Download Coliphages as alternate indicators of fecal contamination in tropical

Coliphages as Alternate Indicators of Fecal
Contamination in Tropical Waters
E.A. HERNANDEZ-DELGADO, M.L. SIERRA, and
G.A. TORANZOS
Department of Biology, University of Puerto Rico, Rio Piedras,
Puerto Rico 00931
ABSTRACT
Strong evidence has recently been found against the use of fecal coliforms as indicator
organisms of fecal contamination in tropical waters due to their indigenous nature in
pristine waters. There is a great need for the development of more rapid, accurate, and
low-cost techniques for determining bacteriological water quality. Coliphages seem to be
a n excellent alternative. We have developed a method to analyze large volumes ofpristine
water, which we compared to a commonly used direct method. It involves the filtration
of water through positively charged filters and a virus-elution step. The eluent is then
mixed with culture medium, a host bacterium, and incubated. We sampled pristine
tropical rivers, water colleeted from bromeliads (epiphytic vegetation), sewage-contaminated waters, and marine waters. Concentrations of indicator bacteria were higher
than recommended levels for recreational waters, including bromeliad waters. Indicators
levels were higher in bromeliad waters than in sewage-contaminated rivers. Phages were
isolated only from waters being used for recreational purposes and from sites known to
be contaminated with domestic sewage, but not from pristine or bromeliad waters. These
results suggest that there is a correlation between the presence of coliphages and fecal
contamination. This further suggests that coliphages may be reliable indicators of fecal
contamination in the environment.
INTRODUCTION
Among some of the world’s greatest public health problems have been
waterborne diseases, especially in tropical and subtropical areas, where
the diversity and severity of these diseases is higher. Three groups of
indicator microorganisms have been used to monitor water for the
possible presence of enteric pathogens. However, evidence has been
recently found against the use of fecal coliforms as indicator organisms
of fecal contamination in tropical waters (Hazen, 1988).
Several studies in rain forest watersheds of Puerto Rico indicated
Environmental Toxicology and Water Quality: An International Journal
Vol. 6, 131-143 (1991)
CCC 1053-4725/91/020131-13$04.00
0 1991 John Wiley & Sons, Inc.
132/HERNANDEZ-DELGADOET AL.
that Escherichia coli could survive, remain physiologically active, and
grow in the environment (Carrillo et al., 1985;Hazen et al., 1987;L6pezTorres et al., 1987,1988; Santiago-Mercadoand Hazen, 1987;Bermudez
and Hazen, 1988; Jim6nez et al., 1989; P6rez-Rosas and Hazen, 1989).
These indicator organisms have also been isolated in other tropical
countries from pristine waters (Feachem, 1974; Oluwande et al., 1983;
Fujioka et al., 1988). See Hazen (1988) for a review.
Fecal coliforms and fecal streptococci have also been isolated from
water accumulated in the axilae of bromeliads (epiphytic flora) in a
Puerto Rican tropical rain forest (Rivera et al., 1988). High natural
productivity of tropical environments probably allows for survival and
regrowth of pathogens and indicators in freshwater. In addition to
the presence of indigenous indicators in the environment, these are
probably only a few of the reasons why only 13 of the 67 river sampling
stations (19%) failed to meet the maximum contaminant levels standards for recreational waters in recent years (less than 100 fecal coliforms/100 mL) (Curtis et al., 1984). None of the sites sampled met raw
source water standards (less than 2 fecal coliforms/100 mL).
There is a great need for the development of more rapid, accurate,
and low-cost techniques for determining microbiological water quality.
Coliphages have been proposed as an alternate and economically reliable indicator of fecal contamination of water (Wentsel et al., 1982;
Grabow and Coubrough, 1986; Sim and Dutka, 1987; Castillo et al.,
1988; Sim et al., 1988; Toranzos et al., 1988) because phage assays
are easier and cheaper to perform than currently used bacteriological
techniques. In addition, they can be quantified in environmental samples within 3-24 h as compared to at least 96 h when performing
bacteriological assays.
One of the many potential applications of bacteriophages is as
indicators of sewage contamination (Gerba, 1987).The ubiquity of coliphages in the feces of warm-blood animals, sewage, and in sewagecontaminated waters suggests that coliphages could reliably indicate
fecal contamination of the environment. Coliphages also exhibit a great
resistance to environmental stress, and thus the longest survival of the
bacteria and viruses excreted in the feces. They are, then, potentially
useful for detecting remote sources of fecal contamination or to indicate
fecal contamination of waters.
Commonly used indicators, including E . coli, are naturally occurring in pristine waters, whereas coliphages have never been detected at these same sites, suggesting the absence of fecal contamination. In Puerto Rico coliphages have only been isolated from streams
and rivers being used for recreational purposes, and from sewage-con-
COLIPHAGES AS FECAL CONTAMINATION INDICATORS/ 133
taminated rivers, estuaries, and beaches, suggesting a correlation between the presence of phages and human fecal contamination.
Although the use of coliphages as indicators of fecal contamination
appears attractive, thorough studies involving statistical comparisons
with the presence of other microbial indicators and pathogens, and
environmental coliphage survival, are needed before they can be seriously considered as meaningful indicators of fecal contamination. The
purpose of this study was to evaluate the reliability of coliphages as
alternate indicators of fecal contamination in tropical surface freshand marine waters in Puerto Rico.
MATERIALS AND METHODS
Study Sites
The Mameyes River watershed located in the Caribbean National Rain
Forest was selected because it offers several different habitats. The
river begins at an elevation of nearly 1000m above sea level. According
to L6pez-Torres et al. (1987), the upper third is in the Caribbean National Rain Forest, and it is characterized by pristine waters in the
upper parts and recreational waters in its lower area. The middle
third receives primary treated domestic sewage from a large housing
development and it is also used for recreation. The lower third passes
through two towns, where it is further polluted by domestic and industrial sewage effluents. This portion of the river is commonly frequented
by fishermen (personal observations). The river empties into the Atlantic Ocean at Fortuna Beach. It is located near Luquillo Beach, Puerto
Rico’s largest public beach.
Sampling Procedures
Grab samples were collected for bacteriological and phage assays in 1L sterile Nalgene plastic containers (Nalge Company, Rochester, NY)
and kept on ice until assayed. PHAGE assays were carried out within
6 h while bacteriological assays were conducted within 24 h.
Bacteriological Analysis
Samples were analyzed by the membrane filtration technique using
m-Endoagar for the isolation of total coliforms, the m-FC medium for
fecal coliforms, and KF-Streptococcus agar for fecal streptococci.Repre-
134/HERNANDEZ-DELGADOET AL.
sentative colonies were confirmed using standard techniques (American Public Health Association, 1985).
Bacteriophage Host
An E . coli C strain (ATCC 15597) was utilized as the phage host.
Previous studies with this strain have shown it to be the most sensitive
host for the isolation of coliphages from the environment.
Phage Assays
A modified adsorption-elution technique was evaluated to isolate
phages from pristine waters. This technique involves the filtration of
large volumes (one liter or larger) of water through a positively charged
filter (Versapor 30S, obtained from AMF-Cuno Division, Meriden,
CT), where phages adsorb. Then, they were desorbed in a small volume
of 3%beef extract + 3% tryptose phosphate broth (pH 8.0). This eluent
was mixed with trypticase soy agar (Scott Laboratories, Fiskeville, RI)
and the phage host cells, and incubated at 37°C for 12 h.
Contaminated water samples were analyzed by a direct method
(Grabow and Coubrough, 1986). Briefly explained: a 100-mL sample of
water was mixed with 100 mL of trypticase soy agar (2 x concentration)
and the host cell, then poured into sterile Petri dishes and incubated
at 37°C for 12 h. Because of the low volume sampled, this is not an
adequate assay for pristine waters, where a large volume is needed to
detect low numbers of phages.
Phage Survival Studies Under Laboratory Conditions
Survival studies were carried out in the laboratory. Briefly explained:
environmental waters were seeded with lo7 plaque-forming units
(PFU) isolated from domestic sewage and the rate of inactivation followed by obtaining samples at increasing time intervals. Phages were
detected as described above.
In Situ Phage Survival Studies
In situ phage survival studies were carried out in a pristine tropical
rain forest river using virus environmental survival chambers (25 mL)
loaded with 0.01- and 0.45-pm pore size filters. Phages isolated from
raw sewage were mixed with sterile water (chamber A), and with river
water containing natural coliform strains (chamber B). MS-2 were
mixed with sterile water and used as a control (chamber C).Aliquots
COLIPHAGES AS FECAL CONTAMINATION INDICATORSI135
TABLE I
Average concentrationsa of coliphages and indicator bacteria in several sampling
stations in the Mameyes River watershed, Puerto Rico
Sampling
station
Bromeliad
waterb
La Mina River
La Mina River
(recreational
area)
La Coca Fall
Mameyes River
(Puente Roto)
Mameyes River
(posteffluent)
Mameyes River
estuary
Fortuna Beach
Total
coliforms
Fecal
coliforms
Fecal
streptococci
1.42 x lo6
4.21 x lo4
8.37 x lo4
0
1.62 x 104
3.22 x 104
5.12 x 103
6.05 x 103
2.30 x 103
6.91 x 103
0
0.17
1.88 X lo3
3.63 x 104
4.66 X lo2
1.07 x 104
1.70 X lo3
6.81 x 103
1
2.14
2.12 x 104
9.09 x 103
2.14 x 104
8.5
1.94 x lo4
3.07 x lo4
1.35 x lo4
1.52 x lo2
3.34 x 104
2.11 x 104
2.00 x 101
Coliphages
37.8
a All concentrations of bacteria and phages are in colony-forming units (CFU)/100
mL and PFU/100 mL, respectively; averages calculated from 10 to 14 samples (about 2
samples/month).
Sites arranged in order ranging from pristine to sewage-contaminated waters.
were taken at different time intervals up to 20 days. River water temperature, pH, conductivity, hardness, dissolved oxygen, and turbidity
were measured for each sampling time.
RESULTS AND DISCUSSION
We have analyzed pristine tropical rivers, water collected from bromeliads (epiphytic vegetation), sewage-contaminated waters, and marine
waters. Total and fecal coliforms levels were almost always higher than
recommended standards of less than 1000 fecal coliforms/100 mL for
recreational waters in all samples. The same was true for fecal streptococci. None of the samples met raw source water standards of less than
2 fecal coliformsll00 mL. Results shown in Table I clearly indicate the
natural presence of indicators in pristine waters as in the upper parts
of the mountains in La Mina River and water from the axilae of bromeliads. In fact, bromeliad waters had higher average concentrations of
total coliforms and fecal streptococci than surface waters. The highest
levels of fecal coliforms were observed at the Mameyes River estuary,
followed by water from the bromeliads. The lower levels of bacteria
ND
lo2
6.24 x
8.90 x lo3
9.0
0
0
6.5
ND
ND
ND
Phages
FS
1.69 x 103
2.21 x lo2
2.20 x 102
June 1989
FC
Tntc
9.25 x lo2
4.10 x 103
TC
7.36 x 104
8.70 x 104
1.69 x 104
3.72 x 104
TC
4.66 x 103
1.08 x 103
1.16 x 103
7.53 x 102
FS
1.67 x 104
1.49 x 105
6.91 x 103
2.91 x 104
July 1989
FC
16.6
0
0
2.5
Phages
a All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL, respectively; averages calculated from 2 samples/
month.
Abbreviations: TC, total coliform; FC, fecal coliform; FS, fecal streptococci; ND, not done; Tntc, too numerous to count.
Sites arranged in order ranging from pristine to sewage-contaminated waters.
Bromeliad waterC
La Mina River
Mameyes River
(Puente Roto)
Mameyes River
(posteffluent)
Sampling
station
TABLE I1
Concentrationsa of coliphages and indicator bacteria in several sampling stations through the months of June and July 1989 in the
Mameyes River watershed, Puerto Ricob
*r
m
0
5
z
F
U
35
8.70 x 104
ND
ND
3.17 x 103
ND
ND
1.01 x 105
ND
ND
Tntc
Tntc
1.17 x 105
1.19 x 103
6.60 x 104
Tntc
2.08 x 104
1.00 x 104
8.00 x 102
2.20 x 103
ND
Tntc
7.00 x 103
4.60 x 10'
1.03 x 103
3.00 x lo2
7.45 x 102
6.27 x lo2
1.46 x
5.39 x
8.5
0
0
0
0
ND
lo2
102
Phages
a All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL, respectively; averages calculated from 2 samples/
month.
For abbreviations, see footnote b of Table 11, except for ND:not done because of a drought that followed Hurricane Hugo, water did
not accumulate in the axilae of bromeliads.
Sites arranged in order ranging from pristine to sewage-contaminated waters.
ND
ND
2
4
2.37 x 103
2.92 x 103
1.32 X 10'
2.92 x 103
2.56 x 103
2.69 x 104
ND
4.00 x 10'
1.50 x 103
ND
5.04 x 103
2.29 x 104
0
0
0.5
1.80 x 105
2.45 x 103
1.81 x 104
1.30 x 104
1.95 X 10'
3.54 x 102
1.11 x 106
1.40 x 104
3.18 x 104
Bromeliad water'
La Mina River
La Mina River
(recreational
area)
La Coca Fall
Mameyes River
(Puente Roto)
Mameyes River
(posteffluent)
Mameyes River
estuary
Fortuna Beach
FS
September 1989
FC
TC
Phages
FS
August 1989
station
FC
TC
Sampling
TABLE I11
Concentrations" of coliphages and indicator bacteria in several sampling stations through the months of August and September 1989
in the Mameyes River watershed, Puerto Ricob
4
w
r
3
ij
ND
3.00 x 105
ND
ND
ND
ND
ND
x 103
4.00 x 104
4.00
1.90 x 103
ND
2.20 x 104
ND
ND
ND
December 1989
FC
FS
1.00 x 105
ND
1.00 x 105
TC
2
5
6
0
0
0
Phages
ND
ND
4.00 x 104
TC
5.35 x 106
ND
1.00 x 104
ND
6.00 x 105
3.05 x 104
1.90 x 105
ND
4.25 x 104
x 103
ND
2.00 x 104
2.00
LOO x 105
1.00 x 103
1.00 x 103
January 1990
FC
FS
3
64
0
2
0
0
Phages
a
All concentrations of bacteria and phages are in CFU/100 mL and PFU/100 mL respectively; averages calculated from 2 samples/
month.
For abbreviations, see footnote b to Table 11.
Sites arranged in order ranging from pristine to sewage-contaminated waters.
Bromeliad watel"
La Mina River
Mameyes River
(Puente Roto)
Mameyes River
(posteflluent)
Mameyes River
estuary
Fortuna Beach
Sampling
station
TABLE IV
Concentrationsa of coliphages and indicator bacteria in several sampling stations through the months of December 1989 and January
1990 in the Mameyes River watershed, Puerto Ricob
+
*r
B
r3
0
E
s*
U
E?
2
iN
E
?
w
COLIPHAGES AS FECAL CONTAMINATION INDICATORS/ 139
TABLE V
Concentrationsa of coliphages and indicator bacteria in several sampling stations in
the month of March 1990 in the Mameyes River watershed, Puerto Ricob
~
Sampling
station
Bromeliad water'
La Mina River
La Mina River
(recreational
area)
Mameyes River
(Puente Roto)
Mameyes River
(posteffluent)
Mameyes River
estuary
Fortuna Beach
~
~
March 1990
TC
FC
FS
Phages
1.42 x 105
4.43 x 104
4.20 x 104
1.60 x 104
2.36 x 104
1.63 x 104
4.10
2.00 x 103
X
lo2
2.00 x 103
0
0
0
1.00
4.35 x 103
7.50
X
lo2
0
3.15 x 104
1.07 x 104
6.00 x lo2
1
1.94 x 104
1.02 x 104
ND
1
3.34 x 104
2.11 x 104
2.00 x 10'
0
xi04
a All concentrations of bacteria and phages are in CFU/100 mL and PFUilOO mL,
respectively; averages calculated from 2 samples/month.
For abbreviations, see footnote b of Table 11.
' Sites arranged in order ranging from pristine to sewage-contaminated waters.
were detected at La Coca Fall. Higher levels of indicator bacteria in
the rivers coincided with heavy rainfall and turbidity (possibly due to
stirring up of the sediments), while the opposite was observed in the
bromeliads, which was probably due to dilution effects. Higher levels
of bacteria from the bromeliads were detected during the dry season
(i.e., March). These results are shown in Tables 11-V, which compare
results between different months for a 10-month period.
Coliphages were isolated only from recreational waters and from
sites known to be contaminated with sewage, but not from bromeliad
and pristine waters. Higher coliphage levels were observed at the
Mameyes River estuary. These waters receive sewage effluents from
two small towns and two primary treatment plants. This suggests that
there may be a correlation between the presence of coliphages and fecal
contamination, and further indicates that coliphages may be reliable
indicators of fecal contamination in the environment.
Coliphage survival studies under laboratory conditions indicated
that total and fecal coliform levels increased over time in both pristine
and sewage-contaminated waters (Tables VI and VII). Coliphage levels
decreased 4 and 3 orders of magnitude for pristine and sewage-contaminated waters, respectively, after 120 h. In addition, the decrease in
140/HERNANDEZ-DELGADO ET AL.
TABLE VI
Coliphage survival under laboratory conditions in pristine river waters
Coliforms
b e r 100 mL)"
Time (h)
pH
Total
0
24
48
72
120
7.0
6.3
5.6
4.2
3.3
4.7 x 104
2.6 x lo7
1.5 x los
1.7 x 10'
1.1 x 106
a
Fecal
6.0
3.4
2.7
1.7
3.0
x 103
x lo6
x lo6
x lo6
x 104
Fecal
streptococci
(per 100 mL)
ND~
105
105
104
103
1.0 x
1.6 x
2.8 x
2.0 x
Coliphages
(per 100 mL)
7.0
2.3
6.0
9.0
3.0
x lo6
x 105
x 104
x 104
x lo2
Total and fecal coliforms and streptococci were naturally occurring in these waters.
ND. not done.
phage concentration was directly proportional to the decrease in the
pH of both types of water.
In order to evaluate phages' survival in surface freshwater, and
to determine if they can replicate in natural coliform strains under
environmental conditions, an in situ survival study was carried out in
a pristine river. No coliphage inactivation was observed during the first
192 h (8 days) of the study (Fig. 1).This was probably due to their
resistance to environmental factors. As environmental parameters did
not fluctuate greatly through the study, they did not appear to have
had a significant effect on phage stability.
After 10 days, a 2 log decrease was observed in the sewage-isolated
coliphage chamber mixed with river water containing natural coliform
strains. It was demonstrated that sewage-bornecoliphages did not replicate under environmental conditions using indigenous E . coli as hosts.
TABLE VII
Coliphage survival under laboratory conditions in sewage-contaminated waters
Coliforms
(per 100 mL)
Time (h)
pH
Total
Fecal
Fecal
streptococci
(per 100 mL)
0
24
7.0
6.6
6.0
5.4
5.4
1.7 X lo6
3.8 x lo7
1.1 x lo8
8.0 x 107
3.0 x lo6
1.1 x lo3
2.1 x lo6
2.7 x lo6
2.0 x 106
1.1 x lo6
ND"
2.0 x 105
9.0 x 104
4.4 x 104
2.1 x 104
48
72
120
a
ND, not done.
Coliphages
(per 100 mL)
7.7
2.4
2.0
3.7
4.7
x
x
x
x
x
106
105
105
104
103
COLIPHAGES AS FECAL CONTAMINATION INDICATORS1141
101
109
-
.
E
108
a
10
U
n
CHAMBER A
CHAMBER B
CHAMBER C
106
10
10’
I . ,
0
2
. ,
4
., ., .
6
8
, . , . ,
10
12
14
1
,
36
.
,
18
. ,
I
20
TIME (DAYS)
Chamber A = Phages isolated from raw sewage + sterile water
Chamber B = Phages isolated from raw sewage + river water
containing indigenous fecal coliforms
Chamber C = MS-2 Phage + sterile water
Fig. 1. Survival of coliphages in a tropical rain forest pristine river.
In previously published studies (Castillo et al., 1988; Grabow and
Coubrough, 1986; Wentsel et al., 19821, all coliphage data were obtained
from small sample volumes; thus if low concentrations of phages were
present, they were not detected due to the low sensitivity of the technique. In our technique, the concentration of 1L of sample down to 50
mL, which can then be easily analyzed, improves on the ability to detect
very low levels of phages. Nonetheless, even concentrating this volume,
it was not possible to detect coliphages except in waters known to
be contaminated with sewage or waters being used for recreational
purposes.
For our filtration-elution method, using water seeded with MS2,
adsorption efficiencies ranging from 93 to 99.8%were obtained. A 43%
elution efficiency was obtained using 3% beef extract + 3% tryptose
phosphate broth (pH 8.0). The method has been tested in pristine river
waters and in tap water samples, where up to 3 L of water were filtered.
In the former, no coliphages were detected, while in the latter a total
of 3 PFUiL were detected.
We have detected coliphages only in sites where there is fecal
contamination. Thus, coliphages seem to have the potential as an excellent alternative to the use of bacteria to indicate fecal contamination
of waters and therefore to predict the possible presence of pathogens.
l&/HERNANDEZ-DELGADO ET AL.
CONCLUSIONS
Total and fecal coliforms levels were always higher than recommended
standards in all samples. They were also constantly isolated from
pristine river waters and water from the axilae of bromeliads,
where no human fecal contamination was detected. Coliphages were
detected only in recreational waters and waters known to be sewage
contaminated. They were never detected in pristine rivers. These
results suggest an association of coliphages with human fecal
contamination.
Phage survival studies under laboratory conditions demonstrated
that phage inactivation was directly proportional t o a decrease in pH,
while in situ survival studies demonstrated that coliphages did not
replicate in indigenous coliform strains under environmental conditions. Highest coliphage inactivation was observed in the sewageisolated coliphages mixed with river water containing resident flora.
Coliphages have the potential of being used as indicators of fecal contamination in aquatic environments in spite of its long survival period
of time, because its dilution in the environment will prevent their
constant detection. In addition, phages did not replicate under laboratory or under environmental conditions in the presence of indigenous
fecal coliform bacteria.
Our filtration-elution method showed an adsorption efficiency
ranging between 93 and 99.8%, and a 43% elution recovery. It proved
to be an excellent method for phage concentration where phage levels
were low (as in tap water) and where other methods (i.e., Grabow and
Coubrough, 1 9 8 6 ) are less sensitive.
Studies are being carried out on the detection of bacterial, viral,
and parasitic pathogens in the environment. We are correlating them
with the presence of coliphages. In addition, we are currently working on molecular techniques for the detection of coliphages in environmental samples, including the use of genetic probes and PCR
techniques.
We acknowledge the technical help of Nayah Harb and Leonor Alicea. Portions
of this work were supported by SUBE no. 2S06RR08102-18, INDUNIV no.
9020539303, and WRRI 14-080001-G1611.
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