Cadmium tolerance and antibiotic resistance in Escherichia coli isolated from waste stabilization ponds
Sova Patra, T K Das*, C Avila, V Cabello, F Castillo, D Sarkar (Paria), Susmita Lahiri (Ganguly) & B B Jana**
International Centre for Ecological Engineering and
*Department of Biochemistry & Biophysics, University of Kalyani, Kalyani 741 235, India Received 23 March 2009; revised 1 February 2012
The incidence pattern of cadmium tolerance and antibiotics resistance by Escherichia coli was examined periodically from the samples of water, sludge and intestine of fish raised in waste stabilization ponds in a sewage treatment plant.
Samples of water and sludge were collected from all the selected ponds and were monitored for total counts of fecal coliform (FC), total coliform (TC) and the population of Escherichia coli, which was also obtained from the intestine of fishes. Total counts of both FC and TC as well as counts of E. coli were markedly reduced from the facultative pond to the last maturation pond. Tolerance limit to cadmium by E. coli tended to decline as the distance of the sewage effluent from the source increased; the effective lethal concentration of cadmium ranged from 0.1 mM in split chamber to 0.05 mM in first maturation pond. E. coli isolated from water, sludge and fish gut were sensitive to seven out of ten antibiotics tested. It appears that holistic functions mediated through the mutualistic growth of micro algae and heterotrophic bacteria in the waste stabilization ponds were responsible for the promotion of water quality and significant reduction of coliform along the sewage effluent gradient.
Keywords: Antibiotic resistance, Cadmium tolerance, Escherichia coli, Waste stabilization pond
Much concern has been generated about the scarcity of quality water especially in the water-scarce developing nations. This leads to the question of rational use of wastewater resource and promotion of integrated water resource management. As a consequence, multiple uses of wastewater have been greatly emphasized in the water scarce and economically less developed nations due to lack of adequate infrastructure for conventional sewage treatment.
Municipal wastewater is an excellent media for a large variety of heterotrophic microbial community involved in biogeochemical cycle and disease causative pathogens. While the biogeochemical cycling bacteria are beneficial and primarily responsible for the decomposition of organic matter, the pathogenic bacteria, on the other hand, pose a threat to public health via food chain. In fact, waste stabilization ponds in the form of man - made wetlands have long been used for reducing microbial
load by the way of rapid die off of most pathogenic forms or anaerobic bacteria, and rapid degradation of organic matter upon exposure to aerobic conditions via mineralizing bacteria1-6.
Waste stabilization ponds have become increasingly important because of the solar energy driven eco- friendly biological process with concerted and coordinated activities of algae-microbes-vegetation- animal association that promote degradation of organic matter and achieve standard water quality criteria before discharging to natural habitats.
Consisting of two subsystems, wastewater-fed aquaculture is an important link to close the loop between the nutrient and biological production functions. Fish raised in the wastewater fed aquaculture system are exposed to pathogenic bacteria, hazardous chemicals, pharmaceutical drugs, antibiotics, heavy metals, etc. Some of the recalcitrant compounds are subject to biomagnification in the food web that begins with microorganisms7. It has been reported the incidence of human health risk with E. coli contamination in paddy fields irrigated with reclaimed wastewater8.
——————
**Correspondent author
Telephone: 091-33-2582 6323 (R), 091-33-2580-9212(O) Fax: 033-2582 8282
E-mail: [email protected]; [email protected]
Bacteria can develop a variety of cellular mechanisms for acquiring resistance against antibiotics through genetic mechanisms9. As antibiotics accumulate in the environment and even persist for up to a year10, this could enhance the resistance of bacteria to antibiotics or drugs. Resistant bacteria has been reported from raw sewage and water11, more than 90% (432) of the 480 strains of Escherichia coli, Pseudomonas sp. and Staphylococcus sp. collected from raw sewage and river Tigris have been reported to be antibiotic resistant12.
Escherichia coli, a gram-negative facultative anaerobic rod (Enterobacteriaceae) normally occurs in the intestine of all animals13. Most strains of E. coli are harmless, but some strain (e.g. Escherichia coli 0157:H7), which serves as indicator of fecal contamination may cause severe illness in human being. As a consequence, there is public health concern about the safety of the wastewater fed ecosystem in terms of E. coli sensitivity to some antibiotics, cadmium tolerance as well as fish health raised in the sewage-fed wetlands. It is reported that antibiotic resistance genes against ampicillin, streptomycin and tetracycline are known to be transferable to other bacteria14, indigenous flora and fish15.
Kalyani sewage treatment plant is fed by municipal wastes that contain various pharmaceutical drug residues discharged from household as well as effluents of some industries producing batteries, dye etc. Therefore, cadmium and some antibiotic residues are common toxicants of sewage fed water9. In view of the public health concern about the safety of fish raised in wastewater fed ponds and ecosystem health and lack of information thereof, it is of particular interest to assess the eco-health of the wastewater-fed ponds and the nature of bacterial isolates particularly Escherichia coli from the intestine of fish raised in wastewater-fed ponds. Though some studies have reported the ecological characteristics and the potentials of biogeochemical cycling bacteria occurring in the wastewater fed aquaculture ponds in the tropical Asian countries3,5,16-19 little is known about the environmental and health risk of fish raised in wastewater-fed systems. This base data is essential for promotion of water conservation through multiple uses of wastewater in irrigation of agricultural crops, aquaponics and in aquaculture. The purpose of the present study is to determine the cadmium tolerance and antibiotic resistance of Escherichia coli isolated from water, sludge and fish raised in various stages of sewage treatment farm. Eight wetland ponds were
used for biological treatment of wastewater along the sewage effluent gradient. Fish grown in the last four maturation ponds were used for tertiary treatment of domestic sewage.
Materials and Methods
The farm site—A sewage-fed fish farm with the facilities of three step ecological treatment in eight wetland ponds was selected for the study. The sewage treatment plant (Kalyani, West Bengal, India) is connected by an underground sewer system, fed mainly by domestic sewage of the township and occasionally by the effluents discharged from some industries producing battery, dye, pharmaceutical drugs, etc. Discharge of pharmaceutical drugs including antibiotics from residential areas of the township is very common and indeed, sewage may become a source of cadmium and antibiotic contamination.
The average rate of sewage discharge is 17 cubic meters/day. The characteristics of sewage water entering the anaerobic pond were as follows: pH, 6.9–
7.2; dissolved oxygen – nil; chemical oxygen demand, 390–720 mg/l; ammonium-N, 52.0–65.0 mg/l;
orthophosphate, 6.0–30.0 mg/l.
From the split chamber 11 cubic meters/day of sewage is passed into conventional treatment plant and then discharged into rivers; the remaining 6 cubic meters/day is treated biologically using waste stabilization ponds and finally discharged into river Ganges.
Ecological treatment in the present wastewater fed farm has been designed to achieve through three- step treatment in series: first step anaerobic ponds achieve a high volumetric rate of organic load removal, in the second stage biological treatment or green treatment is done in the secondary facultative ponds and the tertiary treatment is done in the maturation ponds.
The present sewage fed farm consisted of two anaerobic ponds (26 m × 52 m × 2.5 m), two facultative ponds (64 m × 150 m v 1.5 m) and four maturation ponds (52 m × 156 m × 1.0 m) located in a series. The sewage effluents travel a distance of 400 m from the source of the anaerobic to the outlet point in a zigzag way before being discharged into the natural ecosystem, the river Ganges. The average flow rate of wastewater is 6 millionlitre/day. The average retention time of water in these wetlands was about 7 days, with a mean depth of 1.5 m. This resulted in achieving the standard water quality at the outlet point
for discharge into the natural habitats. The last four maturation ponds or stocking ponds were used for rearing of Indian carp, rohu (Labeo rohita) and tilapia (Oreochromis mossambicus) by local fisher folk without following any specific management protocols.
The wastewater system as well as fish culture practice in the maturation ponds have been described in a earlier communication9. Fish culture was performed using batch cultures for raising the fishes in the growing ponds with periodical harvesting after a grow out period of 4-5 months. Therefore, the fishes were exposed to the contaminants of the wastewater- fed ponds for a limited period of 4-5 months before being harvested. In order to promote decontamination of fishes through depuration, the harvested fishes were kept in clean water ponds for about two weeks prior to sale for human consumption.
Collection of samples—For enumeration of E. coli tolerant to cadmium and resistant to selected antibiotics, six replicate samples of water and sludge (surface layer, 0 - 3 cm depth) were collected aseptically from selected sites (split chamber, anaerobic pond, facultative pond, maturation ponds – 1, and 4) during May 2005 to March 2006, at monthly intervals. Ten live specimens of rohu (Labeo rohia, 60–190 g) and tilapia (Oreochromis mossambicus, 50–150 g) were procured from the test ponds every month, and were placed immediately in plastic bags kept on ice while transported to the laboratory for isolation of E. coli from the intestine of test fish separately.
Preparation of intestine samples—Once in the laboratory, the fishes were externally washed with 70% alcohol and killed by pithing in the brain. The fishes were dissected and 1 g of intestine was taken from each fish aseptically, weighed and homogenized in a glass homogenizer and then transferred to a sterilized vial containing 100 mL sterile (121 °C, 15 min) 0.85 % NaCl prepared in deionized water.
The intestine of each individual fish was used as a separate sample.
The fish were collected from the ponds and were used in the experiment in accordance with national and institutional guidelines for the protection of human subjects and animal welfare.
Enumeration—All the routine procedures described by Atlas20 were followed for culture and enumeration of Escherichia coli. Aliquots of 10–6, 10–7 and 10–9 dilutions were prepared from fish intestine, water and sludge samples respectively by ten fold dilutions.
The suspension of surface sludge (3 cm) was prepared by mixing 1 g of wet sludge in 99 mL sterile distilled water and then samples were used. Each of the six replicate samples of water, sludge and fish intestine was plated in quadruplicate (n=6×4). The specific culture medium (MacConkey Agar) described by Atlas20 was used for the growth of E. coli. The composition of the medium was as follows: agar:
15 g, peptone: 20 g, lactose: 10 g, NaCl: 5 g, Bile salt:
5 g , Neutral red: 0.075 g, pH: 7.4 ± 0.2 at 25 °C, distilled water: 1000 mL.
Conventional spread plate technique under aerobic conditions was used to enumerate the counts of total colony forming unit (cfu) of E. coli at 37 oC after 48 h of incubation. The colonies developed from four plates and six replicate samples (n=6 × 4) were obtained and their mean counts were used in the present study.
Most Probable Number (MPN) technique21 was used to detect both fecal coliform (FC) and total coliform (TC). For total coliform, Lauryl tryptose broth was used as a test media for presumptive test, Brilliant green lactose bile broth for the confirmatory test and McConkey agar plates for the completed test22.
Loss of tolerance to cadmium—The sensitivity of E. coli to different concentrations of cadmium was tested in vitro using the samples of water, sludge and intestine of test fish. A stock solution of 1 mM of CdCl2 was prepared to achieve the desired level of cadmium chloride concentration ranging from 0.05 to 0.9 mM. One ml dilutions of water, sludge or intestine content of fish samples were spread onto the agar plates containing specific recommended culture medium for E. coli with the addition of CdCl2 in the medium in quadruplicate. A set of control plates with CdCl2 added to the medium was also plated in quadruplicate. The plates were incubated at 37 oC for 2 days23 and the overall mean number of colonies per 24 plates (n=6×4) was determined.
Loss of resistance to antibiotics—The antibiotic sensitivity test was performed by the isolates of E.
coli obtained from the samples of water, sludge and the intestine of fish procured from selected ponds of the sewage-fed fish farm using the disc diffusion method24. The commercial antibiotics discs developed by Hi Media Private Limited, India (netilmicin sulphate: 30 µg; colistin: 10 µg; tobramicin: 10 µg;
polymyxin-B: 300 units; amikacin: 30 µg; tetracycline:
10 µg; gentamicin: 10 µg; ticarcillin: 7.5 µg;
imipenem: 10 µg; ciprofloxacin: 5 µg ) were used in the present study.
The antibiotic discs were placed on nutrient agar plates previously seeded with 18 h broth culture of E. coli. The plates were incubated at 37 °C for 48 h, after which zones of inhibition were measured and interpreted accordingly25. Six replicate samples of water, sludge or fish gut collected every time from test ponds was tested in quadruplicate and their mean results were used in the present study. The patterns of resistance of the bacterial isolates to the antibiotics were constructed as per instruction for susceptibility test provided by Hi Media, Private Limited, India.
Analysis of water quality—Parallel with the microbiological samples for enumeration of E. coli, samples of water were also collected from different sites for determination of water quality parameters (temperature, pH, free CO2, total alkalinity, dissolved oxygen, chemical oxygen demand, phosphate-P, ammonium-N, nitrate-N) following the standard
methods21. The mean values of water quality parameters for different dates of observation were used in the present study.
Statistical analysis—The collected data were evaluated using appropriate statistics. A two way analysis of variance (ANOVA) followed by Fisher’s Least Significant Differences (LSD) tests were applied to find the differences in the mean counts of bacteria among the selected sites of the farm as well as in different months during the period of study.
Analysis of variance was carried out using ponds as whole plot and months as sub-plots26. The level of significance was accepted at P < 0.05.
Results
Water, sludge and fish intestine—Site-wise, the colony forming units of E. coli isolated from samples of water tended to reduce (Table 1) by 6-fold from split chamber (2.62 ± 6.77 × 109 / mL) to the last maturation pond (4.2 ± 1.25 × 108/mL). The counts of E. coli were distinctly higher (F4,20 = 720.92;
Table 1—Colony forming units of Escherichia coli occurring in water, sludge and in gut of fish raised in different maturation ponds in a wastewater-fed aquaculture system.
[Values are mean±SE]
Split chamber
Anaerobic pond Facultative pond Maturation pond–1 Maturation pond–4
Fish gut Fish gut
Months
Water
×107/ml Water
×107/ml Sludge
×109/g Water
×107/ml Sludge
×109/g
Fish gut Tilapia
×106/g
Water
×107/ml Sludge
×109/g
Tilapia
× 106/g Rohu
×106/g
Water
×107/ml Sludge
×109/g Tilapia
×106/g Rohu
×106/g May,
05
- - - - - - 94
±1.41 71
±1.25
- 104
±0.47 42
±1.25 36
±1.41
- 76
±1.25 June,
05
- - - - - - 100
±0.94 77
±1.25
- 113
±1.25 50
±0.47 40
±1.63
- 76
±1.25 July,
05
- - - - - - 108
±2.49 84
±0.94
- 95
±0.72 53
±1.25 42
±1.25
- 82
±1.25 Aug,
05
- - - - - - 100
±0.94 108
±1.24
- 98
±0.47 55
±0.82 43
±0.47
- 84
±1.25 Sept.,
05
- - - - - - 103
±0.94 88
±1.63
- 110
±0.94 50
±2.50 39
±0.47
- 72
±0.94 Oct.,
05
230
±2.35 192
±0.94 162
±0.94 134
±2.62 98
±1.41 211
±3.86 99
±2.87 58
±1.41 122
±0.94
- 49
±1.25 32
±1.41 108
±0.47 - Nov.,
05
232
±2.16 191
±1.19 164
±0.47 137
±5.80 98
±0.94 213
±0.82 98
±2.05 62
±1.89 137
±1.25
- 51
±2.62 34
±0.94 96
±1.25 - Dec.,
05
231
±2.16 193
±0.94 163
±2.36 131
±2.50 95
±1.98 213
±0.47 94
±1.41 51
±1.66 128
±2.49
- 47
±0.82 30
±0.47 102
±0.82 - Jan.,
06
232
±5.26 183
±6.28 188
±7.92 143
±3.09 104
±2.84 217
±3.93 100
±1.44 82
±4.28 134
±5.44
- 52
±2.88 41
±3.81 107
±3.60 - Feb.,
06
255
±2.68 192
±8.30 215
±2.13 156
±1.66 118
±1.18 236
±2.13 113
±2.60 98
±1.44 153
±2.62
- 66
±2.37 57
±1.89 127
±3.77 - Mar,
06
262
±6.77 186
±4.12 216
±2.88 163
±2.13 122
±2.60 245
±1.89 118
±4.64 105
±3.09 165
±3.31
- 75
±2.94 64
±0.98 145
±1.60 -
P < 0.05) in the inlet source than in the outlet of sewage effluent. There was considerable variations in the counts of E. coli in different months of observation (F5,20 = 10.048; P < 0.05), being significantly higher in the month of February or March.
The counts of Escherichia coli (0.30 to 2.16 × 1011/g) in sludge were more than double than that occurred in water samples (Table 1). Site-wise, the counts tended to reduce from the source to the outlet of sewage effluent (F3,15 = 109.224; P < 0.05). The counts of E. coli were also significantly higher (F5,15 =4.389;
P < 0.05) in May compared to February or March.
E. coli isolated from the intestine of tilapia (9.6 ± 1.25–16.5 ± 3.31 × 107/g) or rohu (7.2±0.94–1.3±1.25
× 107/g) was significantly less in numbers (F2,10 = 212.63.830; P < 0.05) when the fishes were procured from last maturation pond compared to the facultative pond or first maturation pond. Count differences in different months were significant (F5,10 = 214.0.6696;
P < 0.05).
E. coli occurring in the intestine of tilapia were 35–46% higher in counts than that occurred in rohu (Table 1). This clearly reflected the difference in the feeding habits of two fishes examined; tilapia was a predominantly omnivore consuming detritus of the pond bottom along with greater proportion of bacteria in the diet, while the rohu was a column feeder mainly subsisting on plankton in the water column.
Differences in the feeding habits of two selected fishes were well known27. It is reportedthat the fish tilapia harbored at least 8 g -ve bacteria (Aeromonas hydrophila, A. veronii, Burkholderia cepacia, Chromobacterium violaceum, Citrobacter freundii, Escherichia coli, Flavimonous oryzihabitants and Plesiomonas shigelloides) in the gastrointestinal tract28.
Loss of tolerance to cadmium in E. coli over sewage effluent—E. coli isolated from different selected sites showed clear growth in vitro containing low concentrations of cadmium in the medium (0.05 mM/mL), but failed to grow when the cadmium level in the medium increased and remained at 3 mM (Table 2).
The maximal cadmium concentration at which E. coli failed to grow, tended to decrease as the distance of the effluent from the sewage source increased. The isolated gut E. coli tolerant to cadmium also tended to reduce in counts when the fish was procured from the last maturation pond compared to the facultative pond closer to sewage influent source (Table 2). At the outlet (maturation pond - 4), the effective concentration of cadmium was 0.05 mM and 0.09 mM for E. coli .
Loss of resistance to antibiotics in E. coli over sewage effluent— E. coli isolated from water, sludge and gut of fish raised in facultative pond as well as in all maturation ponds were sensitive to all the ten (netilmicin sulphate, ciprofloxacin, amikacin, gentamicin, tetracyclin, colistin, imipenem, tobramicin, ticarcillin and polymyxin-B) antibiotics tested, whereas those were isolated from the split chamber and anaerobic pond were sensitive to seven antibiotics (netilmicin sulphate, ciprofloxacin, amikacin, gentamicin, tobramicin, ticarcillin and polymyxin-B) and were resistance to imipenem or intermediate to colistin (Table 3).
Fecal coliform (FC) and Total coliform (TC) load—FC counts exhibited a distinct reduction by two orders of magnitude (6.92×106–2.52×104 MPN/
100 mL)from the inlet of the facultative to the last maturation pond. Similar was the trend with TC (7.82×107–1.36×105 MPN/100 mL) (Table 4). The
Table 2—Tolerance level of cadmium (as CdCl2) in Escherichia coli isolated from water, sludge and gut of fish raised in different maturation ponds of the wastewater- fed aquaculture system
Split chamber
Anaerobic pond Facultative pond Maturation pond–1 Maturation pond–4
Fish gut
×106/g
Fish gut
×106/g Medium
containing Cd2+(mM)
Water × 107/ml
Water
×107/ml Sludge
×109/g Water
×107/ml Sludge
×109/g
Tilapia (gut)
× 106/g Water
×107/ml Sludge
×109/g
Tilapia Rohu
Water
×107/ml Sludge
× 109/g
Tilapia Rohu
0.05 + + + + + + + + + + - - - -
0.06 + + + + + + - - + -
0.07 + + + - - - -
0.08 + + +
0.09 + - -
0.1 +
0.2 -
overall reduction was over 99 % in both the cases.
The spatial differences in counts along the sewage effluent gradient were significant (P < 0.05).
Water quality—There was gradual increase in pH of water from split chamber (6.9) to the final outlet pond (8.0). The concentration of free carbon dioxide ranged from 25–132 mg/l during the period of study.
An anaerobic condition was prevailed from split
chamber and anaerobic pond, whereas an aerobic condition with adequate amount of dissolved oxygen (5.03–12.10 mg/l) prevailed in the facultative and last maturation pond. As expected, the amount of COD was maximum in the split chamber (1370 mg/l) and minimum in the last maturation pond (22–150 mg/l).
The level of phosphate also tended to reduce from the source (6.0–30.0 mg/l) to the last maturation
Table 3—Summary of antibiotic sensitivity test for Escherichia coli isolated from water, sludge and the gut of fish raised in different maturation ponds in a sewage fed system. (S: Sensitive; R: Resistance; I: Intermediate)
Antibiotics used in bacteria
Split chamber
Anaerobic pond Facultative pond Maturation pond-1 Maturation pond -4
Water Water Sludge Water Sludge Fish gut (Oreochromis mossambicus
Water Sludge Fish gut (Oreochromis
mossambicus
Water Sludge Fish gut (Oreochromis
mossambicus
Ciprofloxacin S S S S S S S S S S S S
Amikacin S S S S S S S S S S S S
Gentamicin S S S S S S S S S S S S
Netilmicin sulphate
S S S S S S S S S S S S
Tobramicin S S S S S S S S S S S S
Ticarcillin S S S S S S S S S S S S
Polymixin-B S S S S S S S S S S S S
Tetracyclin I S S S S S S S S S S S
Colistin I I I S S S S S S S S S
Imipenem R R R S S S S S S S S S
Table 4—Reclamation efficiency of waste stabilization pond in terms of selected water and sludge quality and some biological parameters.
Physico-chemical parameters Anaerobic pond Facultative pond First maturation pond Last maturation pond
pH 6.9 - 7.2 7.2 - 7.6 6.9 – 7.7 7.4 – 8.0
Dissolved oxygen (mg l-1) 0 - 1.5 6.0 - 12.1 5.3 – 9.8 5.0 – 9.8
Chemical oxygen demand (mg l-1) 390 - 720 137 - 364 94 - 280 22 – 150
Ammonium-N (mg l-1) 52.0 – 65.0 1.3 – 10.2 0.94 – 2.2 0.31 – 0.77
Nitrate-N (mg l-1) - 0.6 – 7.4 0.14 – 0.25 0.1 – 0.17
Orthophosphate (mg l-1) 6.0 – 30.0 0.1 – 0.9 0.05 – 0.4 0.02 – 0.24
Water Cd (mg l-1) 0 – 0.018 0 – 0.018 - 0 – 0.007
Sludge Cd (mg l-1) 0.0017 – 0.007 0.0012 – 0.004 0.0009 – 0.004 0.0012 – 0.004
Gross Primary productivity (g C m-2 d-1) - 5.6 – 67.5 0.68 – 21.12 0.90 – 19.04 No. of Total Phytoplankton (×104 m-3) - 1571 - 37284 629 - 17497 448 - 7720
No. of Total zooplankton (×104 m-3) - 304 - 5340 54 -5075 34 - 514
No. of total benthic invertebrates (m-2) - 1975 - 9456 2027 - 5478 732 - 1772
Fish yield (kg y-1) - - 2196 - 2981 3646 – 3999
Total coliform (TC) 1.10×107 –1.68×108 7.00×105–3.00×107 7.00×104–1.60×107 3.30×104–2.80×105 Faecal coliform (FC) 1.6×106–1.6×107 2.7×105–9.0×106 5.0×104–1.6×105 1.4×104–5.0×104
pond (0.02–0.24 mg/l). The value of ammonium-N was maximum in split chamber (52–65 mg/l) and minimum in the last maturation pond (0.31–
0.77 mg/l). The nitrate-N was also higher in the facultative pond (0.6–7.4 mg/l) compared to the last maturation pond (0.1–0.17 mg/l). Thus, the treatment efficiency increased with improvement in water quality over the sewage effluent gradient (Table 4).
Discussion
Significant reduction in the counts of FC and TC from the source of wastewater to the outlet of the waste stabilization ponds21 was attributed to the holistic functions of the wetland ecosystem contributed by the integrated coordinated activities of biotic complex as well as by the physical treatment and precipitation of the sewage effluent. In the present waste stabilization pond employed, there was greater abundance of blue green algae in the facultative pond and some species of green algae in the maturation ponds19. According to WHO6, wastewater treatment in waste stabilization ponds is green treatment achieved by the mutualistic growth of microalgae and heterotrophic bacteria resulting in highly aerobic condition due to photosynthetic activities of the intense microalgal bloom. As a consequence, the coliform load of the influent water underwent significant reduction. It is reported that production of photosynthetic oxygen during day time may preclude the growth of obligate anaerobes, and exhaustion of dissolved oxygen during night hours may alter the self purification process of the sewage treatment and kill the obligatory aerobic organisms7.
It is well known that bacteria can develop a variety of cellular mechanisms for acquiring resistance against antibiotics, including genetic mechanisms29. In the present study, E. coli isolated from the gut of two fishes (tilapia and rohu) did not develop resistance against any of the ten antibiotics tested.
Likewise, the Pseudomonas sp isolated from the intestine of the tilapia did not have resistance to any of the ten antibiotics tested9. This shows that the fish reared in the maturation ponds were perhaps free from health risk for human consumption after depuration of fish in clean water for two weeks and pretreatment for cooking. Absence of E. coli in the muscle of fish raised in some sewage fed ponds of West Bengal has also been reported30,31.
Loss of resistance to antibiotics and loss of tolerance to cadmium over sewage effluent gradient
seem to be related. Since the genes responsible for both these properties of antibiotic resistance and cadmium tolerance, the genes apparently reside on plasmid DNA. The possibility of loss of plasmid DNA by the bacterium over the gradient could be accounted for these characteristic feature of E. coli growing in sewage fed ponds.
Because the estimated threshold concentration of cadmium, at which inhibition of growth of these bacteria occurred, tended to reduce as the distance from the sewage source increased, this suggests that the promotion of wastewater occurred through ecological treatment by the way of sedimentation, chemical precipitation and chelation with organic loading that caused substantial reduction of cadmium in the water phase along the sewage effluent. Absence of E. coli resistant to all the ten antibiotics tested suggested that the fish were in good health. The achieved water quality at the outlet remained nearly same of the high microbiological standards set for wastewater fish pond water32,6. In the present study, improvement in water quality and treatment performance (Table 4) was clearly reflected in the enhanced fish yield (73 %) in the last maturation pond compared to the first maturation pond or facultative pond.
It becomes apparent that the three step ecological treatments in waste stabilization ponds were conducive not only for the promotion of water quality, but also for the production of safe fish for human consumption. Such low cost eco-technology using waste stabilization ponds appears to be most appropriate in the tropical economically less developed countries where there is great demand for fish protein to combat malnutrition among the poor and need for integrated multiple use of wastewater for water conservation.
Acknowledgement
Thanks are due to CSIR, New Delhi for financial assistance and Dr. R. D. Banerjee for suggestions.
References
1 Indian Council of Agricultural Research (ICAR), Handbook of fisheries and aquaculture (ICAR, New Delhi) 2006, 755.
2 Gerardi M H, Wastewater Bacteia. 1st ed. (Wiley-Interscience, Hoboken, New Jersey) 2006, 272.
3 Jana B B, Sewage-fed aquaculture; the Calcutta model, Ecol Eng, 11 (1998) 73.
4 Shuval H I, Adin A, Fattal B, Rawitz E & Yekutiel P, Wastewater irrigation in developing countries: health effects and technical solutions. Technical Paper No. 51. (World Bank, Washington DC.) 1986,
5 Edwards P, Wastewater-fed aquaculture: state- of- the- art, in: Waste recycling and resource management in the developing world-ecological engineering approach, edited by B B.Jana, R D Banerjee, B Gutersteam and J Heeb (University of Kalyani, India and International Ecological Engineering Society, Switzerland) 2000, 37.
6 WHO (World Health Organization), Guidelines for the safe use of wastewater, excreta and grey water. Vol 3:
Wastewater and excreta use in aquaculture. (World Health Organization, WHO Press, Geneva, Switzerland) 2006, 166.
7 Atlas R M & Bartha R, Microbial ecology. Fundamentals and applications, 4th edn. (Pearson Education, Delhi) 2005, 704.
8 Youn-Joo A, Chun Y, Kwang –Wook J & Jong-Hwa H, Estimating the microbial risk of E. coli in reclaimed wastewater irrigation on paddy field, Environ Monitor Assessment, 129 (2007) 53.
9 Patra S, Das T K, Ghosh S C, Sarkar D & Jana B B, Cadmium tolerance and antibiotic resistance of Pseudomonas sp. Isolated from water, sludge and fish raised in wastewater- fed tropical ponds, Indian J. Exp Biol, 48 (2010) 383.
10 Zuccato E, Calamar D, Natangelo M & Fanelli R, Presence of therapeutic drugs in the environment, Lancet, 355 (2000) 1789.
11 Malik A & Ahmad M, Incidence of drug and metal resistance in E. coli strains from sewage water and soil, Chem. Environ.
Res., 3 (1994) 3.
12 Al-Jebouri M M, A note on antibiotic resistance in the bacterial flora of raw sewage and sewage- polluted river Tigris in Mosul, Iraq, J Appl Bacteriol, 58 (1985) 401.
13 Food processing: Understanding and controlling E. coli contamination (The Hartford Loss Control Department.
USA.) TIPS Series S 190.007.
14 Walia S K, Kaiser A, Prakash M & Chaundhry G R, Self- transmissible antibiotic resistance to ampicillin, streptomycin and tetracycline found in Escherichia coli isolated from contaminated drinking water, J. Environ. Sci. Health A Toxic/Hazard. Subst. Environ. Eng., 39 (2004) 651.
15 Sanyal S, Basu A & Banerjee S, Drug resistance profiles of coliforms from sewage exposed fish, World J. Fish Mar Sci., 3 (2011) 275.
16 Sarkar U K, Bhowmik M L & Pandey B K, Effects of domestic sewage on fish, aquatic ecosystem and public health implications, in Waste recycling and resource management in the developing world-ecological engineering approach, edited by B B. Jana, R D Banerjee, B Gutersteam and J Heeb (University of Kalyani, India and International Ecological Engineering Society, Switzerland) 2000, 79.
17 Datta A K, Roy A K & Saha P K, Comparative evaluation of sewage-fed and feed based aquaculture, in Waste recycling and resource management in the developing world- ecological engineering approach, edited by B B. Jana, R D Banerjee, B Gutersteam and J Heeb (University of Kalyani, India and International Ecological Engineering Society, Switzerland) 2000, 97.
18 Ganguly S, Some biogeochemical cycling bacteria and their activities as indices of reclamation strategies in domestic
sewage-fed fish ponds, Ph.D. Thesis, University of Kalyani, West Bengal, India, 2002.
19 Sarkar D, Ecosystem analysis in domestic wastewater-fed aquaculture farm: Assessment of primary and secondary productivity, Ph.D. thesis, University of Kalyani, West Bengal, India, 2007, 132.
20 Atlas R M, Handbook of media for environmental microbiology (CRC Press. Inc.) 1995, 540.
21 APHA (American Public Health Association), AWWA (American Water Work Association), WEF (Water Environment Federation), Standard methods for the examination of water and wastewater. 20th ed, (American Public Health Association, Washington DC). 1998, 1038.
22 Avila C, Cabello V, & Castillo F, Pathogen and nutrient removal in wastewater stabilization ponds in West Bengal, India, Masters Dissertation, Halmstad University, Sweden, 2006.
23 Nwachukwu S C U, Enhanced rehabilitation of tropical aquatic environment polluted with crude petroleum using Candida utilis,. J Environ Biol, 21 (2000) 241.
24 Bauer A W, Kirby W M M, Sherris J C & Turck M, Antibiotic susceptibility testing by a standard single disc diffusion method. Am J Clinical Path, 45 (1966) 493.
25 Chortyk T O, Severson R F, Cutler H C & Siesson V A , Antibiotic activities of sugar esters isolated from selected Nicotiana sp., Biosci Biotechnol Bioch, 57(1993) 1355.
26 Gomez K A & Gomez A A, Statistical procedures for agricultural research, 2nd edn. (Wiley Interscience publication, New York) 2006, 704.
27 Jhingran V.G. Fish and fisheries of India. (Hindustan Publishing Corporation (India). Delhi) 1988, 666.
28 Molinari L M, Scoari D D, Pedroso R B, Bittencourt N D, Nakamura C V, Nakamura T U, Abreu Filhoi B A D & Filho B P D, Bacterial microflora in the gastro-intestinal tract of Nile tilapia, Oreochromis niloticus cultured in a semi- intensive system, Acta Scientiarum, 25 (2003) 67.
29 Adhikari R P, Kalpana K C, Shears P, & Sharma A P.
Antibiotic resistance conferred by conjugative plasmid in Escherichia coli isolated from community ponds of Kathmandu. Valley, J. Health Popul. Nutr. 18 (2000) 57.
30 Bhowmik M L, Chakrabarti P P, & Chattopadhyay A, Microflora present in sewage-fed systems and possibilities of their transmission, in Waste recycling and resource management in the developing world-ecological engineering approach, edited by B B. Jana, R D Banerjee, B Gutersteam and J Heeb (University of Kalyani, India and International Ecological Engineering Society, Switzerland) 2000, 71.
31 Sanyal S, Basu A & Banerjee S, Occurrence and antibiotic susceptibility among coliform bacteria isolated from sewage exposed fish, Rec. Res Sci Tech, 2 (2010) 42.
32 Blumenthal U J, Peasey A, Ruiz-Palacios G & Mara D D, Guidelines for wastewater reuse in agriculture and aquaculture. Task No. 68, Part-1,(Well. London School of Hygiene & Tropical Medicine, UK,WEDC, Loughborough University, UK) 2000, 34.