Prevalence of diarrhegenic serotypes of Escherichia coli in the Cochin estuary, along west coast of India

Abstract

Prevalence of indicators of faecal pollution such as faecal coliform and faecal streptococci were studied at the Cochin estuary. There was consistently high load of faecal indicator bacteria at all stations. Serotyping of E. coli revealed the presence of diarrhegenic serotypes of E. coli such as enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC) and uropathogenic E. coli (UPEC). The diversity of E. coli serotypes was particularly high as we were able to detect more than 40 serotypes. The E. coli serotypes also showed spatial and temporal variation. Characterisation of faecal streptococci revealed predominance of Streptococcus faecalis subspecies liquefaciens followed by enterococcus group and Streptococcus bovis. Seasonal variation in the prevalence levels of these organisms showed a higher load of the indicator organisms during the monsoon season, especially that of faecal streptococci indicating a higher land run-off during this period. The results of the present investigations reveal the high degree of faecal pollution in the Cochin estuary posing health hazard to those who use this system for fishing and recreation.

Full text

Indian Journal of Marine Sciences
Vol. 33(3), September 2004, pp. 238-242
Prevalence of diarrhegenic serotypes of Escherichia coli in the
Cochin estuary, along west coast of India
A. A. Mohamed Hatha*, Abirosh Chandran & K. M. Mujeeb Rahiman
School of Environmental Sciences, Mahatma Gandhi University, Kottayam-686 008, Kerala, India
Received 9 July 2003, revised 24 May 2004
Prevalence of indicators of faecal pollution such as faecal coliform and faecal streptococci were studied at the Cochin
estuary. There was consistently high load of faecal indicator bacteria at all stations. Serotyping of E. coli revealed the
presence of diarrhegenic serotypes of E. coli such as enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC),
enterohemorrhagic E. coli (EHEC) and uropathogenic E. coli (UPEC). The diversity of E. coli serotypes was particularly
high as we were able to detect more than 40 serotypes. The E. coli serotypes also showed spatial and temporal variation.
Characterisation of faecal streptococci revealed predominance of Streptococcus faecalis subspecies liquefaciens followed by
enterococcus group and Streptococcus bovis. Seasonal variation in the prevalence levels of these organisms showed a higher
load of the indicator organisms during the monsoon season, especially that of faecal streptococci indicating a higher land
run-off during this period. The results of the present investigations reveal the high degree of faecal pollution in the Cochin
estuary posing health hazard to those who use this system for fishing and recreation.
[Key words: Escherichia coli, faecal pollution, estuary, diarrhegenic serotypes]
Introduction
Population explosion and inadequate infrastructure to
properly treat and dispose the sewage has resulted in
the discharge of considerable quantities of untreated
wastes into natural waters. Sewage effluents contain a
wide range of pathogenic microorganisms that may
pose a health-hazard to human populations when they
are discharged into recreational waters1. The
increased pollution of the natural waters in
conjunction with the improved screening techniques
has resulted in the detection of emerging pathogens in
areas affected by sewage discharges2, with subsequent
hazard to public health3. The presence of indicator
organisms in natural waters indicates pollution, but
their absence does not necessarily guarantee quality of
the water4.
Cochin estuary, a typical tropical estuary, had
undergone considerable pollution in the last decade
resulting mainly from the development of satellite
townships all across the estuary. The pollution of the
estuary is mainly of microbial, as the industrial
development had declined in this region. The only
industry which contributes to microbial pollution of
Cochin estuary is seafood industry, as the wastewater
from many factories, which is rich in organic matter,
end up in the estuary. Apart from two previous
studies5,6, no systematic studies have been carried out
on the microbial pollution of this important natural
water body during the last decade. The present study
has been taken up to study systematically the
pollution status of the Cochin estuary with special
reference to indicator bacteria such as faecal
streptococci and Escherichia coli for a period of one
year. Prevalence of diarrhegenic serotypes of E. coli
has been analysed by serotyping the strains. Seasonal
variation in the prevalence level of these organisms as
well as the correlation between the indicator and the
definite pathogen has also been worked out.
Materials and Methods
The water samples were collected monthly from
five different stations (Fig.1) along the Cochin estuary
during November 2001–October 2002. The stations
were selected based on the closeness to satellite
townships and waste input. Two of the stations
(Chittoor and Thevara) were fixed upstream, two in
the central part of the estuary namely Bolghatty and
Marine Science Jetty, and one at the barmouth. Water
samples were collected between 7 and 9 am in sterile
_
______________
*Present address:
Dr. A.A. Mohamed Hatha, Department of Biology, School of Pure
and Applied Sciences, The University of the South Pacific, Private
Mail Bag, Suva, Fiji, Phone: + 679 321 2550, Fax: +679 331
5601, E-mail: abdulla_m@usp.ac.fj, URL: www.usp.ac.fj/biology

HATHA et al.: E. coli IN COCHIN ESTUARY
239
plastic bottles (Tarson, India) one foot below the
surface to get a better representation of the water
column. Water samples were transported to the
laboratory in an icebox and subjected to
bacteriological examination within 4 hours of
collection. The samples were processed for microbial
parameters7 such as faecal coliform (FC), and faecal
streptococci (FS).
A three-tube MPN technique was used to estimate
faecal coliforms using E.C. broth (Hi-media) as the
medium and incubation at 44.5oC. Inocula from tubes
showing growth and gas production were streaked on
Eosine Methylene Blue (EMB) agar for the isolation
of E. coli and incubated at 37oC. Typical E. coli like
colonies were selected, restreaked to ensure purity
and confirmed by indole, methyl red, voges-proskauer
and citrate (IMViC) test. Similarly, MPN of faecal
streptococci were determined by inoculating azide
dextrose broth and incubation at 37oC. Inocula from
tubes showing growth were streaked on Pfizer
selective enterococcus agar and incubated at 37oC.
Typical colonies were isolated, purified and
characterised biochemically into different groups.
Confirmed E. coli cultures were serotyped at National
Salmonella and Escherichia Centre, Central Research
Institute, Kasauli, Himachal Pradesh (India).
Fig. 1⎯Map showing sampling stations.
Results and Discussion
The prevalence of faecal indicator bacteria such as
faecal coliforms and faecal streptococci at different
stations during the period November 2001 to October
2002 is represented in Table 1. The results revealed
that there were considerably high levels of FS and FC
at all stations. The values were higher than those
recorded near the mouth region of Vembanad lake5, of
which Cochin estuary is a part. The values were also
greater than those recorded in Mandovi and Zuary
estuarine complex of Goa8. The higher load is possible,
as the human population has grown considerably in the
last two decades and hence the waste generation.
Though sewage collection systems are there, many
illegal sewers are entering the estuary, especially from
market places. Also there is a practice of dumping
animal carcasses into the estuary at various points.
There are no effective measures to control such
activities, which can result in gross contamination of
the system, once the waste input goes beyond the
critical load of self-purifying capacity of the system.
The mean value of the faecal indicator bacteria for
the entire year revealed that station very close to the
Cochin City, station 3, had maximum FC load. The
load was minimum, though well above the prescribed
limits, at upstream station 1. This again indicates the
population pressure on infrastructure and resultant
increased waste discharge into the system. The mean
FC level for the year at station 5 showed that
considerable removal of FC is taking place as they
reach the Barmouth, which is almost 4 kilometers away
from the stations near to Cochin city and the salinity
varied between 5-20 ppt during the study period.
However, the mean values for the FS level showed an
interesting pattern. The highest mean load was
recorded at stations 3 and 5 indicating better survival of
these bacteria than FC at higher salinities and
chemically dynamic nature of the Barmouth region. FC
values vary considerably between these two stations
and the load was reduced considerably at Barmouth.
The mean FS levels were comparatively lower at the
upstream station 1 as in the case of FC.
The study period has been categorised into three
distinct seasons, such as premonsoon (February–
May), monsoon (June–September) and postmonsoon

INDIAN J. MAR. SCI. VOL. 33, No.3, SEPTEMBER 2004
240
Table 1⎯Prevalence of faecal coliforms and faecal streptococci at different stations during Nov. 2001 to Oct. 2002
Sta.
No. MPN value (per 100 ml) of FC and FS during the study period
Test
organism Nov.
2001 Dec. Jan.
2002 Feb. Mar. Apr. May Jun. Jul. Aug. Sep Oct.
1 FCa1100 1100 1100 1100 1100 1100 11000 110000 4600 9300 2300 46000
FSb1100 1100 1100 1100 1100 140 11000 110000 15000 9300 110000 46000
2 FC 1100 1100 1100 1100 1100 1100 4600 110000 15000 110000 4300 4300
FS 1100 1100 1100 1100 1100 70 11000 110000 110000 24000 110000 110000
3 FC 1100 240 1100 1100 1100 1100 11000 110000 110000 110000 21000 21000
FS 1100 1100 1100 1100 1100 930 11000 110000 110000 46000 110000 110000
4 FC 1100 120 1100 1100 1100 1100 11000 110000 9300 110000 900 900
FS 1100 1100 1100 1100 1100 4600 11000 110000 110000 24000 21000 15000
5 FC 1100 23 1100 1100 1100 1100 11000 110000 46000 9300 9300 15000
FS 1100 1100 1100 1100 1100 2100 11000 110000 110000 46000 110000 110000
aFaecal coliform, bFaecal Streptococci
Table 2⎯Seasonal variation of indicator bacteria at different stations during Nov. 2001 to Oct. 2002
Station no. Premonsoon Monsoon Postmonsoon
FCaFSbFC FS FC FS
1 3575 3335 14580 35360 1100 1100
2 1975 3317 28920 73000 1100 1100
3 3575 3532 54600 77400 813 1100
4 3575 4450 26420 36200 773 1100
5 3575 3825 18120 77400 741 1100
Premonsoon – Feb. to May; Monsoon – Jun. to Sep., Postmonsoon – Oct. to Jan.
aFaecal coliforms (Values are indicative of mean MPN values/100ml for the season)
bFaecal streptococci (Values are indicative of mean MPN values/100ml for the season)
Table 3⎯Different serotypes of Escherichia coli and different groups of faecal streptococci encountered in the Cochin estuary
during Nov. 2001 to Oct. 2002
E. coli serotypes
(n=81) % incidence E. coli serotypes
(n=81) % incidence Faecal streptococci
(n=72) % incidence
O1 4.9 O63 2.5 Streptococcus faecalis
Sub sp. Liquifaciens 65.3
O39 2.5 O19 1.2 Streptococcus bovis 6.7
O173 1.2 O51 2.5 Enterococcus sp. 10.7
O78 1.2 O139 2.5 Unidentified 17.3
O157** 3.7 O86 1.2
O8* 2.5 O116 1.2
O101 7.4 O150 1.2
O91 1.2 O32 1.2
O165 2.5 O135 1.2
O106 1.2 O102 1.2
O22 4.9 O15 1.2
O104 1.2 O29 1.2
O107 1.2 O9 3.7
O25 8.6 O88 2.5
O14 1.2 O80 1.2
O69 2.5 O131 1.2
O117 1.2 O66 1.2
O105 1.2 O30 1.2
O156 3.7 O20*** 1.2
O113 1.2 O2+1.2
O60 1.2 UT 3.7
O33 7.4
*Enterotoxigenic E. coli (ETEC). **Enterohemorrhagic E. coli (EHEC)
***Enteropathogenic E. coli (EPEC), +Uropathogenic E. coli (UPEC), UT – Untypable.

HATHA et al.: E. coli IN COCHIN ESTUARY
241
(October–January), which is a marked feature of the
weather in the study area. The mean value for FS and
FC during each season for each station has been
presented in Table. 2. The results indicated
substantially high level of indicator bacteria, both FS
and FC, at all stations during monsoon months. The
samples collected from stations 3 had the maximum of
both FS and FC, though only of FS high at station 5.
The high load during the monsoon months is
anticipated as the heavy monsoon showers bring in
heavy land run-off into the Cochin estuary, resulting in
increased levels of waste discharge into the system.
Also the inactivating effect of major self-purifying
factor such as sunlight9 is very much reduced during
the monsoon months as the sky is almost always
overcast.
Another important observation made during the
seasonal studies is the significant variation in the FS
and FC levels during the monsoon months, while there
was no significant variation between the mean
prevalence of these organisms during the premonsoon
and postmonsoon periods. FS levels were considerably
higher than FC at all stations during the monsoon
months, which is indicative of the huge land run off
into this receiving system. The high load of FS is most
likely to be from the faecal environment of other
domesticated animals, which reaches the system during
the monsoon months.
The faecal coliform bacteria were characterized
biochemically to see the prevalence of E. coli.
Escherichia coli was isolated consistently from all
stations. Strains of E. coli were serotyped at National
Salmonella and Escherichia Centre (Kasauli, Himachal
Pradesh, India) to see the diversity. The results (Table
3) revealed remarkable diversity of the E. coli strains in
the system. More than 40 different serotypes were
encountered which included potential pathogens such
as enterohemorrhagic E. coli (EHEC),
enteropathogenic E. coli (EPEC), enterotoxigenic E.
coli (ETEC) and uropathogenic E. coli (UPEC). The
EHEC levels were considerably higher and this is for
the first time the isolation of this emerging pathogen is
being reported from the Cochin estuary. The interesting
observation was that EHEC was isolated from the
station nearest to Cochin city, whereas none of the
other stations gave any positive isolation for this
organism during study period. This is suggestive of the
possibility of release of this organism through hospital
waste from many of the hospitals in and around Cochin
city. Though prevalence of faecal indicator bacteria
from Vembanad lake has been reported earlier,
serological characterization and existence of different
serotypes were not reported so far.
Faecal streptococci were also subjected to
biochemical characterization in order to group them
into different species. The results (Table 3) indicated
the predominance of Streptococcus faecalis sub sp.
liquefaciens, followed by enterococcus group and
Streptococcus bovis. Except for the monsoon months,
the FC levels were greater than FS indicating human
source of pollution of the study environment. This is
again reconfirmed by the diversity of the E. coli
serotypes in the system, which showed great diversity.
In conclusion, the results of the present study are
indicative of considerable microbial pollution in the
Cochin estuary. Emerging pathogens such as EHEC,
ETEC and EPEC are thriving in the system, which is
a potential danger to the valuable fish and shellfish
resources of this estuary as well as those who use it
for recreation. Coastal waters are being used for
several purposes such as domestic, industrial and for
aquaculture. The results revealed that monitoring of
faecal indicator bacteria and pathogens in coastal and
estuarine waters could be made mandatory for impact
assessment studies while these resources are used for
the above-mentioned purposes.
Acknowledgement
The present study has been carried out as a part of
the DST FAST TRACK project (SR/OY/LS-18/2001)
funded by Department of Science and Technology
(DST), Govt. of India. The financial assistance to
A.A.M. Hatha to carry out the project is thankfully
acknowledged.
References
1 Borrego, J. J & Figueras, M.J., Microbiological quality of
natural waters, Microbiologia, 13 (1997) 413-426.
2 Morinigo, M.A., Munoz, M.A., Cornax, R., Martinez-
Manzanares, E., & Borrego, J.J., Presence of indicators and
Salmonella in natural waters affected by outfall waste water
discharges, Water Sci. Technol., 25 (1992) 1-8.
3 Moe C L, Waterborne transmission of infectious agents, in:
Manual of environmental microbiology, edited by C.J. Hurst,
G.R. Knudsen, M.J. McInerney, L.D. Stetzenbach and M.V.
Walter (American Society for Microbiology, Washington,
D.C.) 1997, pp. 135 -162.
4 Dutka, B.J., Coliforms are an inadequate index of water
quality, J. Environ. Health., 36 (1973) 39-46.
5 Lakshmanaperumalsamy, P., Chandrasekharan, M., Bright
Singh, I. S. & Chandramohan. D., Microbial indicators and
pathogens at the mouth region of Vembanad lake, Bull. Dept.

INDIAN J. MAR. SCI. VOL. 33, No.3, SEPTEMBER 2004
242
Marine Sci. Uni. Cochin., 12 (1981)103-119.
6 Chandrika, V., Studies on bacterial indicator pathogen
Vibrio paraheamolyticus in Cochin backwaters, Ph.D thesis,
Cochin University of Science and Technology, India, 1983.
7 Anon., Standard methods for the examination of water and
waste water, 19th Ed. (American Public Health Association,
Washington, D.C.) 1995, pp. 61-83.
8 Row, A., Microbiological studies in the Mandovi-Zuari river
system, Indian J. Mar. Sci., 10 (1981) 189-191.
9 Abirosh Chandran & Hatha, A.A.M., Survival of Escherichia
coli in a tropical estuary, S. Pac. J. Nat. Sci., 21 (2003)
41-46.