of natural waters.

23.9 STANDARDS AND CRITERIA FOR

INDICATORS TABLE 23.9 Drinking Water Criteria of the European
Union
Bacterial indicators such as coliforms have been used for Tap water
the development of water quality standards. For example, Escherichia coli 0/100 ml
the U.S. Environmental Protection Agency (EPA) has set a
Fecal streptococci 0/100 ml
standard of no detectable coliforms per 100 ml of drinking
Sulfite-reducing clostridia 0/20 ml
water. A drinking water standard is legally enforceable in
the United States. If these standards are violated by water Bottled water
suppliers, they are required to take corrective action or they Escherichia coli 0/250 ml
may be fined by the state or federal government. Authority Fecal streptococci 0/250 ml
for setting drinking water standards was given to the EPA in Sulfite-reducing clostridia 0/50 ml
1974 when Congress passed the Safe Drinking Water Act. Pseudomonas aeruginosa 0/250 ml
Similarly, authority for setting standards for domestic waste-
water discharges is given under the Clean Water Act (see From European Union, 1995.

Table 20.1). In contrast, standards for recreational waters

and wastewater reuse are determined by the individual
states. Microbial standards set by various government bod-
ies in the United States are shown in Table 23.8. Standards has been used to develop criteria is that of recreational
used by the European Union are given in Table 23.9. swimming. Epidemiological studies in the United States
Criteria and guidelines are terms used to describe rec- have demonstrated a relationship between swimming-asso-
ommendations for acceptable levels of indicator micro- ciated gastroenteritis and the densities of enterococci (Fig.
organisms. They are not legally enforceable but serve as 23.8) and fecal coliforms. No relationship was found for
guidance indicating that a potential water quality problem coliform bacteria (Cabelli, 1989). It was suggested that a
exists. Ideally, all standards would indicate that an unac- standard geometric average of 35 enterococci per 100 ml be
ceptable public health threat exists or that some relation- used for marine bathing waters. This would mean accept-
ship exists between the amount of illness and the level of ing a risk of 1.9% of the bathers developing gastroenteritis
indicator organisms. Such information is difficult to acquire (Kay and Wyer, 1992). Numerous other epidemiological
because of the involvement of costly epidemiological stud- studies of bathing-acquired illness have been conducted.
ies that are often difficult to interpret because of confound- These studies have shown slightly different relationships
ing factors (see Chapter 29). An area where epidemiology to illness and suggested that other bacterial indicators were
Chapter | 23 Indicator Microorganisms 495

70
TABLE 23.10 Guidelines for Recreational Water
Total GI Quality Standards
60 y  24.2x 5.1
r  0.82, p  0.001 Country or Regime Criteria or standarda
gastrointestinal illness (GI) (%)
Swimming associated rate for

50 agency (samples/time)
U.S. EPA 5/30 days 200 fecal coliforms/100 ml
40 10% to exceed 400/ml
Freshwaterb
33 enterococci/100 ml
30 126 E. coli/100 ml
Marine watersb
35 enterococci/100 ml
20
European 2/30 daysc 500 coliforms/100 ml
Highly credible GI Economic 100 fecal coliforms/100 ml
10 Community 100 fecal streptococci/100 ml
y  12.2x  0.2
r  0.75, p  0.001 0 Salmonella/liter
0 enteroviruses/10 liters
0
1 10 100 1000 Ontario, 10/30 days 1000 coliforms/100 ml
Mean enterococcus density 100 ml 1 Canada 100 fecal coliforms/100 ml

FIGURE 23.8 Dose–response relationships produced by the work of From Saliba, 1993; U.S. EPA, 1986.
a
Cabelli et al. (1982). All bacterial numbers in geometric means.
b
Proposed, 1986.
c
Coliforms and fecal coliforms only.

more predictive of illness rates (Kay and Wyer, 1992).

These differences probably arise because of the different
sources of contamination (raw versus disinfected waste-
water), types of recreational water (marine versus fresh),
types of illness (gastroenteritis, eye infections, skin com- TABLE 23.11 A Comparison of Arithmetric and
plaints), immune status of the population, length of obser- Geometric Averages of Bacterial Numbers in Water
vation, etc. Various guidelines for acceptable numbers of
indicator organisms have been in use (Table 23.10), but MPNa Log
there is no general agreement on standards.
2 0.30
The use of microbial standards also requires the develop-
ment of standard methods and quality assurance or quality 110 2.04
control plans for the laboratories that will do the monitoring. 4 0.60
Knowledge of how to sample and how often to sample is 150 2.18
also important. All of this information is usually defined
1100 3.04
in the regulations when a standard is set. For example, fre-
quency of sampling may be determined by the size (number 10 1.00
of customers) of the utility providing the water. Sampling 12 1.08
must proceed in some random fashion so that the entire 198  arithmetic average 1.46  log x antilog x  29
system is characterized. For drinking water, no detectable
29  geometric average
coliforms are allowed in the United States (Table 23.8).
However, in other countries some level of coliform bacteria a
MPN, most probable number.
is allowed. Because of the wide variability in numbers of
indicators in water, some positive samples may be allowed
or tolerance levels or averages may be allowed. Usually,
geometric averages are used in standard setting because
x  anti log(log x ) (Eq. 23.2)
of the often skewed distribution of bacterial numbers. This
prevents one or two high values from giving overestimates
of high levels of contamination, which would appear to be where N is the number of samples, x is the geometric aver-
the case with arithmetic averages (see Table 23.11). age, and x is the number of organisms per sample volume.
Geometric averages are determined as follows. As can be seen, standard setting and the development
of criteria is a difficult process and there is no ideal stan-
log x 
∑ (log x) (Eq. 23.1) dard. A great deal of judgment by scientists, public health
N officials, and the regulating agency is required.
496 PART | VI Water- and Foodborne Pathogens

23.10 MICROBIAL SOURCE TRACKING by matching the utilization pattern of a bacterium, such as
E. coli or enterococci, on a suite of antibiotics or carbon
One area of current focus in environmental microbiology is sources with those of isolates from a source culture library.
source tracking. Source tracking is a procedure that is devel- In general, library-dependent methods have the advantage
oped to allow identification of the source of microbial fecal of being quantitative, highly sensitive, and reproducible
contamination. Once the source is identified, control mea- and can be used to classify isolates from multiple sources
sures can be developed and applied to eliminate the con- (Fong and Lipp, 2005). Their major drawbacks are that they
tamination. A variety of microbial source tracking methods are time-consuming, they require a large isolate database,
have been developed to try to determine the role of human which may be geographically specific, and they have higher
versus animal sources, and specific discharges into surface false-positive rates than library-independent methods.
and groundwaters (see Information Box 23.1). These meth- Library-independent methods do not require the sample
ods can be divided into two basic groups, genotypic or phe- to be compared to a database. For example, a library-inde-
notypic, and take advantage of molecular techniques (see pendent genotypic analysis might use bacterial host-specific
Chapter 13) either independently or in conjunction with markers of bacteria. The use of bacterial host-specific
culture-based analysis. Genotypic methods differentiate markers does not require a reference library or a cultiva-
sources through genetic patterns of bacteria in the source tion step and testing can be done by PCR (Fong and Lipp,
sample whereas phenotypic methods differentiate sources 2004). For example, source identification using host-
through antibiotic resistance or carbon utilization patterns. specific molecular markers using terminal-restriction frag-
These two methods can be further divided into those that ment length polymorphism (T-RFLP) has been reported
require a library of bacterial isolates of known origin and using the enteric anaerobic bacteria genera Bacteriodes–
those that do not (Table 23.12). For example, a library- Prevotella and Bifidobacterium (Bernhard and Field, 2000;
dependent phenotypic method might differentiate sources Bernhard et al., 2003). Enterotoxin biomarkers using

Information Box 23.1 Comparison of Methods for Microbial Source Tracking in Aquatic Environments

Method Advantages Disadvantages

Genotypic, library based
Ribotyping Quantitative; highly sensitive and Large isolate database required, geographically specific;
reproducible; classifies isolates labor-intensive and time-consuming; high percentage of
from multiple sources inconclusive results
Pulsed field gel electrophoresis Sensitive, discriminative, and Labor-intensive and time-consuming; may be too sensitive
reproducible; quantitative for discriminating multiple sources
Phenotypic, library based
Antibiotic resistance analysis Rapid; classifies isolates from Large isolate database required, geographically specific;
multiple animal sources isolates have to show antibiotic resistance to be typed;
antibiotic resistance traits are not stable; no consensus
on contamination and dose of antibiotics used
Library and culture independent
(bacterial host-specific markers)
Terminal restriction fragment Rapid and easy to perform; no Survival and distribution of molecular markers in aquatic
length polymorphism database or cultivation required; environments are not well studied; expensive equipment;
high accuracy in differentiating currently applicable to only a limited number of host
human and nonhuman sources groups
Direct measurement of
host-specific viruses
PCR for viral pathogens Library independent and directly Nonquantitative in conventional PCR; requires more
relates to health risk; rapid and sensitive detection methods; limited knowledge
straightforward; detects conserved of prevalence of animal-specific viruses in aquatic
regions of a viral genome; may not environments; serotyping is expensive and time-
have geographical limits consuming
PCR and phage typing (e.g., F Subgroups are well correlated to Serotyping is expensive and time-consuming; low survival
RNA coliphage) sources; straightforward in marine and tropical waters; may proliferate in sewage;
exceptions in association of coliphage subgroup and host
group have been noted
From Fong and Lipp, 2005. © American Society for Microbiology. Used with permission.
Chapter | 23 Indicator Microorganisms 497

species-specific PCR primers to amplify toxin genes in

TABLE 23.12 Classification of Genotypic and Pheno- E. coli have been found capable of differentiating vari-
typic Methods ous domestic animal sources (Khatib et al., 2002). A simi-
lar approach using enteroccoci surface protein (ESP) in
Method type Library Library independent Enterococcus faecium has also been reported useful for differ-
dependent entiating human and animal sources of fecal pollution (Scott
Genotypic Ribotyping Host-specific molecular et al., 2005). Male-specific coliphages can also be used in
markers (PCR) this way because they belong to four groups or serotypes
Repetitive Enterotoxin biomarkers
that can differ in their occurrence in humans and animals
intergenic DNA (PCR)
sequences Terminal restriction (Stewart-Pullaro et al., 2006). Another approach is the
(rep-PCR) fragment length direct measurement of viral pathogens and bacteriophages
Pulsed field gel polymorphism (T-RFLP) with different host groups, for example, hepatitis A virus
electrophoresis analysis of total or F RNA coliphage (Fong and Lipp, 2005). Library-
(PFGE) bacterial community
independent methods have the advantage that they are rapid
Phenotypic Multiple antibiotic F RNA coliphage and have high accuracy in differentiating human and non-
resistance serotyping human sources. Disadvantages include the requirement for
Carbon source
expensive equipment for the analyses and the fact that we
profiling
do not have a good understanding of how well each host-
From NRC, 2004. © National Research Council. Used with permission. specific marker actually survives in the environment.
Figure 23.9 shows a flow chart illustrating the different
approaches that can be taken in microbial source tracking.

Cultivation-Independent
Sample Cultivation-Dependent
Library-Independent

Concentrate for processing

(can be stored at 20°C) Library-Independent Library-Dependent
Direct
sample Extract Target Verification
analysis nucleic acids Isolate or Enrich Target Organism(s)
(possible
but not
common) Extract Phenotypic Genotypic
Phage
PCR nucleic Analyses Analyses

Confirmation
Viruses using host Toxin Gene Antibiotic Carbon
Microbial Specific Bacteria infection PCR Resistance Utilization
(e.g., Entero-,
Community (e.g., Bacteriodes,
Adeno-,
(DGGE, T-RFLP) Biflodobacterium,
viruses, or Direct cell PCR
Streptococcus, Extract nucleic
coliphages) Blot and
Rhodoccus) acids
hybridize with
Pick plaques do gene specific Rep-PCR RADP
RNAse test probe
Restriction
AFLP enzyme digestion
Serotype Genotype Analysis

PFGE Ribotyping

Blot and hybridize with rRNA

gene probe
FIGUGE 23.9 Schematic representation of microbial source tracking methods. See Chapter 13 for an explanation of the techniques shown.
PCR, polymerase chain reaction; Rep PCR, repetitive-intergenic DNA sequence PCR; DGGE, denaturing gradient gel electrophoresis; T-RFLP,
terminal-restriction fragment length polymorphism; PFGE, pulsed field gel electrophoresis; RAPD, randomly amplified polymorphic DNA;
AFLP, amplified fragment length polymorphism. Adapted from Microbial Source Tracking Guidance Document, U.S. Environmental
Protection Agency.
498 PART | VI Water- and Foodborne Pathogens

It should be noted that these are relatively new approaches European Union (EU). (1995) Proposed for a Council Directive concern-
and although many potential source tracking methods have ing the quality of water intended for human consumption. Com (94)
been tested, none have been accepted by the regulatory 612 Final. Offic. J. Eur. Union 131, 5–24.
Fong, T. T., and Lipp, E. K. (2005) Enteric viruses of humans and animals
community and questions still remain about the temporal
in aquatic environments: health risks, detection, and potential water
and geographic stability of traits and genetic sequences.
quality assessment tools. Microbiol. Mol. Biol. Rev. 69, 357–371.
Food and Drug Administration (2005). National Shellfish Sanitation
Program. “Guide for the Control of Molluscan Shellfish. 2005.”
QUESTIONS AND PROBLEMS Washington, DC.
Geldreich, E. E. (1978) Bacterial populations and indicator concepts in
1. What are some of the criteria for indicator bacteria? feces, sewage, storm water and solid wastes. In “Indicators of Viruses
2. What is the difference between standards and criteria? in Water and Food” (G. Berg, ed.), Ann Arbor Science, Ann Arbor,
3. Why are geometric means used to report average MI, pp. 51–97.
concentrations of indicator organisms? Geldreich, E. E., and Kenner, B. A. (1969) Comments on fecal strep-
4. Calculate the arithmetic and geometric averages for tococci in stream pollution. J. Water Pollut. Control Fed. 41,
the following data set: fecal coliforms/100 ml on R336–R341.
different days on a bathing beach were reported as 2, Gleeson, C., and Gray, N. (1997) “The Coliform Index and Waterborne
3, 1000, 15, 150, and 4000. Disease,” E and FN Spon, London, UK.
5. Define coliform and fecal coliform bacteria. Why are Havelaar, A. H., Hogeboon, W. M., Furuse, K., Pot, R., and Horman, M. P.
(1990) F-specific RNA bacteriophages and sensitive host strains in
they not ideal indicators?
faeces and wastewater of human and animal origin. J. Appl. Bacteriol.
6. Why have coliphage been suggested as indicator
69, 30–37.
organisms? Kay, D., and Wyer, M. (1992) Recent epidemiological research leading
7. What are two methods that can be used to detect to standards. In “Recreational Water Quality Management, Volume
indicator bacteria in water? 1. Coastal Waters” (D. Kay, ed.), Ellis Harwood, Chichester, UK,
8. What is the difference between library-independent pp. 129–156.
and -dependent source tracking methods? What are Khatib, L. A., Tsai, Y. L., and Olson, B. H. (2002) A biomarker for the
the major disadvantages of both methods? identification of cattle fecal pollution in water using the LYIIa toxin
9. What is the difference between a fecal indicator gene from the enterotoxigenic Escherichia coli. Appl. Environ.
organism and process indicator? Give an example Microbiol. 59, 97–104.
of each. LeChevallier, M. W., Seidler, R. J., and Evans, T. M. (1980) Enumeration
and characterization of standard plate count bacteria in chlorinated
and raw water supplies. Appl. Environ. Microbiol. 40, 922–930.
LeChevallier, M. W., Cameron, S. C., and McFeters, G. A. (1983) New
REFERENCES AND RECOMMENDED medium for improved recovery of coliform bacteria from drinking
READINGS water. Appl. Environ. Microbiol. 45, 484–492.
LeChevallier, M. W., Cawthen, C. P., and Lee, R. G. (1988) Factors
Ashbolt, N. J., Grabow, W. O. K., and Snozzi, M. (2001) Indicators of promoting survival of bacteria in chlorinated water supplies. Appl.
microbial water quality. In “Water Quality: Guidelines, Standards and Environ. Microbiol. 54, 649–654.
Health” (L. Fewtrell, and J. Bartram, eds.), IWA Publishing, London, National Research Council (NRC) (2004) “Indicators of Waterborne
pp. 289–315. Pathogens.” Washington, DC.
Bernhard, A. E., and Field, K. G. (2000) Identification of nonpoint Payment, P., and Franco, E. (1993) Clostridium perfringens and somatic
sources of fecal pollution in coastal waters by using host-specific coliphages as indicators of the efficiency of drinking water treat-
16S ribosomal DNA markers from fecal anaerobes. Appl. Environ. ment for viruses and protozoan cysts. Appl. Environ. Microbiol. 59,
Microbiol. 66, 1587–1594. 2418–2424.
Bernhard, A. E., Goyard, T., Simonich, M., and Field, K. G. (2003) A Pepper, I. L., and Gerba, C. P. (2004) “Environmental Microbiology:
rapid method for identifying fecal pollution sources in coastal waters. A Laboratory Manual,” Second Ed. Academic Press, San Diego.
Water Res. 37, 909–913. Pepper, I. L., Gerba, C. P., and Brusseau, M. L. (1996) “Pollution
Cabelli, V. (1978) New standards for enteric bacteria. In “Water Pollution Science,” Academic Press, San Diego.
Microbiology” (R. Mitchell, ed.), Vol. 2, Wiley-Interscience, New Reasoner, D. J., and Geldreich, E. E. (1985) A new medium for enumera-
York, pp. 233–273. tion and subculture of bacteria from potable water. Appl. Environ.
Cabelli, V. J. (1989) Swimming-associated illness and recreational water Microbiol. 49, 1–7.
quality criteria. Water Sci. Technol. 21, 13–21. Saliba, L. (1993) Legal and economic implication in developing criteria
Cabelli, V. J., Dufour, A. P., McCabe, L. J. and Levin, M. A. (1982) and standards. In “Recreational Water Quality Management” (D. Kay,
Swimming associated gastroenteritic and water quality. Am. J. and R. Hanbury, eds.), Ellis Harwood, Chichester, UK, pp. 57–73.
Epidemiol. 115, 606–616. Scott, T. M., Jenkins, T. M., Lukasik, J., and Rose, J. B. (2005) Potential
Charoenca, N., and Fujioka, R. S. (1993) Assessment of Staphylococcus use of a host associated molecular maker in Enterococcus faecium
bacteria in Hawaii recreational waters. Water Sci. Technol. 27, as an index of human fecal pollution. Environ. Sci. Technol. 39,
283–289. 283–287.
Chapter | 23 Indicator Microorganisms 499

Stewart-Pularo, J., Daugomah, J. W., Chestnut, D. E., Graves, D. A., Toranzos, G. A. (1991) Current and possible alternative indicators of fecal
Sobsey, M. D., Scott, G. I. (2006) F RNA coliphage typing for contamination in tropical waters: A short review. Environ. Toxicol.
microbial source tracking in surface waters. J. Appl. Microbiol. 101, Water Qual. 6, 121–130.
1015–1026. USEPA. (1986) United States Environmental Protection Agency. Ambient
Tartera, C., and Jofre, J. (1987) Bacteriophage active against Bacteroides water quality. Criteria—1986. EPA440/5–84–002. Washington, DC.
fragilis bacteriophage as indicators of the virological quality of water.
Water Sci. Technol. 18, 1623–1637.
Tartera, C., Lucena, F., and Jofre, J. (1989) Human origin of Bacteroides
fragilis bacteriophage present in the environment. Appl. Environ.
Microbiol. 55, 2696–2701.