United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-89/019 Mar. 1990
&EPA Project Summary
Protozoan Sources of
Spontaneous Coliform
Occurrence in Chlorinated
Drinking Water
Emmett B. Shotts, Jr. and Richard E. Wooley
The spontaneous occurrence of
conforms in chlorinated drinking
waters has resulted in concern over
their potential source and mecha-
nism(s) of Introduction into water de-
livery systems.
Previous observations related to
protozoa! resistance to chlorine
coupled with the ingestion of
bacteria by this group of organisms
prompted an in-depth study of
bacterial-protozoan interaction in the
presence of chlorlnatlon.
Bacterial organisms used were
either from previous "spontaneous
coliform outbreaks" or potential
human pathogens. The protozoans
utilized were laboratory adapted and
wild strains of protozoans found
naturally In water reservoirs. These
organisms were established In axenic
cocultures and the effect of various
concentrations of chlorination and
exposure were determined.
It was found that, alone, the
bacteria were killed by from 0.25-1
mg/L of chlorine; however, when
ingested by protozoans, these same
bacteria survived in significant con-
centrations from levels of chlorina-
tion 4 to 10 times the above dose.
These findings provide a possible
and plausible explanation of how
bacteria may survive chlorination and
appear in water delivery systems. It
further suggests a need for increased
awareness in developing improved
methods of processing drinking
water that include the removal of
both viable and dead protozoans from
entering the distribution system.
This Project Summary was devel-
oped by EPA's Risk Reduction Engi-
neering Laboratory, Cincinnati, OH, to
announce key findings of the research
project that Is fully documented in a
separate report of the same title (see
Project Report ordering Information at
back).
Introduction
The spontaneous occurrence of coli-
forms in chlorine-treated drinking water
has generated a great deal of concern,
particularly in areas where reservoir water
is sand filtered and chlorinated. Prior
work on this problem addressed the
production of chlorine resistant clones of
bacteria and also the inability to readily
isolate chlorine-injured bacteria. It was
the purpose of this investigation to
related the sparing and enhancement
qualities noted in Legionella-Tetrahymena
interaction into this emerging problem in
water delivery systems. This was ac-
complished by evaluating the bacterial
(coliforms isolated from outbreaks)-proto-
zoan (both laboratory and wild strains)
relationship to determine if ciliated
protozoans that naturally occur in water
impoundments could provide protection
against chlorination for engulfed bacteria,
thus allowing the bacteria to be released
spontaneously by the protozoan at a later
time in the water distribution system. To
achieve this, techniques were used that
involved enumeration of bacteria follow-
ing protozoal engulfment with subsequent
-------
chlorination. Chlorination was done on
aliquots both before sonication and after
sonication, respectively, thus providing
appropriate controls. From this research,
it was determined that protozoa did
provide protection against chlorination to
engulfed coliform bacteria, in some
cases, for at least 24 hr from initial
exposure of 2-4 mg/L of chlorine
although protection to exposures of 20
mg/L of chlorine was noted in some
interactions.
Materials and Methods
This investigation examined the
effects of free and combined chlorine
upon selected protozoan-bacterial cocul-
tures that might be found in drinking
water resources. Comparative inactiva-
tion studies were done on bacteria alone
and in cocultures with protozoans under
chlorine demand-free conditions that
simulated those of routine water treat-
ment. Selected coliforms previously
recovered from "spontaneous occur-
rences" in water distribution systems
were obtained from the Risk Reduction
Engineering Laboratory, USEPA, Cin-
cinnati, OH. These include Klebsiella
pneumonia, K. oxytoca, Enterobacter
cloacae, E. agglormerans, Citrobacter
freundii and Escherichia coli. Other
organisms subsequently used included
Campylobacter jejuni, Legionella gor-
manii, Salmonella typhimurium, Yersinia
enterocolitica, and Shigella sonneii.
These bacteria were studied in
coculture with Tetrahymena pyriformis
(primary organism used because of its
ease in handling) (ATCC 30327), Acan-
thamobea castellanii (ATCC 30234), and
Bodo edax (ATCC 30903). Axenic
cultures of the latter resulted in limited
success; subsequent engulfment studies
were not fruitful and it was removed from
the study. Near the end of the study, a
wild strain of Cylidium was studied in
coculture with £ coli to establish that the
relationship extended beyond laboratory
strains of protozoans.
All studies were carried out using
chlorine demand free buffer (CDFB) and
were considered as chlorine demand
free (CDF) when the chlorine con-
centration was less than 0.01 mg/L free
available chlorine as measured by DPD
colorimetric method read in a B & L
Spectronic 2000 spectrophotometer.* All
chlorine estimations were standardized
to chlorine quality control samples from
"Mention of trade names and commercial products
does not constitute endorsement or recom-
mendation for use
USEPA, Environmental Monitoring Sys-
tems Laboratory, Cincinnati.
Subsequent study of the experimental
components (i.e., protozoa and bacteria
alone) were done prior to coculture
studies where concentrations of 1 x 104
of the respective bacteria and protozoan
were allowed to interact.
Following exposure to various con-
centrations of chlorine at time intervals,
bacterial viability and numbers were
determined by plate counts using mT7
agar where possible to permit maximum
recovery of possible chlorine-damaged
cells.
Prior to plating, the chlorine containing
suspension of coculture was neutralized
with sodium thiosulfate, centrifuged, and
resuspended in sterile culture medium
for sonication to release the bacteria
from the protozoa.
Samples were obtained and observed
for engulfment, vacuole formation, and
multiplication by electron microscopy
(EM).
Results
As noted in Table 1, a great variability
existed among bacteria isolated as to
their survival within respective protozoan
hosts. It was readily established that all
bacteria unprotected by protozoan
engulfment were dead at 1 mg/L of
chlorine or less. The protozoa, on the
other hand, were viable after 2 to 5 times
this chlorine dose. These data indie
that although Tetrahymena was killec
2 mg/L of chlorine, the bacteria con
ued to replicate within the protozc
Similarly, the same thing happened
the Acanthamoeba, and, although re
tance to chlorine was not demonstra
in the wild type Cylidium, viable bact<
were present after 24 hr. It should
noted that in no case were more than
10 organisms noted ingested in
protozoan, yet large percentages
bacterial survival were obtained p
viding evidence of bacterial replicat
within both the viable and dead prc
zoans.
Discussion and Conclusions
These studies have demonstrated
alternate explanation to chlorine res
tance as a mechanism of bactei
introduction into water delivery syster
This study further demonstrates tl
although protozoans may be killed duri
chlorination, viable bacteria may still
introduced into water delivery systems
These occurrences have been denru
strated not only with isolates frc
previous spontaneous coliform occi
rences but also with several prims
human pathogens of gastrointestir
disease and also with Leg/one
gormanii. In the latter case, the inti
duction of viable organisms into t
delivery system with subsequent temp*
Table 1. Relative Survival of Selected Bacteria Engulfed by Protozoans and Exposec
Varying Amounts of Chlorination for 24 Hours.*
Protozoans*
Bacteria&
K. pneumonia
K. oxytoca
Ent. cloacae
Ent. agglormerans
Cit. freundii
Esc. coli
Camp, jejuni
Leg. gormanii
Sal. typhimurium
Yer. enterocolitica
Shig. sonneii
Tetrahymena
pyriformis
2/20*
1120
0.51100
2/70
1/30
4/60
0.25/10
4/50
2/65
4/100
41100
Acanthamoeba
castellanii
10/80
4/20
2/80
2/100
0/0
1/100
-@
—
Cylidium
spp.
-
:
—
-
—
'Percentage survival of engulfed bacteria at highest chlorine concentrations following 24
exposure.
Tetrahymena resisted 1 mg/L chlorine for 24 hr; Acanthamoeba resisted 10 mg/L chlorine
24 hr.
&AII bacteria (alone) were killed at 1 mg/L chlorine except for C. jejunt and Ent. cloacae (0
mg/L chlorine).
* First number denotes highest concentration of chlorine in mg/L showing surviving bacterii
24 hr; second number represents the percentage of the original bacterial inoculum (1 x I
surviving.
@Not done
-------
ature selection in hot water tanks could
provide an explanation related to the
spread of this disease in bath waters. In
other studies, L pneumophila has been
shown to act similarly.
From the standpoint of gastrointestinal
disease, it is suggested that, without
further processing, drinking water that is
sand filtered and chlorinated for human
use presents a potential source of
infection. This risk could be reduced by
further processing of the water prior to
distribution.
In summary, a mechanism present in
nature has been demonstrated whereby
bacterial-protozoan interaction negates
usual methods of water treatment for
many supply systems and, despite
chlorination, presents the possibility of
the introduction of measurable amounts
of potentially pathogenic bacteria into
drinking water delivery systems.
The full report was submitted in
fulfillment of CR812833-01 by the Uni-
versity of Georgia Research Foundation
under the sponsorship of the U.S. Envi-
ronmental Protection Agency.
-------
Emmett B. Shotts, Jr. and Richard E. Wooley are with the University of Georgia,
Athens, GA 30602.
Eugene W. Rice and Harry 0. Nash are the EPA Project Officers (see below).
The complete report, entitled " Protozoan Sources of Spontaneous Coliform Oc-
currence in Chlorinated Drinking Water,"(0rder No. PB 89-190 292IAS; Cost:
$13.95, sub/ect to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officers can be contacted at:
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
MALTY
Official Business
Penalty for Private Use $300
EPA/600/S2-89/019
u.s.OFFICIAL MAIL:
U.S.POSIASf]"
s 0 .2 5 :
S00S°E»VIB PMTECTICH.
CHICAGO
------- |