American Water Works Association
Parti
Sunday Seminars Through
Poster Presentations
in
Presented at the AWWA
Water Quality Technology Conference .
November 10-14,1991 .« Orlando, Florida'
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PATHOGENS IN DRINKIBS MATER - ARE THERE ANY NEW ONES?
Donald J. Reastmer, Chief
Microbiological Treatment Branch, DWRD, RREL •
U.S. Environmental Protection Agency
ABSTRACT
Since 1976 three newly recognized human pathogens have become
familiar to the drinking water industry as waterborne disease agents.
These are: the legionnaires disease agent, Leoionella pnemnophila
and related species; and two protozoan pathogens, Giardia 1amblia
and CrvptosDO.rJLdj.un! parvuiB. both of which form highly disinfectant
resistant cysts that are shed in the feces of infected individuals.
The question frequently arises - are there other emerging waterborne
pathogens that may pose a human health problem that the drinking water
industry will have to deal with? This paper will review the current
state of knowledge of the occurrence and incidence of pathogens
and opportunistic pathogens other than Leglonella, Giardia and
Cryptosooridium in treated and untreated drinking water. Bacterial
agents that will be reviewed include Aergagnoas. Pseudomonas.
Campylobacter. Hvcobacterium. Yersjnja and Plesjomonas. Aspects of
detection of these agents including detection methods and feasibility
of monitoring will be addressed.
INTRODUCTION
During the past 15 years three specific human pathogenic micro-
organisms, Leqionena. Giardia and Cryptosporidiunu have been shown
to be waterborne and have caused waterborne disease outbreaks.
Only Leqionella and Giardia are currently regulated under the SDWA
Amendments of 1986. These organisms pose: 1) a potential health
threat to the general population and; Z) along with several
opportunistic pathogens, may pose a significant waterborne health
threat to an estimated 30 million Americans who have reduced or
impaired immune function (1)- 1° addition to these microorganisms, a.
variety of enteric viruses are known to be a major cause of human
waterborne disease outbreaks. These include Poliovirus, Coxsackievirus
A and B, Echovirus, Rotavirus, Adenovirus, and Hepatitis A virus. In
most cases of waterborne viral disease, drinking water that received
complete treatment was implicated (53% of reported isolations), while
26% was associated with water that received only disinfection and 15%
was associated^with untreated water (2). The identification of viral
agents that cause waterborne outbreaks is problematic due to the fact
that some of these agents can't be propagated in the laboratory. For
those that can be propagated, large sample volumes must be examined
to detect the organism. Propagation and identification of the organism
takes several days. In addition, by the time the outbreak has been
recognized, it is usually long after the contamination event that
caused the outbreak and water containing the agent is no longer
available. This problem is common to all waterborne microbiological
agents.
Legionella has already been mentioned, but what about other
bacteria? What other bacterial agents can cause human health problems
through exposure to water? tfe are familiar with traditional waterborne
bacterial pathogens such as Vibrio cholera (cholera agent), and
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Salmonella tvohi (typhoid fever agent), but these organisms are easily
Inactivated by currently used treatment and disinfection practices.
Are there other waterborne bacterial disease agents that we should
be concerned with? What about other viruses, protozoans, fungi and
algae? The following is a quick survey of non-traditional nricrobio-
logical agents that have, or may have, an adverse impact on the water
consumer's health.
Bacteri al Haterborne Agents
There are a variety of waterborne bacteria that are of concern
from a human health perspective. These are the opportunistic pathogens
that can be found as part of the heterotrophic bacteria flora of
aquatic systems. The problens with most of these bacteria is that they
are not obviously pathogenic to "healthy" humans and there is a
tendency to discount their importance as agents of human disease. In
general, they cause apparent disease only in susceptible individuals
who are immunologically compromised. Also, as sanitary nicrobiologists
we tend to be preoccupied with those disease agents that involve
gastrointestinal tract upsets, rather than waterborne disease agents
that are involved in wound infections; eye, ear, nose and throat
infections; or generalized disseminated infections in the body.
Finally, for many of the opportunistic pathogens, specific selective
media for enumeration and isolation do not exist, making it difficult
to directly and easily monitor for their presence and density in water.
The bacteria generally considered opportunistic pathogens that
are frequently found in drinking water are shown in Table 1. Some
organisms in this list may be considered frank or primary pathogens,
meaning that they are capable of being the primary disease causing
agent rather than & secondary invader. Table 2 is a list of bacteria
that includes some previously recognized, but still emerging
opportunistic organisms and some new candidates. Table 3 shows new
or emerging pathogens in the viral, protozoa and blue-gresn algal
groups.
Of the older/emerging opportunistic bacteria, the organisms of
most concern appear to be Aeromonas spp., Camoylobacter. 4-fnethylura-
belliferyl-B-D-glucuronide (HUG) - negative Escherichia coll such as
serotype 01S7:H7, and Hvcobacterimn spp. However, it is difficult
to assess the real human health risk of exposure, to these and other
opportunistic organisms. The following factors make it difficult to
assess the health implications of these organisms, }) The lack of data
on the occurrence and densities of these organisms in U.S. water
supplies. 2} The lack of data on infective dose required to establish
infection. 3) The lack of data on the incidence of human disease
caused by waterborne exposure to these organisms. 4) The interactive
effects of exposure to mixed types and densities of these organisms.
5) The range of susceptible individuals in the exposed population.
6) The effectiveness of treatment processes and post-disinfection for
control of these agents, individually and collectively. 7} The need
for good detection methodologies that would permit adequate
surveillance and monitoring for these organisms,
Detection and enumeration methods for any of the waterborne
pathogens and opportunistic pathogens remain a significant impediment
to our ability to accurately assess the occurrence and-potential health
impact of these organisms.
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Table 4 provides density estimates obtained from the literature
for selected opportunistic pathogens in drinking water.
Table 5 summarizes some of the available information on the
sensitivity of eaerging and raw candiate opportunistic pathogens to
disinfection by chlorine. Essentially no information is available on
the sensitivity of these organisms to alternative disinfectants such as
chlorine dioxide, and ozone. Presumably most of the organisms in Table
5 would be more sensitive to ozone than they are to chlorine, chlorine
dioxide or chloramines. However, such work remains to be done.
Aer omona.s_spp.
For lactose fermenting Aeromonas spp., detection methods are
similar to those for coliforas. A few additional biochemical tests
are required to differentiate'and identify these organisms. As a
general comment, it appears that Aeromonas spp. are not found in
significant densities in treated and disinfected drinking water,
although they have been found in higher numbers in water that was
disinfected only. They may gain entry, however, through post-treatment
contamination events, or some may survive treatment and disinfection
and regrow under suitable conditions in some areas of the distribution
system. Aeromonads are adversely affected by copper concentrations
above 10 jsg/L (23). In the Netherlands, limits on Aeromonas densities
in drinking water have been set at maxi'mura values of 20 CFU/1QO ml in
water leaving the treatment plant, and 100 CFU/100 ml in distribution
waters.
Non-lactose fermenting aeromonads can be isolated by the use of
selective media containing starch related compounds and inhibiting
agents such as ampicillin, sodium lauryl sulfate, sodium desoxycholate,
and others (5). .
For surface water .supplies, water temperature is an important
factor in the occurrence of Aeromonas in drinking water since the
highest numbers reported occurred during the summer. Aeroraonad
concentrations may increase in the distribution water, responding to
loss of disinfectant residual and to assimilable organic carbon in the
water. Aeromonads can also utilize a wide range of -biopolymers that
may be available in distribution biofilm so control of biofilm in the
distribution systen may be important for control of aeromonads.
Hascher, et aj.. (32) did not find seasonal variations in
Aeroaonss spp, in well water and in the distribution systems. They
concluded that nearly 50% of the Aeromonas spp. they tested could
be presumed to be enterotoxin - producers.
Campy!obacter
Campylobacter jejuni causes human diarrheal disease throughout
the world and there is a large reservoir consisting of most of man's
domestic animals. Many outbreaks are food and nilk related.
Isolations of Campvlobacter from treated and disinfected,
unrecontaminated drinking water have not been reported. Interest in
the occurrence of these organisms has been related to their occurrence
in surface waters and, therefore, the potential for their occurrence in
untreated or inadequately treated drinking water. .Municipal water
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supplies contaminated with C. .ie.iuni caused major outbreaks each
involving more than 2000 people (33,34) and an outbreak in Illinois
affected 78 persons (62%) from 34 of 201 households (24). Another
outbreak that involved 94 people was due to Camovlobacter je.luni
contamination of a hospital water supply (35).
Escherichia coli 0157:H7
This organism is an atypical fecal coliform in that it does not
hydrolyze the fluorogenic substrate HUG used in recently developed
specific substrate tests for total coliforms and E. coli. £. coli
0157:H7 is better known as a foodborne (meat and milk) pathogen, but it
has been implicated in at least two waterborne disease outbreaks
(8,36,37). It causes hemorrhagic colitis, hemolytic uremic syndrome,
and is a leading cause of kidney disease in children (38). In all
waterborne outbreak cases due to E. coli 0157:H7, the water supply was
undisinfected ground or surface water that became fecally contaminated.
Detection of E_. coli 0157:H7 cannot be readily done using the
standard membrane filter procedure with m-FC medium and incubation
at 44.5°C for 24 h. because the organism doesn't grow well at this
temperature. Since E. col i 0157:H7 does not ferment sorbitol, total
coliform isolates that give a sorbitol negative reaction on MacConkey-
sorbitol medium are presumptive E. coli 0157:H7. Final identification
is done by use of the API 20 E system and confirmed by serotyping
for 0:H antigens.
Plesiomonas shiqelloides
This organism is closely related to Aeromonas hvdroohila: both
are member of the family Vibrionaceae. P. shiqelloides has been
found in fresh water environments (lakes and rivers), fish and seafood
and animals (39). It has been implicated in cases of traveller's
diarrhea and consumption of raw oysters (40,41). There appears to
be no consensus concerning whether or not Plesiomonas shioelloides
is a gastrointestinal pathogen for the normal host, but it is an
opportunistic pathogen for the immunocompromised host. The organism
has also been associated with extra-intestinal illnesses such as
bacteremia, meningitis, cholecystitis, osteomyelitis, and pseudo-
appendicitis. In spite of this, the organism apparently has a low
pathogenic potential (42). It's potential as a waterborne disease
agent has not been assessed and occurrence data in treated drinking
water are lacking.
Detection of £. shiqelloides involves the same basic methodology
as for Aeromonas hvdroohila; £. shioelloides is also oxidase negative.
Isolation can be accomplished using antibiotic supplemented agar media
such as Salmonella-Shioella agar and KacConkey's agar. Identification
of lactose non-fermenting, oxidase-positive, potential Plesiomonas
isolates can be accomplished by use of a biochemical identification
system such as the API-20 E. A medium (KS5) for selective isolation
of P. shiqelloides was reported by Sakata and Todaka (43). To date,
however, the use of this medium for direct recovery of P. shiqelloides
from water, including drinking water, has not been evaluated.
Hvcobacterium SDD.
Hycobacteria have probably been 'emerging' as opportunistic
pathogens in potable water longer than any other genus or group of
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bacteria. Their emergence has been accelerated in recent years by
their occurrence as disease agents in AIDS patients and the Isolation
of Hycobacterium spp. from chlorinated water sources in patient care
areas in hospitals. (11,26). Diseases caused by these organisms range
from skin granulontas, to .chronic pulmonary mycobacteriosis, to
disseminated disease in AIDS patients or in patients with underlying
hematologic disorders. In the latter patients, a case fatality rate up
to 73% nay occur (11].
The occurrence and incidence of mycobacteria in water, including
potable water, is well documented and was reviewed by Collins, el al..
(29). In the time period since that review additional work has shown
that potable hot water systems in hospitals contain higher concentra-
tions of H. avium than cold water systems (26). Sixty-nine percent of
hot water {T = 52°C to S7°C) sites sampled contained H. avium at
densities that ranged from 1.0-500 CFU/100 ml (average 141 CFU/100 ml).
By comparison only 17% of samples from cold water sites contained M.
aviuB at densities 1.0-2.0 CFU/100 ml (average 1.5 CFU/100 ml). In
; addition to the recovery of H, avium serotype 4 from distribution
water, this serotype has been recovered from a majority of clinical
specimens of patients with AIDS in Massachusetts, indicating the
likelihood of waterborne transmission. Aerosolized water (showerheads,
etc.) containing mycobacteria provides opportunity for inhalation
exposure, in addition to ingestion exposure.
The use of low level chloranination (1 ng/L chloramine residual)
before the first customer may select for mycobacteria because they
have been found to be resistant to chlorine at this concentration (12).
The mycobacteria are, in general, more resistant to disinfection
than other types of bacteria. Havelaar, et al. (25) reported that a
free available chlorine concentration (hypochlorous acid, HOCL) of 1
mg/L was required to assure low mycobacteria levels in bathing water.
Susceptibility of mycobacteria to disinfectants has not been fully
evaluated. Pelletier, et aj,. (12) found that free chlorine at 1.0
rag/L gave 5 logs of inactivation of a variety of clinical and
environmental strains of mycobacteria in an 8 hour period, but free
chlorine at 0.15 mg/L had essentially no bactericidal effect on the
same strains.
Detection and enumeration of mycobacteria in water requires the
use of membrane filtration and selective and inhibitory media. After
sample filtration, the membrane filter say or may not be treated to
reduce the background of other organisms. Such treatment usually
involves the use of sodium hydroxide, hypochlorite or oxalic acid
followed by neutralization before culturing the treated membranes.
Alternatively, the water sample may be treated using a selective agent
such as'cetylpyridinium chloride {0.04% final concentration) for 24
hour before membrane filtration of the water sample (44).
The major problem in examination of water for mycobacteria
is that an extended time period may be needed to culture the organisms
(depending on the species); up to 30 days at 37°C on a medium such as
Middlebrook-Cohn 7H10 agar. The plates nust be sealed in a container
to prevent dehydration of the medium during incubation. Detailed
methodology is available elsewhere (44,45,46).
New Candidate Organisms - Bacteria
Anaerobiosoirillum succinciproducens is an anaerobic or micro-
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aerophillc, motile, spiral shaped rod that has been Isolated froa
diarrhea! feces, the genus flnaerobiosoirillua was first described
in 1976 by Davis, et at.(47) and there have been few reports of these
organisms producing disease in humans (3). This organism has been
associated with human diarrhea and bactereraia. Malnick, et al_. (3)
reported the developaent of a median for the isolation of this organism
from diarrhea! feces and blood of humans. On this medium, the organism
produces uniform, dark-blue, low convex shaped colonies, 1-2 mm
diameter, in 48 h. incubation. The medium was used to survey human,
canine and feline feces for the presence of anaerobiospirillumlike
organisms, Because these organisms are anaerobes, isolation requires
anaerobic incubation at 37°C for 48 h. Previously, the organisms
were isolated from Skirrow campylobacter medium. Halnick, et al.
found no AnaerobiosoiriTlum spp. in 527 normal human fetal samples
and obtained only one isolate from one of 100 selected diarrhea!
samples. Anaeroblosplrillum spp. were isolated from rectal swabs
of 7 of 10 cats and 3 of 10 dogs. These results clearly show that
Anaerobic- spirillum spp. are not part of the normal fecal flora of
humans.
No data is available on the occurrence of these organisms in
water or on their susceptibility to water treatment disinfection
processes. Currently used methods for bacterial analysis of potable
water would not detect these organisms up because the media used are
not appropriate, and anaerobic rather than aerobic incubation is
required.
Helicobacter pylori
He! 1cobacter pylori (formerly Campy!obacter pylori) is an
organism that causes the majority of human gastritis cases and is
associated with human duodenal ulcers. Infection with the organism
may also contribute to an increased risk of gastric carcinoma.
Persons at risk for gastric carcinoma have been shown to have a high
prevalence of H. pylon' infection. Little information is available
about reservoirs of the organism, routes of transmission, survival
in the environment, occurrence in water, and susceptibility to control
by water treatment processes.
H- pvlori infection is widespread among Peruvian adults fay the
age of 30 and Klein, et aj.., {9} reported that municipal water supply
•appeared to be an important source of infection among Lima school
children. Mai, et a],., (10) reported that H. pylori may survive in
the environment in a dormant but viable state. Using stepwise
increases in nutrient concentration and alteration of physico-chemical
factors, they demonstrated reversion of H. pylori from the dormant
stage back into dividing and infective cells. This information
suggests the possibility of water as an environmental reservoir for
this organism. The fecal oral route of infection is suspected but
not yet proven.
The detection of H. pylori by cultural Eethods is difficult
because the organism is fastidious and 3 or more days incubation are
required for isolation. If 1t enters the dorsant state, recovery using
cultural techniques may be even more difficult. The association of
H. pylori with the risk of gastric carcinoma raises interesting
questions concerning the possibility of other opportunistic organisms
having some association with gastrointestinal carcinomas.
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Vibrio fluvial 1s
This organism is described in the literature as a marine
bacterium. Host cases of gastroenteritis caused by V. fluvial ii have
been associated with consumption of seafood. The isolation of 1,
fluvial is from potable distribution waters from two systems is,
therefore, surprising (48). Isolates of this organism were obtained
from m-Endo medium, apparently in the absence of total coliforms.
It is not known whether or not the identification of this organism
was serologically confirmed by appropriate serotyping techniques.
V. fluv ial is is the name given' to organisms which were previously
designated as Group F or EF-6. .
V. fluv ialls isolation from fresh surface water and sediment was
reported by Venkateswaran, et a] . (49). To isolate total vibrios from
the water, they used a membrane filter and pad preenrichment with
alkaline peptone water for 6 hr at 30°C. This was followed by transfer
of the membrane filters to thiosul fate-citrate-bile salt-sucrose (TCBS)
agar and incubation at 30°C for 18-20 h. Subsequent to isolation, the
organisms were biochemically identified.
Other than these two reports, very little is known of the
occurrence and distribution of V. fluv ial is in freshwaters that serve
as drinking source waters, or in potable waters. Presumably, the
methodology of Venkateswaran, et al. (49) could be used for surveying
for the presence of these bacteria in surface and potable waters.
This organism was not included in the tables because there have
been only a few episodes of waterborne disease attributed to these
organisms, primarily to X. enterocolitica. These outbreaks have
involved untreated water and only a small number of individuals. A
review of Y. enterocolitfca prepared by Schiemanri (50) summarized the
current state of knowledge about this organism.
Other Hicrobial Groups
Enteric Viruses
Due to the variety of viruses that compose this group of micro-
organisms, and the concern about human disease caused by waterborne
exposure, there has been a significant research effort over the past
several years. These efforts ave been primarily directed at developing
information on inactivation of enteric viruses by drinking water
disinfectants. Much less emphasis has been placed on surveying water
supplies for occurrences of enteric viruses, but some such studies have
been done. A recent article by Hurst (51) reviewed the literature
for studies on the occurrence of enteric viruses in natural freshwaters
and their removal by conventional drinking water processes. Recently,
Payment (52) found no human enteric viruses in 100 L volumes of 162
finished drinking water samples.
Regarding the occurrence of emerging or new human enteric viruses
that may be waterhorne, rotaviruses are a common cause of acute
non-bacterial gastroenteritis in infants and children (535- Rotavirus
has been detected in contaminated drinking water and wastewater in
Mexico (54).
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Bloch, et al (55) reported the cell culture isolation of
hepatitis A virus (HAV) from contaminated well water that was
implicated in a coimon source outbreak that resulted in 35 cases of
'hepatitis A. This study provided new information on the stability of
HAV in untreated water since the water samples from which the virus was
isolated were obtained three months after the onset of symptoms in the
index case. In addition, water concentrates were stored 8 months at
4°C before they were inoculated into cell culture.
The effectiveness of conventional water treatment processes and
disinfection in the removal and inactivation of enteric viruses
suggests that very little enteric waterborne disease occurs in people
who consume water that consistently receives full treatment. However,
when breakdowns occur in the treatment and/or disinfection processes,
waterborne illness risk is increased.
A considerably greater risk exists for those people who consume
untreated groundwaters that are subject to surface water contamination.
This is, and will continue to be, a topic of major concern because of
the potential for viral contamination of groundwaters. Because of the
low infectious doses for many viruses (1-2 infectious units) and the
fact that dissemination of infectious viruses may occur without"
detection, it is probable that there is much greater occurrence
of waterborne viral disease in the population than is presently
recognized. Efforts currently underway to use genetic probes to detect
viruses in water may provide the means to more easily and reliably
detect enteric viruses in all types of water, including drinking water.
The key question, however, is - when viruses are detected by gene probe
methods, what does it mean? How can one tell from probe detection
methods whether the viruses were viable and infective, or if they were
inactivated, but the UNA or RNA was intact?
Pathogenic Protozoa
The two enteropathogenic protozoa that have been in the limelight
as waterborne agents for the past several years are Giardia and Crypto-
sporidium. It is likely they will continue to receive attention
because of their resistance to inactivation by disinfection and the
recent waterborne outbreaks attributed to them. What about other
protozoa that are enteropathogenic? Are there any and are they
waterborne? The answer is maybe!
BlastocYstis hominis
The history of this organism has been one of conflicting
descriptions of it nature and pathogerncity. This protozoon has proven
difficult to classify taxonomically. It has been described as an
intestinal yeast and was first named Blastocvstis enterocola in 1911;
in 1912 the name was changed to £. hominis. However, it has many
characteristics that are protozoan in nature, and it was reclassified
in 1976.' It may eventually be placed in its own niche within the
protozoa. This is based on the diversity of its shapes and sizes,
unique physiology and some of its reproductive modes. Also, shared
RNA sequences with other eucaryotic cells confirm that B. hominis is a
protozoan. Since reclassification in 1S76, it has been placed in the
family Sporozoa, but several minor characteristics indicate a better
assignment would be to the Sarcodina. A complete description of the
organism, its characteristics and current classification was given by
Zierdt (15).
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In fecal samples, this complex protozoan appears in a variety of
cell forms that range from yeast size (ca, 7 /m) to giant cells of
20-40 im. The predominant hollow spherical form of this organism has
been easily confused with unrelated cells from many animals. Many
degenerated tissue cells, vegetable cells, 2nd many artifacts resemble
B. hominis. Hence, it will be extremely difficult to detect in en-
vironmental samples. The organism is strictly anaerobic and has no
cyst form, but may have glycocalyx (capsule). This suggests that it
may be much more susceptible to disinfection than are Giardia cysts and
Cryptosporl.dium oocysts,
The most frequent complaint of blastocystosis patients is intense
abdominal discomfort accompanied with pain. Diarrhea is not standard
and constipation is common; a variety of other symptoms such as vomit-
ing and fatigue may occur. Many mild cases of the disease resolve in
about 3 days without treatment and asymptomatic cases are often seen,
The occurrence and distribution of fi. hominis in water and the
environment is unknown. Isolation of B. hominis has been confirmed
only in humans, monkeys, apes, pigs and possibly guinea pigs. The
incidence rates of B. hotninis in humans range from 5.3% in Saudi
Arabia (56) to 16% in New York (57).
Hicrosporidia
Another group of pathogenic protozoa that may bear watching are
the microsporidia. These are obligate intracellular parasites found in
many vertebrates including man. Of the microsporidia two forms are
enteric parasites, Encephalitozoon cuniculi and Enterocytozapn biemisi.
These organisms have ellipsoid shaped spores approximately 2.5 by
1.5 pi for £. cuniculi and 1.5 by 0.5 pn for E. bienusi. There are
known fecal-oral transmission mechanism cycles for these organisms
similar to Giardia and Crvptosporidium. These organisms may be latent
parasites in many people, which would explain why, with highly immuno-
compronrised people (AIDS patients), the microsporidian parasites are
being seen more frequently. At present, there is no effective drug
therapy.
There is no information available regarding their occurrence and
distribution in water or other environmental samples. Likewise, the
effectiveness of conventional treatment and disinfection processes for
removal and inactivation of the cysts of these organisms is unknown.
Blue fireen Alcrae fCyanobecteria)
An alga has been associated with traveler's diarrhea in travelers
to the Carribean, Hexico, South America, India, or Southeast Asia,
and several AIDS patients (17). The organism observed in stool
specimens was described as resembling a coccidian oocyst or fungal
spore, or a "large CryptosDoridium". Electron microscopy revealed cell
characteristics that indicate the organism may be a species of blue
green algae similar in structure to ChloreTla. .The immunologically
competent patients with traveler's diarrhea complained of nausea,
vomiting, anorexia, weight loss and explosive, watery diarrhea. In
most patients, the symptoms cleared within two weeks along with a
decrease in the number of cyst!ike bodies in the stools. The
The organisms may have been acquired by consumption of improperly
filtered water.
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An outbreak of diarrhea! illness occurred in 21 people who lived
or worked in a hospital dormitory in Chicago, Illinois (58). The
outbreak occurred following transient failure of a building water tank
pump. Examination of patient stools for parasites resulted in the
observation of coccidian-like bodies in 9 of 17 stools. Light
microscopy examination of water tank sediment revealed occasional
coccidian-like bodies. The Centers for Disease Control identified the
organism as a cyanobacterium or cryptosporidium-like organism.
The specific identify of the organism, its occurrence and distri-
bution in water and its susceptibility to removal and inactivation by
water treatment process is unknown,
There have been numerous reports of wildlife, livestock and
pet deaths due to algal toxins in water (59). Cyanobacteria (blue
green algae) are known to produce several toxins, including neuro-
toxins, hepatotoxins and contact irritants. The unusual aspect of the
diarrheal illness outbreaks cited above is that the diarrhea] illnesses
were caused apparently by cyanobacterium infection rather than by
ingestion of toxins elaborated and released into the water by the
cyanobacteria, but this is by no means certain.
Conclusions
A variety of microbial opportunistic pathogens and primary
pathogens may be found in water used as drinking source water. Many of
these organisms have also been isolated from treated drinking water,
particularly the bacterial opportunistic pathogens. Essentially all of
the other microbial opportunists have been found in potable water that
was contaminated due to inadequate treatment or breakdowns in the
treatment processes.
Perhaps none of the organisms covered herein pose a significant
or high health risk to most exposed healthy individuals. However,
accumulating evidence indicates that all of these organisms can be
severe health risks for imraunocoiBprotaised individuals. The population
of inmunocompromised individuals in the U.S. population is steadily
increasing, partially due to the aging of the population, and partially
due to the increasing number of people receiving medical treatment for
long term diseases such as AIDS and cancer. It seems likely that we
will be seeing an increasing emergence of diseases due to waterborne
opportunistic bacterial pathogens in people less iraunologically com-
promised than are AIDS patients.
He usually focus on single species of organisms when talking
about determining the causative agent in a disease outbreak. It is
possible, perhaps probable, that in evaluating human health risks due
to drinking water, we should be considering the collective impact of
simultaneous and repeated exposures to variable levels of several
opportunistic bacteria, rather than focusing on individual organisms.
The collective adverse health impact »ay be far greater than the
impacts of individual organisms would suggest.
In a recent study by Payment, gt al_. (60), excess gastro-
intestinal illness was found in consumers of treated drinking water
compared to consumers who drank the sane water that was further
treated by reverse-osmosis. However, it was also suggested that
heterotrophic bacteria which grew in drinking water produced by
reverse osmosis filtration units, and were enumerated on R2A medium
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incubated at 35°C, may be associated with low numbers of gastro-
intestinal illnesses (61). Heterotrophic bacteria? densities in the
treated distribution water ranged from 5.2 x 10l to 4.3 x 102 CPU/ml
(35°C) in one system and from 2.2 x 10l to 9.5 x if CFU/ml (35°C) in
the other system. Heterotrophic bacterial densities in the R-0 water
ranged from 0 to 107 CFU/ml, but'most contained between i04-105 CFU/ml.
Significant differences between the exposure experienced by the
consumers of treated distribution water versus consumers of R-0 treated
water were: 1} treated distribution water contained lower densities of
organisms/ml, but a greater diversity of types of bacteria based on
organisms isolated and identified (14); and 2} R-0 water contained high
densities of organisms, but of very limited diversity, sometimes only
one bacterial type or species at a time (48). Overall, data from
studies by Payment, et aj.. suggest that they saw increased illnesses
in R-0 water consumers caused by ingestion of water containing high
densities of one .or a few types of bacteria, and in consumers of
treated distribution water by ingestion of water containing low
bacterial densities but having higher diversity of bacterial types.
An alternative possibility is that undetected enteric viruses may
have been responsible for the observed or reported illesses, not the
heterotrophic bacteria.
Thus, the collective impact of exposure to many kinds of
bacteria, some of which are opportunistic pathogens, in drinking water
may result in measurable disease, just as exposure to an infective
dose of a single species can result in disease.
Are there any new pathogens in drinking water? The answer is
yes! So-e are new in the traditional usage of the term new. Others
are new in the sense of dawning recognition of pathogenic potential of
organisms that, individually and collectively, have not been considered
pathogenic in the past. :
ACKNOWLEDGEMENTS
I thank Dr. Eugene W, Rice and Mr. Terry Covert for their review
and comments on this manuscript, and Mrs. Elizabeth Creamer for her
able assistance in the preparation of this manuscript.
DISCLAIMER
This paper has been reviewed in accordance with the U.S. Environ-
mental Protection Agency's peer and adminsitrative review policies and
approved for presentation and publication.
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524
-------
Table 1. Opportunistic Bacterial Pathogens Isolated
from Drinking Water
Acinetobacter spp.
Achromobacter xvlosoxidans
Aeromonas hvdrophila*
Bacillus spp.
Camovlobacter spp.*
Citrobacter spp.
Enterobacter aeroqenes
E. aoglomerans
E. cloacae
Flavobacterium meningosepticum
Hafm'a alvei
Klebsiella pneumoniae*
LeoioneTIa pneumoohila*
Horaxella spp.
Hvcobacterium spp.
Pseudomonas aeruginosa*
Pseudomonas spp. (non-aeruginosa)
Serratia fonticola
S. Tiouifaciens
S. marcescens
Stachvlococcus spp.*
Vibrio fluvial is*
* indicates the organism may be a primary (frank) pathogen
525
-------
Table 2. Summary of Emerging and New Potentially Waterborne
Opportunistic Pathogens
Organism Old/Emerging New Candidates
Anaer_QblospJ_rJ lltmt sued n 1 c |producens ?
Aeroffionas IPJK X
CampYloba^ter. spp. X
EjjchgrJchla c.o.1,1 0157;H7 X
He.11<;ftha.c.t.C-C Mlfid. ' ? •
Mycopacterium spp. X
Plesjomonas shlgelloldes ?
Vibrio fluvial is ?
Reference
3
4
5
6
7
8
9
10
11
12 •
13
14
-------
Table 3, Summary of New or Emerging Potential Waterborne Mieroblal Pathogens
Viruses
Protozoa
Rotawiruses (Group A)
Blastoc-vstls homj.nls.
Mlcrosporldia
New Candidate
X
X
15
16
Algae
Blue Green Algae
(Cyanobacteria)
17
-------
in
N
00
Table 4. Densities of Selected Opportunistic Pathogens
in Drinking Mater
pjflatLjsfl
Aeromonas hydroohjla
Aerononas spp.
spbr1..a
ELaiiojMUHs liUaallaidas
CMfiKlfiMfitej: spp.
Hveobacterluni spp.
M- flprdgriaB, M- avlum. H. fortultum
DensltoJ-CFU/Muiiel
100 - 150/100 ml
10 - 80/100 ml (mains water)
300/100 ml (storage tank)
600 - 800/L (ground water)
0 - 100/L (deep ground water)
0 - 3,300/100 ml
1 - 1,900/100 ml
1 - >H6/100 ml
<1 - >100/100 ml
<1 - 16/100 ml
«. *
- HA **
1.5/100 ml (cold water)
111/100 ml (hot water)
200-500/100 ml (unchlor. ground water) '
1 - <80/L (tapwater)
1 - >500/100 ml
Reference
18
19
If
4
(1
20'
21
18
22
23
24
12
M
25
26
* -, no Information available
** NA, not available, detection usually by enrichment, not direct isolation.
-------
Table 5. Disinfectant Sensitivity of Emerging and New Candidate
Waterborne Opportunistic Pathogens
Organism
An a ero b i o s pi r 11 1 urn syce 1 n_i c iproducen s
Cafflpjjflbacter, spp.
Disinfectant
Any
Free chlorine; >0.3 mg/L
Free chlorine; 0.1 mg/L
Free chlorine
Comments on
Survival lime
Sensitivity
unknown.
Short, increases
with decreased
residual .
>4 logs removal in
5 win (? 4°C,
Sensitive; rapidly
Reference
18
5
27
28
Eschierichia coll 0157:H7
PAesiofflonaji shigelloides
Hvcobacterium spp.
Any
Any
Freo chlorine; 1.0 mg/L
inactivated unless
high level of
organic contami-
nation present.
Sensitivity probably
similar to other
enteric bacteria.
Unknown, but suspected
to be more sensitive
than Aerojnonas.
3 Mvcobacterlum spp.
were eliminated in
fl hrsj.
12
01
8
-------
Table 5 (Cont'd.) Disinfectant Sensitivity of Emerging and New Candidate
Waterborne Opportunistic Pathogens
Hycobacterium spp. (Cont'd,}
llejicobacter pylori
Rotavlrus, human
Blastocvstis hoffiinis
DJsJnfectant
Free chlorine; 0.15 mg/L
Chlorine; 0,1-1,0 mg/L
Free chlorine; 0.3 and
0.7 mg/L
Comments on
Survi'vaLTlrne Reference,
No bactericidal effect. "
Inverse relationship
between disinfectant
residua] and CFU/100 ml.
3 species survived 29
7 days.
Survived 60 min. 30
Any
Any
Chlorine, free, 0.75 mg/L
Any
Sensitivity unknown, but probably
similar to that of vegatatlve
enteric bacteria cells.
Sensitivity unknown.
Approx. 5% survival after
60 minutes,
Sensitivity unknown.
31*
Blue-Green Algae
(Cyanobacteria)
Any
Sensitivity unknown
* Excellent review of disinfectant inactivation of viruses and other health related microorganisms 1n water.
-------
TECHNICAL REPOHT DATA
fftesjt nsdhstoacti&u on At nvtn? btfoft tomfttl?"
1. REfORTNO.
EPA/600/A-937022
2.
I, TiTLt AND SUBTITLE
!. REPORT OATS
Pathogens In Drinking Hater - Are There Any New Ones?
t. f£«f ORMSHQ ORGANIZATION COOS
7. AUTHORISJ
Donald J. Reasoner
3. PERFORMING ORGAM5ZAT1CN REPORT NO.
I. f ERFORMJNG ORGANIZATION NAME AND ADDRESS
Chief, Microbiological Treatment Br.
Drinking Water Res. Div., RREL
U.S. EPA, Cincinnati, Ohio 45268
10. PROGRAM ELEMENT MO.
II. CON TRACT/GRANT WO.
12. SPONSORING AGENCY NAME AND ADDRESS
Risk Reduction Engineering Laboratory—Cincinnati, OH
Office of Research--and Development
US Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
published paper
14. SPONSORING AGENCY CODE
EPA/600/14
is. SUPPLEMENTARY NOTES project Officer = Donald Reasoner (513) 569-7234
Proceedings of the American Water Works Association, Water Quality Technology
Conference, Orlando, FA, 11/10-14/91, 5:509-530
12. ABSTRACT
Since 1976 three newly recognized human pathogens have become familiar
to the drinking water industry as waterborne disease agents. These are:
the legionnaires disease agent, Leqionella pneumophila and related
species; and two protozoan pathogens, Giardia lamblia and Cryptosporidium
parvum, both of which form highly disinfectant resistant cysts that are
shed in the feces of infected individuals. The question frequently
arises - are there other emerging waterborne pathogens that may pose a
human health problem that the drinking water industry will have to deal
with? This paper will review the current state of knowledge of the
occurrence and incidence of pathogens and opportunistic pathogens other
than Leqionella, Giardia and Cryptosporidium in treated and untreated
drinking water. Bacterial agents that will be reviewed include
Aeromonoas, Pseudomonas, Campvlobacter, Hycobacteriunu Yersinia and
Plesiomonas. Aspects of detection of these agents including detection
methods and feasibility of monitoring will be addressed.
7.
KEY WORDS AND DOCUMENT ANAS.YSSS
DESCRIPTORS
b.lDENTIFlERS/OPEH ENDED TERMS
c. COSATi Fie!d/Croup
protozoa
viruses
bacteria
Drinking water quality,
bacterial occurrence,
opportunistic pathogens,
pathogens
S. DISTRIBUTION STATEMENT
Release to Public
CFA F«m 2220-1 {R«T. X-773 PHEVJOUS COITION is OBSOLETE
IS. SECURITY CLASS [Tlia Report!
Unclassified
21. NO. OF PAGES
24
20. SECURITY CLASS iTJiispsjr}
Unclassified
22. PRICE
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