Environ. Sci. Technol. 2010, 44, 6475–6480
Environmental Reservoirs for Enterotoxigenic Escherichia coli in South Asian Gangetic Riverine System GULSHAN SINGH, POORNIMA VAJPAYEE, SIYA RAM, AND RISHI SHANKER* Indian Institute of Toxicology Research (C.S.I.R.), Post Box No. 80, Mahatma Gandhi Marg Lucknow -226001, U.P., India
Received February 6, 2010. Revised manuscript received June 30, 2010. Accepted July 2, 2010.
Forecasting diarrheagenic E. coli contamination of aquatic resources to prevent outbreaks largely depends on rapid and accurate diagnostic testing in a few hours. Real-time PCR is widely used for quick culture-free quantitative enumeration of pathogenic bacteria in environmental samples. In this study, real-time PCR in molecular beacon format was used for detection and culture-free quantitative enumeration of enterotoxigenic Escherichia coli (ETEC) harboring LT1 gene in a sewage-impacted south Asian Gangetic riverine system. The quantitative budget for ETEC in surface water was observed to vary significantly (DMRT, p < 0.05) among the sites. Aquatic flora (Eichhornia crassipes, Potamogeton crispus, Potamogeton pectinatus, Ranunculus sceleratus, Polygonum glabrum, Pontederia cordata, Najas indica and strands of Spirogyra spp.) collected between sites 1 and 9 exhibited significant high levels of ETEC in comparison to their representatives collected from pristine area. The level of ETEC harboring LT1 gene observed in leafy vegetables cultivated along the banks was in the following order: mint leaves > coriander > spinach > methi leaves. The study suggests that the aquatic flora and cultivated leafy vegetables in the south Asian Gangetic riverine system are environmental reservoirs for enterotoxigenic Escherichia coli.
Introduction The worldwide contamination of surface and potable waters by diarrheagenic E. coli due to addition of untreated sewerage has been demonstrated (1, 2). High mortality and morbidity were found associated with diarrheal infections during waterborne outbreaks. In the developing world, surface water is used for irrigation and other domestic uses. The frequency of documented diarrheal outbreaks of illness associated with consumption of raw fruits, vegetables, and surface water has increased in recent years (3, 4). In several instances, foodborne illnesses have been traced to contaminated irrigation water or poor or unsanitary post-harvest practices (5). Potential pre-harvest contamination sources of vegetables include soil, manure, human, farm animal feces and irrigation water (6-9). All of these factors can influence both the composition of the indigenous microbial flora as well as the survival and growth of human pathogens on raw vegetables. * Corresponding author phone: 91+ 0522 -2613786/2614118/ 2627586, ext. 237; fax: 91+ 0522-2611547; e-mail: rishishanker1@ rediffmail.com. 10.1021/es1004208
2010 American Chemical Society
Published on Web 07/15/2010
Hence, irrigation water quality is of significant importance as it may be responsible for carrying microorganisms from the field to the fork (10, 11). Besides, aquatic flora growing in the river impacted by sewage discharge might also serve as environmental reservoirs for pathogenic bacteria (12, 13). Enterotoxigenic Escherichia coli (ETEC) is regarded as a major cause of E. coli mediated diarrhea worldwide in humans, affecting mainly children and travelers (14). In the developing world, an estimated 650 million cases of ETEC infections occur each year, resulting in ∼800,000 deaths, mostly in young children (15). ETEC secretes at least one of two types of enterotoxins (heat-labile, LT; and heat-stable, ST enterotoxins) encoded by LT1 and ST1 genes, respectively (14). The heat-labile enterotoxins of E. coli are oligomeric toxins classified into two major groups (LTI and LTII). LTI is expressed by E. coli strains that are pathogenic for both human and animals. The LT1 gene commonly present in strains associated with human illness has been frequently observed in ETEC recovered from surface waters of India and other south Asian countries contaminated by fecal wastes of human origin (4, 14, 16). The forecasting of diarrheagenic E. coli contamination in aquatic resources to prevent outbreaks largely depends on rapid, sensitive, and accurate diagnostic testing in a few hours. Real-time quantitative PCR (Q-PCR) is a current powerful highly sensitive technology which allows monitoring of the amplification of target gene in real time through fluorescence obviating post PCR handling. Recently, real-time PCR assays targeting LT1 gene based on different chemistries has been reported for quick detection of ETEC in water samples (16, 17). Aquatic vegetation and leafy vegetables cultivated in riverine systems could be important environmental reservoirs of ETEC. A few studies suggested that aquatic flora and leafy vegetables could serve as nonpoint sources of human bacterial pathogens (9, 12, 18). Nonpoint sources of fecal indicator bacteria (e.g., Escherichia coli) derived from environmental reservoirs in a variety of habitats may enhance the problem several fold (18). Aquatic flora and vegetables cultivated along the banks of the river receiving domestic effluents may be important reservoirs of ETEC in the riverine environment and possibly serve as nonpoint sources for ETEC. Hence, global surveillance of environmental reservoirs and identification of possible nonpoint sources of ETEC viz. aquatic flora, leafy vegetables, etc., is required to prevent water- and foodborne outbreaks caused by diarrheal diseases. Therefore, in this study, aquatic flora of a sewage-impacted south Asian river flowing through northern Gangetic plains and f leafy vegetables cultivated in the riverine system and irrigated by surface water of the selected river were screened for culturefree enumeration of ETEC using real-time PCR assay in molecular beacon format.
Materials and Methods Primers and Probe. For specific detection of ETEC harboring LT1 gene in surface water, aquatic flora and leafy vegetables molecular beacon (MB: 5′-CACGCCCGGGACTTCGACCTGAAATGTTGGCGTG-3′, position: 1062, Tm: 58.3 °C) and its corresponding primers (AF: 5′-GGCAGGCAAAAGAGAAATGG3′ AR: 5′-TTGGTCTCGGTCAGATATGTG-3′, position: 9961145, product size 150 bp) were adopted from Ram et al. (17). It has been reported that twenty strains of E.coli exhibiting LT1 gene were positive with adopted molecular beacon based real-time PCR assay and no amplification of the target gene was observed by Ram et al. (17) in E.coli strains reported to be negative for the target gene such as Vibrio cholerae and other bacterial strains lacking LT1 gene. VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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The MB based quantitative real-time PCR could detect 2 CFU/ mL (lowest detection limit) of the reference strain (E. coli MTCC 723 procured from Microbial Type Culture Collection at Institute of Microbial Technology (IMTECH), Chandigarh, India). The results reported by Ram et al. (17) were reproducible during the present study (data not shown). Culture-Free Quantification of ETEC in Riverine Environment. For culture-free detection and quantitative enumeration of ETEC in riverine environment, a sewageimpacted perennial river (Gomti) of northern India has been selected. Nine sites exhibiting distinct anthropogenic activities were selected in the vicinity of Lucknow city for the collection of surface water, aquatic flora and leafy vegetables cultivated on banks (Supporting Information S2, Figure S1). Three quadrants (0.5 m each) were set at each site and all the plants including algal mats were collected and placed in separate plastic bags. Three water samples (1 L each) from each quadrant at each site were also collected in sterilized bottles. Water (n ) 9), aquatic macrophytes (E. crassipes (Marts.) Solms., Potamogeton crispus L., Potamogeton pectinatus L., Ranunculus sceleratus L., Polygonum glabrum Willd., Pontederia cordata L., Najas indica (Willid.) Cham.: n ) 9 for each spp.) and strands of Spirogyra spp. (n ) 9) were also collected from a pristine environment receiving no fecal pollutant. Samples of leafy vegetables (Methi: Trigonella foenumgraecum L., Fabaceae; Spinach: Spinacia oleracea L., Amaranthaceae; Coriander: Coriandrum sativum L., Apiaceae; and Mint: Mentha arvensis L., Lamiaceae, n ) 10 for each vegetable) growing along the banks of the river Gomti at site 8 (Chandiamau) and irrigated by river water were also collected to elucidate the possibility of these vegetables as nonpoint sources of ETEC. Physico-chemical chatacteristics of the river Gomti on the day of sampling were determined at all the selected sites. Temperature (°C) of the river water was measured by thermometer. Electrical conductivity (µS cm-1) was measured by conductivity meter (Systronics 361, India). Total solids (TS), total suspended solids (TSS), total dissolved solids (TDS), phosphates (PO4), chlorides (Cl), and sulphates (SO4) were determined as per methods described in APHA (19). Total coliform and fecal coliform levels in surface water collected from each site were determined by most probable number (MPN) method as per APHA (19). Plant samples (roots/leaf/algal mat) collected from each site including the pristine site were processed for culturefree enumeration of ETEC. Bacteria adhering to plant samples (10 g) were released in 100 mL of phosphate-buffered saline through repeated wash by vigorous shaking (2 min at 220 rev min-1) on a refrigerated rotary shaker (INNOVA 4230, New Brunswick, NJ) and 45 s centrifugation at 2000 rpm (653g). Bacterial density (number of total coliforms and fecal coliforms) in aquatic flora was determined at each site using an aliquot of wash solution (phosphate buffered saline containing bacteria released from plants/algae) through MPN method as per APHA (19). Aliquots (100 mL) of phosphate buffered saline containing bacteria released from plants and 100 mL of each surface water sample collected from each site in the river Gomti were concentrated to 500 µL by repeated centrifugation at 18 000g for 10 min (4 °C). Finally, purified DNA template was prepared as per Ram et al. (17). Four leafy vegetables (Methi: Trigonella foenum-graecum L., Fabaceae, n ) 10; Spinach: Spinacia oleracea L., n ) 10, Amaranthaceae; Coriander: Coriandrum sativum L., n ) 10, Apiaceae; and Mint: Mentha arvensis L., Lamiaceae, n ) 10) cultivated along the banks at site 8 and irrigated by the river water were also investigated for bacterial contamination (FC and TC) using the same method as for the aquatic flora. 6476
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In the present study, real-time PCR assay for culture-free quantitative enumeration of ETEC in surface water, aquatic flora, and leafy vegetables using molecular beacon targeting LT1 gene was performed using iCycler (BIO-RAD, USA) realtime PCR instrument and QuantiTect Multiplex PCR No ROX kit (Qiagen, Germany) as per protocol described by Ram et al. (17). Briefly, the reaction mixture contained 2× QuantiTect Multiplex PCR No ROX Master Mix (25 µL), primers corresponding to probe (0.4 µM, each) for LT1 gene, probe (0.2 µM), and 5 µL of DNA template from each sample of surface water, aquatic plant/alga, and leafy vegetable in a final volume of 50 µL. The real-time PCR program for the target gene was as follows: initial denaturation at 95 °C for 3 min and then 45 cycles at 95 °C for 20 s, 55.8 °C for 30 s, and 72 °C for 30 s. The standard curve generated from serially 10-fold diluted culture of E. coli MTCC 723 (2 × 106 down to 2 × 10-1 CFU/ mL) was used for quantitative enumeration of ETEC in environmental samples. The sample was considered negative if the fluorescent signal did not increase within 45 cycles. Statistical Analyses. PCR amplification efficiencies and detection sensitivities among different experiments, slopes of the standard curves were calculated by performing a correlation and regression analysis through iCycle iQ RealTime Detection System Software Version 3.0A. Amplification efficiency (E) was estimated by using the slope of the standard curve and the formula E ) (10-1/slope) - 1. A reaction with theoretical 100% efficiency will generate a slope of -3.322. One way analysis of variance in randomized complete block design was used to compare the quantitative level of total coliforms, fecal coliforms, and ETEC in surface water at nine sites in river Gomti and different leafy vegetables. Further, Duncan’s multiple range test was performed to differentiate between the means (20). Student’s t test was used to compare the ETEC load in aquatic flora and flora collected from pristine site (21). A correlation coefficient (r) was calculated for water quality parameters to determine correlation between ETEC levels in water (20).
Results Physico-Chemical Characteristics of the River Gomti Water. Results reveal that the temperature, pH, and electrical conductivity of the surface water of the river Gomti at different sampling locations ranged 20.1-20.6 °C, 7.72-8.00, and 562.1-788.2 µS cm-1, respectively (Table 1). However, total solids, total suspended solids, total dissolved solids, dissoloved oxygen, biochemical oxygen demand, phosphates, sulphates, and chlorides ranged 260-460, 240-418, 20-42, 1.2-9.8, 5.3-45, 1.5-9.1, 70-210, and 10.4-40.3 mg/L, respectively (Table 1). Bacteriological Quality of Surface Water, Aquatic Flora, and Leafy Vegetables. Surface water collected from all the sampling sites in the river Gomti exhibit significantly (oneway ANOVA, p < 0.05) high level of the total coliforms and fecal coliforms (Table 2). In this study, total coliform and fecal coliform levels at different sampling locations varied significantly (DMRT, p < 0.05). It was observed that site 1 located upstream of the river Gomti exhibited the lowest number of total coliforms and fecal coliforms, whereas site 5 located in the middle of Lucknow city exhibited maximum bacterial contaminants (Table 2). The aquatic flora growing in the river Gomti possess high concentrations of the total coliforms and fecal coliforms (Table 3). Similarly, leafy vegetables irrigated by the water of the river Gomti were also contaminated by the total coliforms and fecal coliforms (Table 4). However, the level of the contamination varied significantly (DMRT, p < 0.05) in leafy vegetables investigated in this study (Table 4). Culture-Free Quantitative Enumeration of ETEC in Surface Water, Aquatic Flora, and Leafy Vegetables. It was observed that the surface water of the river Gomti exhibited
a Site 1: Ghaila bridge; site 2: upstream of Gaughat; site 3: Gaughat; site 4: Shaheed Smarak; site 5: Laxman mela ground; site 6: La Martinier College; site 7: Pipraghat; site 8: Chandiamau; site 9: Indira Jal Setu. Values [mean (n ) 9) ( SD] are in mgL-1 otherwise stated. Temp: temperature; EC: electrical conductivity; TS: total solids; TSS: total suspended solids; TDS: total dissoloved solids; DO: dissolved oxygen; BOD: biochemical oxygen demand; PO4: phosphate; SO4: sulphates; Cl: chloride.
20.2 ( 0.22 7.60 ( 0.20 775.2 ( 40 460.0 ( 15.60 418 ( 17.1 42.0 ( 2.10 2.4 ( 0.12 35 ( 1.35 7.0 ( 0.32 186.0 ( 7.46 40.3 ( 1.77 20.2 ( 0.36 7.75 ( 0.16 714.2 ( 17 300.0 ( 12.20 278 ( 11.0 22.0 ( 0.90 3.2 ( 0.15 30 ( 1.32 1.5 ( 0.06 210.0 ( 9.56 35.4 ( 1.58 20.5 ( 0.33 7.77 ( 0.18 713.0 ( 32 260.0 ( 10.30 242 ( 6.6 24.0 ( 1.10 2.8 ( 0.13 35 ( 1.51 2.7 ( 0.12 160.0 ( 6.13 32.6 ( 1.53 20.1 ( 0.20 7.72 ( 0.16 562.1 ( 15 310.0 ( 15.30 288 ( 11.5 22.0 ( 0.80 9.6 ( 0.32 5.6 ( 0.20 1.7 ( 0.09 78.0 ( 3.20 13.5 ( 0.58 20.2 ( 0.24 7.84 ( 0.21 622.3 ( 18 260.0 ( 8.00 240.0 ( 8.8 20.0 ( 0.60 9.4 ( 0.39 5.3 ( 0.25 1.5 ( 0.05 70.0 ( 2.80 10.4 ( 0.45 temp (°C) pH EC (µS cm-1) TS TDS TSS DO BOD PO4 SO4 Cl
20.5 ( 0.29 8.00 ( 0.22 582.1 ( 19 400.0 ( 17.80 370 ( 16. 9 30.0 ( 1.50 9.8 ( 0.45 6.7 ( 0.39 2.1 ( 0.11 100.0 ( 3.72 18.5 ( 0.87
20.6 ( 0.31 7.72 ( 0.25 695.0 ( 25 278.0 ( 11.30 253 ( 10.0 25 ( 0.90 1.2 ( 0.04 40 ( 1.88 5.3 ( 0.22 162.0 ( 7.11 25.5 ( 1.25
21.1 ( 0.27 7.75 ( 0.30 788.2 ( 27 308.0 ( 14.50 277 ( 12.8 31 ( 1.20 1.2 ( 0.05 45 ( 2.07 9.1 ( 0.42 168.0 ( 7.22 30.2 ( 1.31
20.3 ( 0.35 7.78 ( 0.31 707.1 ( 22 280.0 ( 9.80 260 ( 10.2 20.0 ( 0.70 2.2 ( 0.11 25 ( 1.20 2.2 ( 0.10 155.0 ( 7.14 34.5 ( 1.68
9 8 7 6 sampling site
5 4 3 2 1 parameters
TABLE 1. Physico-Chemical Characteristics of the River Gomti Water at Selected Sites on the Day of Samplinga
high level of ETEC exhibiting LT1 gene (Table 2). However, the quantitative budget for ETEC varied significantly (DMRT, p < 0.05) among the sampling locations (Table 2). Sites 5 and 7 located in the middle of Lucknow city exhibit no significant difference in ETEC level (DMRT, p > 0.05). Aquatic flora (E. crassipes, P. crispus, P. pectinatus, R. sceleratus, P. glabrum, P. cordata, and strands of Spirogyra spp. collected between sites 1 and 9 exhibited significantly (student’s t-test) high levels of ETEC in comparison to their representatives collected from pristine area (Table 3). E. Crassipes exhibited the highest level of ETEC followed by Polygonum glabrum, Spirogyra spp., R. sceleratus, P. cordata, P. crispus, P. pectinatus, and Najas indica (Table 3). ETEC were absent in the aforementioned species collected from the pristine environment. The concentration of ETEC observed in surface water of the river Gomti exhibit significant positive correlation with the levels of total solids, total dissolved solids, and total suspended solids in water (Supporting Information, Table S1). Similarly, a significant negative correlation between water pH and ETEC level was recorded in the present study (Supporting Information, Table S1). However, positive correlation between other parameters (phosphates, sulphates, chlorides, and biochemical oxygen demand) and ETEC levels detected in water samples by the culture-free method was insignificant (p > 0.10 at df ) 7). ETEC concentrations at different sites exhibit insignificant (p > 0.10 at df ) 7) correlation with total and fecal coliforms level in water of the river Gomti. It was observed that the four leafy vegetables exhibit significantly (DMRT p < 0.05) different loads of ETEC (Table 4). Mint leaves exhibit maximum number of ETEC harboring LT1 gene followed by coriander, spinach, and methi leaves (Table 4).
Discussion The river Ganga and its tributaries, the major source of water supply in the Gangetic planes of northern India are overburdened as a result of rapid urbanization and population growth. Gangetic riverine systems are impacted by sewage discharge from nearby unauthorized residential colonies (7). The river Gomti, a tributary of the river Ganga is a major source of domestic water supply to a population of about 3.5 million and reported to receive 450 million liters per day of untreated domestic wastewater in Lucknow city (22). The organic pollution level in an aquatic source is usually measured and expressed in terms of BOD and declined DO contents. Therefore, low DO, high BOD, and EC values in surface water between sites 4 and 9 of the river Gomti indicate deterioration in water quality due to addition of organicmatter-rich domestic pollutants. Fecal bacteria recorded at all the sampling stations exceeded the standards set by regulatory authorities for surface water reservoirs to be used for drinking and recreational purposes (23, 24). Earlier it has been reported that wastewater from poultry and dairy farms, hospital wastes, and animal and human carcasses contributes pollution to the river Gomti (22). The Gomti river exhibits EC values between 562.1 and 788.2 µS cm-1. According to Olsen’s classification a river exhibiting EC values between 250 and 1000 µS cm-1 is classified eutrophic (25). In the present study, EC values at all the selected sites of the river Gomti exceed this range. Hence, the river Gomti is eutrophicated and supports the growth of aquatic macrophytes and algal flora. Aquatic flora may be an important and significant nonpoint source of indicator bacteria in environmental waters. Aquatic macrophytes form strands in polluted eutrophicated water bodies impacted by urban sewage. Certain reports demonstrate occurrence and perhaps even growth of indicator bacteria in aquatic macrophytes (18, 26, 27). Indicator bacteria adhering to aquatic macroVOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Quantitative Enumeration of Total Coliforms, Fecal Coliforms, and ETEC Exhibiting LT1 Gene in Surface Water Collected from a Sewage-Impacted River sampling sitea
total coliformb (MPN/100 mL)
fecal coliformb (MPN/100 mL)
2.2 × 105 ( 88 × 102 1.6 × 108 ( 48 × 104 d 4.3 × 107 ( 172 × 102 b 1.6 × 1010 ( 8 × 108 a 1.4 × 1012 ( 7 × 1010 f 5.4 × 106 ( 216 × 103 e 9.2 × 106 ( 276 × 103 g 2.4 × 106 ( 96 × 103 h 1.6 × 106 ( 48 × 103
1 2 3 4 5 6 7 8 9
1.7 × 105 ( 68000 3.7 × 107 ( 148000 e 2.3 × 106 ( 92 × 103 a 1.3 × 109 ( 455 × 105 c 1.3 × 107 ( 52 × 104 h 7.8 × 105 ( 3120 d 5.4 × 106 ( 216 × 103 f 3.2 × 105 ( 12 × 103 g 1.8 × 105 ( 9 × 104
ETEC/100 mLb 255 × 102 ( 11.53 1108 × 102 ( 27200 c 898 × 103 ( 40318 b 1008 × 103 ( 42336 d 284 × 103 ( 1100 g 104 × 102 ( 312 d 269 × 103 ( 9415 a 1480 × 103 ( 74000 h 30770 ( 1230
i
i
f
c
b
e
a Site 1: Ghaila bridge; site 2: upstream of Gaughat; site 3: Gaughat; site 4: Shaheed Smarak; site 5: Laxman mela ground; site 6: La Martinier College; site 7: Pipraghat; site 8: Chandiamau; site 9: Indira Jal Setu. b Mean (n ) 9) ( SD. One-way ANOVA, p < 0.05. Identical superscripts denote no significant (p > 0.05) difference between the means according to Duncan’s multiple range test (DMRT).
TABLE 3. Quantitative Enumeration of Total Coliforms, Fecal Coliforms, and ETEC Exhibiting LT1 Gene in Aquatic Flora and Surrounding Surface Water in a Sewage-Impacted River a
quantitative enumeration of microbial density in aquatic florab
sampling site 1 2 3
4 5 6 7 8 9
name of the plant
c
P. crispus P. pectinatus E. crassipes R. sceleratus Pontederia cordata E. crassipes P. crispus P. pectinatus E. crassipes E. crassipes P. crispus E. crassipes R. esculantus N. indica Polygonum glabrum Spirogyra spp.
total coliformd (MPN/100 g)
fecal coliformd (MPN/100 g)
ETEC/gd
16 × 106 ( 48 × 104 16 × 106 ( 32 × 103 13 × 106 ( 11 × 103 16 × 109 ( 608 × 106 16 × 106 ( 64 × 104 12 × 108 ( 36 × 104 16 × 106 ( 32 × 104 24 × 107 ( 72 × 105 11 × 107 ( 22 × 105 23 × 107 ( 92 × 105 47 × 105 ( 141 × 103 23 × 105 ( 46 × 103 16 × 107 ( 64 × 105 16 × 106 ( 48 × 104 22 × 105 ( 88 × 103 16 × 106 ( 32 × 104
9.3 × 104 ( 3720 24 × 104 ( 7200 9 × 106 ( 360000 48 × 104 ( 9600 39 × 104 ( 11700 4 × 105 ( 8000 16 × 105 ( 48000 21 × 104 ( 8400 9 × 107 ( 1800000 16 × 106 ( 320000 7.8 × 105 ( 31200 11 × 104 ( 2200 24 × 106 ( 720000 35 × 104 ( 10500 11 × 105 ( 22000 16 × 104 ( 6400
666 ( 29 300 ( 11 0040 ( 815 612 ( 24 798 ( 33 150 × 103 ( 43215 745 ( 24 304 ( 10 96730 ( 3860 625210 ( 25002 757 ( 24 62148 ( 1276 812 ( 37 191 ( 8 4950 ( 199 1458 ( 65
a
Site 1: Ghaila bridge; site 2: upstream of Gaughat; site 3: Gaughat; site 4: Shaheed Smarak; site 5: Laxman mela ground; site 6: La Martinier College; site 7: Pipraghat; site 8: Chandiamau; site 9: Indira Jal Setu. b Mean (n ) 9) ( SD. c No ETEC was recorded in each plant species collected from pristine environment. d Student’s t test: p < 0.05; sterile Milli-Q water served as negative control.
TABLE 4. Quantitative Enumeration of Total Coliforms, Fecal Coliforms, and ETEC Exhibiting LT1 Gene in Leafy Vegetables Irrigated by the Surface Water of a Sewage-Impacted River name of the leafy vegetable Methi (Trigonella foenum-graecum L., Fabaceae) Spinach (Spinacia oleracea, Amaranthaceae) Coriander (Coriandrum sativum, Apiaceae) Mint (Mentha arvensis, Lamiaceae)
total coliform (MPN/100 g)
fecal coliform (MPN/100 g)
25 × 106 ( 5 × 105 38 × 106 ( 114 × 104 b 94 × 106 ( 235 × 104 a 350 × 108 ( 112 × 107
12 × 104 ( 2880 21 × 104 ( 5460 b 58 × 105 ( 185600 a 240 × 105 ( 48000
d c
ETEC/ga 109 ( 4.73 260 ( 10.50 b 361 ( 16.52 a 12597 ( 54.05
d
d
c
c
a Mean (n ) 10) ( SD. One-way ANOVA, p < 0.05. Different superscripts denote significant differences in ETEC load between the leafy vegetables according to Duncan’s multiple range test (DMRT). The botanical names and family of each leafy vegetable has been given inside parentheses. ETEC was absent in samples collected from pristine site.
phytes could be released frequently into streamwater due to wave action along the banks. Further, plants containing indicator bacteria may detach from their natural strands naturally or by anthropogenic activities, could float several miles downstream along the course of the river, and get transported to distant destinations. Diarrheal disease caused by ETEC is the main cause of death in infants and small children in developing countries (28, 29). Despite the potential public health threat from water- and foodborne ETEC, regulatory authorities in the developing world rely exclusively on “indicators” of fecal pollution (e.g., fecal coliform bacteria or generic E. coli) for determination of water quality. This 6478
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fails to provide a clue on presence of pathogenic forms of E. coli viz., ETEC present in the contaminated source. Therefore, the need for surveillance of water reservoirs and potential nonpoint sources for pathogenic E. coli including ETEC in the developing world has been realized. We report here occurrence of ETEC in aquatic macrophytes and an alga Spirogyra spp. growing in a river contaminated by sewage using a culture-free approach consisting of highly accurate and sensitive molecular beacon based quantitative real-time PCR assays targeting LT1 gene. Prevalence of ETEC in aquatic flora indicates that aquatic plants including algae may serve as reservoirs of ETEC in natural water resources.
The possibility of long-term survival of ETEC in aquatic flora also has important ecological and public health implications. Warm humid conditions during summer months (JuneSeptember), availability of nutrients due to the addition of sewage, decomposing roots, and aging leaves are potential factors that support the hypothesis that pathotypes of E. coli could actively grow in a tropical riverine environment (4). Occurrence of ETEC in aquatic flora supports the view that aquatic plants could serve as secondary habitat for indicator bacteria due to the availability of nutrients from the decomposing tissue. Further, these plants could also act as nonpoint sources of ETEC and other pathogenic bacteria to pristine distant locations. Besides, the enterotoxigenic E. coli levels in impacted waters may remain high due to frequent inputs from aquatic flora by wave action. This could potentially influence the water quality in the affected areas. It has been reported that E. coli could survive, thrive, and multiply in green alga mats for long periods (26, 27) The environmental and potential public health implications caused by survival and persistence of E. coli and Enterococci in dried Cladophora mats has been demonstrated (27). The observations made in the present study concur with previous observations that populations of indicator bacteria can naturally occur and may even grow in habitats such as soil, water, and plants (26, 27, 30). The survival and maintenance of E. coli populations in tropical and subtropical fresh waters because of the combination of high temperatures and the availability of nutrients has been reported (31). The riverine environment in India is conducive to microbial growth because of warm humid conditions with cyclic periods of wet and dry weather and eutrophication (7). A significant positive correlation between TS, TSS, and TDS contents and ETEC level reveals that increase in solids favor the growth of enterotoxigenic E. coli. It has been reported that organic nutrients adsorbed at surfaces of particles facilitate bacterial growth in aquatic environment (32, 33). It has been observed that other water quality parameters including levels of total coliforms and fecal coliforms in surface water exhibit no significant correlation with the ETEC levels in water. The number of pathogenic bacteria in surface water is influenced by a variety of factors (pathogen occurrence in livestock and wildlife, differences in agricultural practices, wastewater composition, and differences in survival or transport characteristics between the pathogens and indicator organisms) that are difficult to quantify in a riverine environment (34). In this study, we have also enumerated ETEC for the first time from the leafy vegetables (another potential nonpoint source) cultivated along the banks of the river Gomti. The present study demonstrates that four leafy vegetables cultivated along the banks of a perennial north Indian river contaminated by ETEC exhibit high levels of ETEC. The irrigation water for these fields was solely derived from the sewage-impacted river. These contaminated vegetables may serve as an important nonpoint source of the ETEC and may be responsible for carrying pathogens from the field to the fork. Several other studies have also found that raw vegetables irrigated with wastewater or surface water impacted by sewage exhibit enteric bacterial pathogens and are considered to be a public health hazard (8, 11, 35, 36). The present study concludes that addition of untreated sewage in a surface water resource poses high risk of waterand foodborne diarrheal diseases to a large population. Gangetic riverine systems exhibit environmental reservoirs of ETEC in the form of aquatic flora and leafy vegetables cultivated and irrigated by sewage-impacted water. Further, aquatic flora growing in the polluted river concentrates ETEC and possibly other bacterial pathogens due to continuous wave action and may serve as a nonpoint source of ETEC. Besides, surface water of these water resources exhibit poor
quality as irrigation water due to presence of ETEC. The present study suggests that the agricultural produce in fields irrigated by the river water impacted by raw sewage discharge may contain high levels of ETEC and possibly other bacterial pathogens. Therefore, these ETEC contaminated vegetables may also serve as nonpoint sources of ETEC together with other bacterial pathogens.
Acknowledgments Authors P.V. and G.S. contributed equally to this work. This work was supported by CSIR Network Project NWP-17. The financial assistance to G.S. (P.A.), S.R. (SRF), and P.V. (WOSB) from CSIR and DST, Government of India, New-Delhi, respectively, is acknowledged. We are grateful to Dr. Richa Mishra, U. P. Pollution Control Board, Lucknow for the analysis of phosphate and sulphate contents in river water. We declare that we have no competing financial interests. IITR manuscript no. 2746.
Supporting Information Available A map depicting sampling locations and description of sampling sites (Figure S1, S2); correlation coefficients calculated to find correlation between water quality parameters and ETEC levels in water (Table S1). This information is available free of charge via the Internet at http:// pubs.acs.org/.
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