Occurrence of Shiga-toxigenic and enteropathogenic E. coli contaminations and assessment of water quality in the southern coastal belt in Sri Lanka Open Access

Sri Lanka is a tropical country located in the Indian Ocean with a larger coastal belt around the country. The coastal belt of Sri Lanka plays a significant role in the national economy as a hotspot for tourism and fishing-related anthropogenic activities. The decline of coastal water quality in Sri Lanka has become a severe drawback owing to accelerated population growth, fast urbanization, and industrialization (Weerasekara et al. 2015; Pal & Tiwari 2023). The land-based waste discharges into coastal waters adversely impact the quality of coastal waters (Alam et al. 2021; Manage et al. 2022; Wijerathna et al. 2023a). In this study, we mainly focused on observing the occurrence of Shiga-toxigenic and enterohaemorrhagic Escherichia coli contaminations in the southern coastal belt of Sri Lanka. Additionally, physicochemical water quality parameters were measured to evaluate the quality of sampled coastal water, as nutrients such as nitrates and phosphates play a critical role in microorganisms' growth, survival, and proliferation. Nutrient limitations also hinder bacterial growth in aquatic ecosystems (Gregory et al. 2017; Wijerathna et al. 2024a, 2024b).

Changes in the pH, conductivity, dissolved oxygen (DO), and nutrients such as N-nitrate, N-nitrite, N-ammonia, total phosphate, total nitrogen, and chemical oxygen demand (COD) lead ocean waters to polluted levels (Fahmy 2003; Wijerathna et al. 2023b). The contamination of coastal waters with faecal matter renders the water microbiologically unsafe, leading to gastroenteritis and other non-gastrointestinal diseases (Hamilton 2016).

Coliforms are a broad group of bacteria in the family Enterobacteriaceae that are Gram-negative and facultative anaerobic (Some et al. 2021). Among the faecal coliforms, E. coli is a prevalent indicator of faecal pollution (Pendergraph et al. 2021; Liyanage et al. 2024). E. coli strains are an ordinary and nearly universal component of the lower gut microbiota in humans and animals. Although E. coli is generally considered a commensal organism, it can also act as a pathogen associated with diarrhoeal diseases and extra-intestinal infections (Chaudhuri & Henderson 2012). Disease-causing E. coli strains are categorized as diarrhoeagenic E. coli (DEC) and further classified into six distinct pathotypes based on specific virulence characteristics, namely, enteropathogenic E. coli (EPEC), Shiga-toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC) (Ori et al. 2019). Enterohaemorrhagic Escherichia coli (EHEC) is considered a subset of STEC (Newell & La Ragione 2018), and both EHEC and STEC are well-known foodborne pathogens that are widely distributed (Mahagamage et al. 2019; Azimirad et al. 2021) and associated with exposures in sewage-contaminated coastal bathing waters (Leonard et al. 2018).

The E. coli O157 is a subset of STEC, EHEC and EPEC that can produce Shiga toxins as STEC and form attaching and effacing lesions on epithelial cells as EPEC and EHEC (O'Sullivan et al. 2007). The E. coli O157 serotype is regarded as the most virulent serotype of E. coli, which potentially causes serious lethal diseases such as haemorrhagic colitis (HC), haemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and even death, in addition to frequent diarrhoea and food poisoning (Ekici & Dümen 2019).

The E. coli O157 strain has been detected using the effacement gene, eae, and Shiga-toxin-producing genes such as stx1 and stx2 (Saxena et al. 2015; Bandara et al. 2024). E. coli O157 virulence factors can produce Shiga-toxin 1 and Shiga-toxin 2 or both, which are encoded by the stx1 and stx2 genes of the Shiga-toxin(stx) family and which damage the Vero cells (Saxena et al. 2015). The distinct subtypes of both stx1 and stx2 are identified and particularly in stx1 includes stx1a, stx1c, and stx1d subtypes whereas stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g subtypes are included in stx2 (Saxena et al. 2015). Moreover, Shiga toxins are mainly responsible for the cytopathic effects on vascular endothelial cells of the colon, kidneys, central nervous system, and other organs (Biernbaum & Kudva 2022). Additionally, the subtypes Stx2a, Stx2c, and Stx2d in the Shiga-toxin family have been associated with distinct clinical manifestations in human infections, including bloody diarrhoea and HUS (Brandal et al. 2015).

Studies show that the proliferation of E. coli O157 in wastewater holding sites is declining, and the strain is disappearing in less than 4 weeks. Still, when introduced into the natural environment, E. coli O157 can survive for extended periods (Gelting et al. 2015).

In history, severe E. coli O157 outbreaks occurred in different countries like the United States, Japan, Canada, the UK, and Africa and research detected E. coli O157 strain in food and water (Effler et al. 2001; Pennington 2010; Heiman et al. 2015; Verhaegen et al. 2016). Only a few studies have been conducted globally on identifying E. coli O157 strains in coastal environments (Richards et al. 2016; Swinscoe et al. 2018; Kotsiri et al. 2019; Barberi et al. 2020).

In literature, E. coli O157 in shellfish from French coastal environments was evaluated by detecting stx genes (Gourmelon et al. 2006). Further in that study, the homogenized whole shellfish and digestive gland tissue enrichments were used, and the strains were identified through a PCR protocol and colony DNA hybridization with a stx-specific gene probe. Also, a triplex polymerase chain reaction (PCR) was used to detect stx1, stx2, and eae genes in E. coli strains (Bennani et al. 2011). Moreover, the E. coli O157 outbreak associated with spinach and provided compelling evidence supporting the role of water as a transmission medium for the E. coli O157 strain was examined (Gelting et al. 2011).

Additionally, several epidemiological and molecular genotyping studies have identified the correlation between HUS, HC diseases, and diarrheal diseases with O157 strains with stx (1/2) genes (Pradel et al. 2008; Kargar & Homayoon 2015).

Only a limited number of studies have been completed in Sri Lanka on this subject. In the literature, a study was conducted to identify pathogenic E. coli strains, especially STEC, in chicken meat and edible poultry organs in Sri Lanka (Ranasinghe et al. 2022). Also, in another study carried out in Sri Lanka, Shiga-like toxin-producing E. coli was found in 28% of calves with diarrhoea but in only 4% of healthy calves, similar to previous research for STEC in cattle farms (Suthienkul et al. 1990). Surprisingly, there is a gap in the literature regarding identifying E. coli O157 strains in Sri Lankan coastal waters, particularly concerning tourism and recreation.

No available literature exists on identifying E. coli O157 strain in Sri Lankan coastal waters and seafood. Therefore, to the authors' knowledge, no data are available on disease cases due to the contamination of coastal waters and seafood by pathogenic E. coli strains. But in India, Antony et al. (2021), Kamala et al. (2022), and Chakraborty (2018) conducted studies on pathogenic E. coli strains in coastal waters and seafood. In Antony et al. (2021), five pathogenic E. coli serotypes were identified, and enterohaemorrhagic E. coli O157 strains were isolated from shellfish and sediment samples on the southwest coast of India. Therefore, conducting more comprehensive research on the prevalence of these pathogenic E. coli strains in Sri Lankan coastal waters and seafood, particularly shellfish harvested from these waters, is essential, as recreational and fishing activities are commonly associated in Sri Lankan coastal waters.

The Sarakkuwa to Mirissa coastal belt was selected for the present study, being popular coastal locations for tourism and recreation, including Colombo, the country's commercial capital city (Manage et al. 2022). Stx1, stx2, and eae genes were used as virulence genes to detect the E. coli O157 strain, including Shiga-toxigenic and enteropathogenic E. coli, which is the causative factor for severe gastroenteritis. Additionally, the water quality parameters of the same water sample were measured to ensure the coastal water quality status in aid of tourism and recreational purposes.

Water quality parameters, water temperature, pH, DO, and electrical conductivity (EC) were measured using a thermometer (Immersion, Philip Harris, and England), pH meter (330 I/ Set, WTW Co., Weilheim, Germany), DO meter (HQD portable multimeter -HACH – HQ 40D) and a conductivity meter (340A-Set 1), respectively, at the site itself. Chemical parameters such as nitrate (as NO), nitrite (as NO), ammonia (as NH₃), total nitrogen, total phosphorous and COD were measured in the laboratory using standard methods for the examination of water and wastewater published by the American Public Health Association (APHA). The closed reflux method was used to determine COD (APHA 2012).

The membrane filtering method determined the total coliform (TC) and E. coli count per 0.1 dm³ of the water samples. Samples were filtered through 0.45-μm filter papers (Whatman Cat No: 7001 0004, D-47 mm), and then the filter papers were kept on the MLGA (Membrane Lactose Glucuronide Agar) plates and incubated at 37 °C for 18 ± 2 h (SLSI, 2013).

The green color colonies obtained from MLGA agar plates were again streaked on Eosin Methylene Blue (EMB) agar plates, and the colonies developed on EMB agar were further identified as coliforms or faecal coliforms (E. coli) using cultural characteristics, morphology, and biochemical tests. For faecal coliforms, colonies with a green metallic sheen were Gram-stained, and the IMVIC test was carried out to identify the colony as E. coli. The colony-forming units (CFUs) of E. coli per 100 mL water were determined from confirmed colony isolates (Section 2.4).

Five colonies were randomly selected from each EMB plate. For plates with ≤5 colonies, all the isolates were selected for further purification. All the selected colonies were subjected to confirmatory tests, which included Gram staining and IMVic biochemical tests to confirm those colonies as presumptive E. coli. Following incubation, a single colony was selected and streaked further on EMB agar to obtain pure isolates, which were then used to determine virulence potentials.

A single selected colony of bacteria was cultured overnight in 5 mL of Luria–Bertani (LB) liquid medium at 28 °C and 100 rpm (Liyanage & Manage 2017). The bacterial cells were then harvested by centrifugation at 12,000 rpm for 2 min and subjected to DNA extraction, starting from the cell lysis step described by Kim et al. (2010). Finally, the DNA sample was stored at −20 °C until the PCR was done.

The extracted DNA was used as the template DNA in different real-time PCR assays to identify genes associated with virulence in three E. coli pathotypes. The various genes tested and the associated pathotypes are shown in Table 1.

Positive controls used in the PCR assays included the O157 strain, including E. coli bacteria. All controls were obtained from the Medical Research Institute (MRI) in Sri Lanka. Reaction mixtures without DNA used as negative controls were also included in each PCR assay. All PCR assays were performed in a thermal cycler (Bioer 865031, USA).

Eae, stx1, and stx2 genes were selected to identify E. coli O157 strain and forward and reverse primers for each gene were obtained. To prepare stock solutions with 100 μM concentrations, all primers were first dissolved in a specified volume of PCR water. Primer working concentrations were 10 μM. For each PCR, the master mixture was prepared as follows (Table 2).

Exact volumes were added to an Eppendorf tube, respectively, and the final volume of the master mix for a single DNA sample was 20 μL. With 20 μL of the master mix, 5 μL of DNA sample was added to a PCR tube. It was mixed using a vortex.

Each 20 μL reaction was performed using desalted primers (Millipore Sigma, USA) and with the following modifications: 0.5 μM final concentration for all primers, MgCl₂ supplemented up to a final concentration of 0.5 mM, and 5 U of GoTaq DNA polymerase (Abia et al. 2017). PCR assays were performed in a thermal cycler and subjected to a PCR cycle, as mentioned in Table 3.

The principal component analysis (PCA) test was conducted using R software to find the correlation between the E. coli count and the water quality parameters in each location. The selected 22 locations were divided into five districts: Gampaha, Colombo, Kalutara, Galle and Matara.

The measured results for both physical water quality parameters, including pH, temperature (°C), DO (mg/L), EC (mS/cm) and chemical water quality parameters that include N-Nitrate (mg/L), N-Nitrite (mg/L), N-Ammonia (mg/L), total nitrogen (mg/L), total phosphate (mg/L) and COD (mg/L) are detailed in Table 4.

Table 4

Physical water quality parameters for Sarakkuwa to Mirissa coastal belt

Location .	pH .	Temperature (°C) .	DO (mg/L) .	(mg/L) .	(mg/L) .	N-NH3 (mg/L) .	TN (mg/L) .	TP (mg/L) .	COD (mg/L) .
Sarakkuwa	7.76 ± 0.05	29.73 ± 0.11	7.56 ± 0.00	1.61 ± 0.39	0.07 ± 0.00	0.51 ± 0.05	2.19 ± 0.01	0.80 ± 0.50	624 ± 0.05
Dikovita	7.91 ± 0.06	29.23 ± 0.05	7.72 ± 0.05	0.81 ± 0.15	0.03 ± 0.00	0.59 ± 0.08	1.43 ± 0.01	0.92 ± 0.10	673 ± 0.05
Pamunugama	8.00 ± 0.01	29.83 ± 0.15	7.69 ± 0.01	1.24 ± 0.14	0.02 ± 0.00	0.19 ± 0.03	1.45 ± 0.05	0.47 ± 0.06	598 ± 0.06
Galle face	7.47 ± 0.39	26.33 ± 0.49	7.64 ± 0.25	0.37 ± 0.04	0.00 ± 0.00	0.27 ± 0.16	0.64 ± 0.06	3.17 ± 0.14	653 ± 0.06
Wellawatta	7.36 ± 0.26	26.53 ± 0.23	6.87 ± 0.42	3.48 ± 0.01	0.64 ± 0.17	0.19 ± 0.09	4.31 ± 0.09	0.21 ± 1.13	661 ± 0.05
Dehiwala	7.49 ± 0.45	26.93 ± 0.57	7.40 ± 0.13	0.39 ± 0.04	0.00 ± 0.00	0.11 ± 0.07	0.50 ± 0.03	0.94 ± 0.04	679 ± 0.06
Mt.Lavinia	7.79 ± 0.33	27.00 ± 0.56	7.28 ± 0.15	0.31 ± 0.02	0.00 ± 0.00	0.25 ± 0.10	0.56 ± 0.09	0.07 ± 0.03	608 ± 0.08
Ratmalana	7.67 ± 0.43	27.47 ± 0.15	7.31 ± 0.08	0.30 ± 0.09	0.06 ± 0.06	0.17 ± 0.10	0.53 ± 0.08	0.88 ± 0.63	652 ± 0.05
Moratuwa	8.10 ± 0.03	27.13 ± 0.25	7.38 ± 0.04	0.37 ± 0.05	0.04 ± 0.03	0.44 ± 0.19	0.85 ± 0.09	0.72 ± 0.16	693 ± 0.05
Panadura	8.01 ± 0.10	28.20 ± 0.10	7.10 ± 0.72	0.41 ± 0.03	0.03 ± 0.03	0.04 ± 0.07	0.48 ± 0.04	0.70 ± 0.31	611 ± 0.05
Wadduwa	8.12 ± 0.05	28.00 ± 0.10	7.50 ± 0.04	0.44 ± 0.01	0.01 ± 0.01	0.07 ± 0.09	0.52 ± 0.03	0.77 ± 0.30	584 ± 0.05
Kalutara	8.05 ± 0.05	28.43 ± 0.15	7.51 ± 0.05	0.38 ± 0.03	0.00 ± 0.00	0.13 ± 0.05	0.51 ± 0.02	0.72 ± 0.57	603 ± 0.02
Benthota	8.05 ± 0.04	26.07 ± 0.06	7.78 ± 0.04	2.45 ± 0.06	0.04 ± 0.02	0.20 ± 0.11	2.69 ± 0.06	0.06 ± 0.04	681 ± 0.02
Ambalangoda	7.90 ± 0.19	26.63 ± 0.15	7.12 ± 0.97	0.44 ± 0.20	0.04 ± 0.00	0.30 ± 0.06	0.78 ± 0.08	0.40 ± 0.09	619 ± 0.05
Hikkaduwa	7.80 ± 0.05	26.62 ± 0.28	7.62 ± 0.22	1.07 ± 0.13	0.06 ± 0.01	0.35 ± 0.19	1.48 ± 0.07	0.35 ± 0.11	667 ± 0.02
Rathgama	8.00 ± 0.05	27.27 ± 0.31	7.77 ± 0.02	1.88 ± 0.22	0.05 ± 0.00	0.89 ± 0.23	2.82 ± 0.03	0.46 ± 0.17	657 ± 0.05
Ginthota	7.96 ± 0.10	29.27 ± 0.29	7.65 ± 0.10	1.44 ± 0.16	0.12 ± 0.08	1.39 ± 0.41	2.95 ± 0.07	0.35 ± 0.12	643 ± 0.02
Unawatuna	8.03 ± 0.02	28.07 ± 0.23	7.92 ± 0.30	1.07 ± 0.24	0.03 ± 0.02	0.03 ± 0.01	1.13 ± 0.07	0.51 ± 0.18	634 ± 0.05
Galle	7.98 ± 0.05	28.77 ± 0.31	7.53 ± 0.13	1.06 ± 0.25	0.03 ± 0.01	0.21 ± 0.03	1.30 ± 0.08	0.44 ± 0.14	626 ± 0.08
Koggala	8.05 ± 0.03	28.57 ± 0.21	7.74 ± 0.04	0.74 ± 0.05	0.02 ± 0.01	0.06 ± 0.04	0.82 ± 0.03	0.46 ± 0.13	602 ± 0.07
Weligama	7.96 ± 0.02	28.60 ± 0.17	7.69 ± 0.12	0.30 ± 0.19	0.04 ± 0.02	0.14 ± 0.07	0.48 ± 0.03	3.17 ± 1.21	662 ± 0.05
Mirssa	7.84 ± 0.19	29.60 ± 0.40	6.64 ± 0.54	1.91 ± 0.46	0.03 ± 0.02	0.67 ± 0.32	2.61 ± 0.04	0.92 ± 0.14	567 ± 0.01
Range	7.36 ± 0.26–8.12 ± 0.02	26.07 ± 0.06–29.83 ± 0.15	6.64 ± 0.54–7.92 ± 0.30	0.30 ± 0.19–3.48 ± 0.01	0 ± 0.00–0.64 ± 0.17	0.03 ± 0.01–1.39 ± 0.41	0.48 ± 0.04–4.31 ± 0.09	0.06 ± 0.04–3.17 ± 1.21	584 ± 0.02–679 ± 0.01
SLSI standards
For bathing and contact recreation water	–	–	5 mg/L	10 mg/L	–	–		0.7 mg/L	10 mg/L
Suitable for aquatic life	6.0–9.0	–	5 mg/L	10 mg/L	–	pH < 7.5–0.94 mg/L	0.4 mg/L	15 mg/L
						7.5 ≤ pH < 8.5–0.59 mg/L
						8.5 ≤ pH – 0.22 mg/L

Location .	pH .	Temperature (°C) .	DO (mg/L) .	(mg/L) .	(mg/L) .	N-NH3 (mg/L) .	TN (mg/L) .	TP (mg/L) .	COD (mg/L) .
Sarakkuwa	7.76 ± 0.05	29.73 ± 0.11	7.56 ± 0.00	1.61 ± 0.39	0.07 ± 0.00	0.51 ± 0.05	2.19 ± 0.01	0.80 ± 0.50	624 ± 0.05
Dikovita	7.91 ± 0.06	29.23 ± 0.05	7.72 ± 0.05	0.81 ± 0.15	0.03 ± 0.00	0.59 ± 0.08	1.43 ± 0.01	0.92 ± 0.10	673 ± 0.05
Pamunugama	8.00 ± 0.01	29.83 ± 0.15	7.69 ± 0.01	1.24 ± 0.14	0.02 ± 0.00	0.19 ± 0.03	1.45 ± 0.05	0.47 ± 0.06	598 ± 0.06
Galle face	7.47 ± 0.39	26.33 ± 0.49	7.64 ± 0.25	0.37 ± 0.04	0.00 ± 0.00	0.27 ± 0.16	0.64 ± 0.06	3.17 ± 0.14	653 ± 0.06
Wellawatta	7.36 ± 0.26	26.53 ± 0.23	6.87 ± 0.42	3.48 ± 0.01	0.64 ± 0.17	0.19 ± 0.09	4.31 ± 0.09	0.21 ± 1.13	661 ± 0.05
Dehiwala	7.49 ± 0.45	26.93 ± 0.57	7.40 ± 0.13	0.39 ± 0.04	0.00 ± 0.00	0.11 ± 0.07	0.50 ± 0.03	0.94 ± 0.04	679 ± 0.06
Mt.Lavinia	7.79 ± 0.33	27.00 ± 0.56	7.28 ± 0.15	0.31 ± 0.02	0.00 ± 0.00	0.25 ± 0.10	0.56 ± 0.09	0.07 ± 0.03	608 ± 0.08
Ratmalana	7.67 ± 0.43	27.47 ± 0.15	7.31 ± 0.08	0.30 ± 0.09	0.06 ± 0.06	0.17 ± 0.10	0.53 ± 0.08	0.88 ± 0.63	652 ± 0.05
Moratuwa	8.10 ± 0.03	27.13 ± 0.25	7.38 ± 0.04	0.37 ± 0.05	0.04 ± 0.03	0.44 ± 0.19	0.85 ± 0.09	0.72 ± 0.16	693 ± 0.05
Panadura	8.01 ± 0.10	28.20 ± 0.10	7.10 ± 0.72	0.41 ± 0.03	0.03 ± 0.03	0.04 ± 0.07	0.48 ± 0.04	0.70 ± 0.31	611 ± 0.05
Wadduwa	8.12 ± 0.05	28.00 ± 0.10	7.50 ± 0.04	0.44 ± 0.01	0.01 ± 0.01	0.07 ± 0.09	0.52 ± 0.03	0.77 ± 0.30	584 ± 0.05
Kalutara	8.05 ± 0.05	28.43 ± 0.15	7.51 ± 0.05	0.38 ± 0.03	0.00 ± 0.00	0.13 ± 0.05	0.51 ± 0.02	0.72 ± 0.57	603 ± 0.02
Benthota	8.05 ± 0.04	26.07 ± 0.06	7.78 ± 0.04	2.45 ± 0.06	0.04 ± 0.02	0.20 ± 0.11	2.69 ± 0.06	0.06 ± 0.04	681 ± 0.02
Ambalangoda	7.90 ± 0.19	26.63 ± 0.15	7.12 ± 0.97	0.44 ± 0.20	0.04 ± 0.00	0.30 ± 0.06	0.78 ± 0.08	0.40 ± 0.09	619 ± 0.05
Hikkaduwa	7.80 ± 0.05	26.62 ± 0.28	7.62 ± 0.22	1.07 ± 0.13	0.06 ± 0.01	0.35 ± 0.19	1.48 ± 0.07	0.35 ± 0.11	667 ± 0.02
Rathgama	8.00 ± 0.05	27.27 ± 0.31	7.77 ± 0.02	1.88 ± 0.22	0.05 ± 0.00	0.89 ± 0.23	2.82 ± 0.03	0.46 ± 0.17	657 ± 0.05
Ginthota	7.96 ± 0.10	29.27 ± 0.29	7.65 ± 0.10	1.44 ± 0.16	0.12 ± 0.08	1.39 ± 0.41	2.95 ± 0.07	0.35 ± 0.12	643 ± 0.02
Unawatuna	8.03 ± 0.02	28.07 ± 0.23	7.92 ± 0.30	1.07 ± 0.24	0.03 ± 0.02	0.03 ± 0.01	1.13 ± 0.07	0.51 ± 0.18	634 ± 0.05
Galle	7.98 ± 0.05	28.77 ± 0.31	7.53 ± 0.13	1.06 ± 0.25	0.03 ± 0.01	0.21 ± 0.03	1.30 ± 0.08	0.44 ± 0.14	626 ± 0.08
Koggala	8.05 ± 0.03	28.57 ± 0.21	7.74 ± 0.04	0.74 ± 0.05	0.02 ± 0.01	0.06 ± 0.04	0.82 ± 0.03	0.46 ± 0.13	602 ± 0.07
Weligama	7.96 ± 0.02	28.60 ± 0.17	7.69 ± 0.12	0.30 ± 0.19	0.04 ± 0.02	0.14 ± 0.07	0.48 ± 0.03	3.17 ± 1.21	662 ± 0.05
Mirssa	7.84 ± 0.19	29.60 ± 0.40	6.64 ± 0.54	1.91 ± 0.46	0.03 ± 0.02	0.67 ± 0.32	2.61 ± 0.04	0.92 ± 0.14	567 ± 0.01
Range	7.36 ± 0.26–8.12 ± 0.02	26.07 ± 0.06–29.83 ± 0.15	6.64 ± 0.54–7.92 ± 0.30	0.30 ± 0.19–3.48 ± 0.01	0 ± 0.00–0.64 ± 0.17	0.03 ± 0.01–1.39 ± 0.41	0.48 ± 0.04–4.31 ± 0.09	0.06 ± 0.04–3.17 ± 1.21	584 ± 0.02–679 ± 0.01
SLSI standards
For bathing and contact recreation water	–	–	5 mg/L	10 mg/L	–	–		0.7 mg/L	10 mg/L
Suitable for aquatic life	6.0–9.0	–	5 mg/L	10 mg/L	–	pH < 7.5–0.94 mg/L	0.4 mg/L	15 mg/L
						7.5 ≤ pH < 8.5–0.59 mg/L
						8.5 ≤ pH – 0.22 mg/L

Results are presented as the mean value of the triplicated samples ± standard deviation.

View Large

According to the recorded chemical water quality values for N-nitrate, N-nitrite, N-ammonia, total nitrogen, total phosphate and COD were in the range of 0.30 ± 0.19–3.48 ± 0.01 mg/L, 0 ± 0.00–0.64 ± 0.17 mg/L, 0.03 ± 0.01–1.39 ± 0.41 mg/L, 0.48 ± 0.03–4.31 ± 0.09 mg/L, 0.06 ± 0.04–3.17 ± 1.21 mg/L and 584 ± 0.02–679 ± 0.01 mg/L, respectively.

The recorded physical water quality parameters for temperature, pH and DO ranged between 26.07 ± 0.06–29.83 ± 0.15 °C, 7.36 ± 0.26–8.12 ± 0.02 and 6.64 ± 0.54–7.92 ± 0.30 mg/L, respectively. The previously observed average physicochemical values for Temperature, pH and DO from Negombo to Mirissa coastal belt were 28.89 ± 1.5 °C, 7.41 ± 1.5–9.64 ± 1.6 and 7.66 ± 2.78 mg/L, respectively (Manage et al. 2022).

Table 5 explains the CFU values obtained from MLGA plates for the Sarakkuwa to Mirissa coastal belt. Based on the recorded number of CFU, the total coliforms were >200 CFU for more than 77% of samples, and the lowest CFU value was recorded as 24 ± 6 at the Wadduwa site. Further, the maximum CFU (157 ± 2) of faecal coliform was recorded at the Wellawatta site, whereas the lowest value (5 ± 5) was recorded at the Koggala site.

The studied coastal belt included key locations vital for tourism and recreation, highlighting the significance of ensuring safe water quality (Liyanage et al. 2014). This study involved detecting the occurrence of Shiga-toxigenic and enteropathogenic E. coli contaminations and assessing water quality in the southern coastal belt of Sri Lanka. As physicochemical water quality parameters, temperature, pH, DO, conductivity, N-nitrate, N-nitrite, N-ammonia, total nitrogen, total phosphate, and COD were measured.

The N-nitrate and N-nitrite concentrations remain within the Sri Lanka Standards Institution (SLSI) standard values for water suitable for aquatic life, bathing, and contact recreational water. The N-ammonia values of Ginthota and Mirissa, 2.39 and 0.67 mg/L, exceeded the SLSI standard values of the considered two categories. As the second most-produced chemical globally, ammonia is vital in various industries, contributing economic value across sectors such as fertilizers, energy storage, and explosives (Edwards et al. 2023; Idroos et al. 2023). The Ginthota estuary was there, and Ginganga carries effluents and land-based sewage to the coastal waters. In Mirissa, the Mirissa fisheries harbour is where many boat activities occur. So, oil and other chemicals from boats may contaminate the coastal waters. The SLSI standard values for total phosphate for water suitable for aquatic life, bathing, and contact recreational water are 0.4 and 0.7 mg/L. Except for the total phosphate values of the water taken from Wellawattta, Hikkaduwa, and Ginthota, other values exceeded the value of 0.4 mg/L, the standard value for water suitable for aquatic life.

The COD values of all locations exceeded the maximum COD value suitable for aquatic life, bathing, and recreational activities. The COD is a crucial pollution factor in environmental water quality assessments, as it indicates the concentration of organic materials in the water. Plenty of organic materials generated from anthropogenic activities are finally directed to the ocean; thus, the COD of coastal waters has increased. Similar COD values for the selected coastal stretches have been recorded in much of the previously published literature (Weerasekara et al. 2015; Manage et al. 2022; Jayawardhane et al. 2023; Pathmalal et al. 2023). High levels of COD and faecal coliforms in coastal waters are strong indicators of human-induced pollution, often resulting from poor waste management, industrial discharges, agricultural run-off, and recreational activities. These pollutants harm the health of aquatic ecosystems, pose risks to human health, and can negatively impact the economy, especially in regions dependent on tourism and fisheries.

Faecal coliforms are important factors when considering microbial water quality, as they act as indicators for faecal contamination, as are enterococci (WHO 2021). Faecal coliforms were detected in all considered coastal locations. In Wellawatta, the faecal coliform CFU was assumed to be high because the Wellawatta canal carries domestic and septic effluents of the residents who live near the canal banks. In this study, our focus was to detect EHEC and STEC in the coastal waters of the selected locations through the detection of E. coli O157 genotype, a pathogenic E. coli. As E. coli is a coliform bacterium, it's important to confirm which faecal coliforms are E. coli before estimating the E. coli O157 concentration, and the obtained results are indicated in Table 5.

The pathogenic E. coli O157 genotype was detected in 12 out of 22 beach locations where faecal coliforms were isolated. The E. coli O157 strain has been detected using the E. coli attachment and effacement gene, eae, and Shiga-toxin-producing genes such as stx 1 and stx 2 (Saxena et al. 2015). Table 6 describes where positive E. coli O157 isolates were obtained and for each (eae, stx 1, stx 2) gene type. According to the PCR results, the presence of virulent genes stx1 was detected in the coastal water samples taken from Ambalangoda, Hikkaduwa, Rathgama and Weligama beach locations, whereas stx2 was identified in the coastal water samples taken from Galleface, Mt. Lavinia, Ginthota, Unawatuna, Koggala, and Mirissa beach locations. Stx (1/2) can produce Shiga toxins. Although some E. coli O157 strains may include stx genes, they may not cause HUS and other diarrhoeal diseases unless they have other virulence factors, such as eae, iha, hlyA, etc.; i.e., only at two sites (Table 6).

These virulent factors are located on pathogenicity islands, which include locus of enterocyte effacement (LEE) and non-LEE-encoded (Nle) effectors (Bolton 2011). These nleA, nleB, nleC, nleD, nleE, nleG, etc., have been identified in strains that cause human infections, and they are involved in various functions, such as preventing phagocytosis, interfering with host innate immune responses, and hindering cell division, etc. (Balière et al. 2016).

Additionally, the eae virulent gene, responsible for the attaching and effacing effect, was detected in the water samples taken from Dehiwala, Mt. Lavinia, Rathgama, and Galle Beach locations. Eighteen types and nine subtypes of the eae gene, including α, α2, β1, β2, β3, γ1, γ2, δ, ε, ε2, ε3, ε4, ζ, η, η2, θ, ι, ι2, κ, λ, μ, ν, ξ, ο, π, ρ and σ, have been recorded in the GenBank database. The eae gene, encoded by the LEE region in E. coli O157, produces an outer membrane protein called intimin, which has a molecular size of 94 to 97 kDa. Intimin is responsible for adherence to the intestinal mucosa, forming unique intestinal lesions known as attaching and effacing intestinal lesions (Saxena et al. 2015; Govindarajan et al. 2020). Enterohaemolysin, encoded by hly A, and flagellar H7, encoded by fliCh7 are other virulent factors for E. coli O157:H7.

The literature mostly focused on eae, stx1 and stx2 as they are the most common virulent associated with the E. coli O157 genotype. In addition, the other virulence factors, such as hly A associated with both EHEC and STEC whist, fliCh7, rbf0157, ast, aat, etc., are associated with STEC. But these virulence factors are not as common in the environment as the studied virulence genes, eae, stx1 & stx2 (Hounkpe et al. 2023).

Overall, presumptive Shiga-toxin-producing E. coli (STEC) was detected at ten out of 22 sampling locations (∼46%) along the southern coastal belt of Sri Lanka, whilst presumptive enterohaemorrhagic E. coli (EHEC) was identified in 12 out of 22 (∼55%) sampling locations.

The concurrent presence of stx1, stx2, or eae, synergistically or individually, signifies the E. coli O157 genotype (Werber et al. 2003; Liyanage et al. 2024), i.e., only at Mt. Lavinia and Rathgama (Table 6). Consequently, among the bacterial isolates of 22 distinct beach locations, the E. coli O157 strain was identified in 12 beach locations, namely Galleface, Dehiwala, Mt.Lavinia, Ambalangoda, Hikkaduwa, Rathgama, Ginthota, Unawatuna, Galle, Koggala, Weligama and Mirissa. Notably, all these beach locations where the E. coli O157 strain was detected serve as prominent destinations for tourism and recreational activities for residents and international visitors.

As depicted in Figure 2, the E. coli O157 strain has been identified across all the selected beach locations spanning from Ambalangoda to Mirissa within the Southern province. There are plenty of tourism and recreational activities, hotels and restaurants, fishing harbours like Hikkaduwa, Ambalangoda, Dodanduwa, Galle, Mirissa etc., as well as estuaries like Maadu Ganga river estuary, Madampe river estuary, Hikkaduwa lake, Rathgama lake, Ginganga, Mahamodara lake etc. which carries sewage effluents to the sea are located in Ambalangoda to Mirissa coastal areas. This indicates a strong potential for pathogenic E. coli O157 in the Ambalangoda to Mirissa coastal areas rather than the Sarakkuwa to Ambalangoda coastal area. Tourism in the area from Sarakkuwa to Ambalangoda is much lower compared to the region from Ambalangoda to Mirissa. Additionally, along the coastline south of Ambalangoda, beach recreational activities, beachside restaurants, beach parties, etc., have increased. This is because the beaches in this area have gained more attention from tourists, as they are surrounded by some of the most attractive and eye-catching beaches, and thus, the prevalence of E. coli O157 has likely increased.

This observation underscores the compromised water quality of these areas, signifying potential challenges for tourism and recreational activities.

EHEC and STEC in coastal waters pose a risk of gastrointestinal diseases to beach swimmers, surfers, and others who ingest contaminated coastal water. Additionally, when pathogenic E. coli strains are present in coastal waters, there is a risk of entering these pathogenic strains and the presence of antimicrobial resistance (Leonard et al. 2022) within aquatic organisms, particularly those consumed as seafood by humans. The pathogenic E. coli can reach the human gastrointestinal tract through this pathway, potentially causing severe illness.

Pathogenic E. coli are responsible for severe illnesses such as HUS, haemolytic colitis (HC), and TTP. Since tourism is a major source of foreign income in Sri Lanka, pathogenic strains and increasing cases of related diseases could negatively impact the tourism industry. Based on the findings, we recommend the implementation of continuous monitoring programmes for coastal waters to ensure the protection of public health and to maintain environmental integrity to minimize exposure to pathogenic E.coli. It is also crucial to ensure proper wastewater treatment, especially for nutrient-rich waste, before being discharged into coastal waters. Adequate waste treatment, stricter industrial and agricultural run-off regulations, and improved sanitation systems are essential to improving coastal water quality and reducing these risks.

Several mitigation strategies can be adopted to reduce contamination levels of pathogenic E. coli further and improve water quality in the studied coastal regions. These include enhancing existing wastewater treatment systems, developing new methods for improving the management of agricultural run-off and stormwater systems, raising awareness among local communities and stakeholders, and implementing new ecosystem restoration programmes.

E. coli bacteria were detected in all water samples collected from each of the 22 locations from Sarakkuwa to the Mirissa coastal belt. The E. coli O157 genotype was identified in 12 out of 22 coastal locations, namely, Galleface, Dehiwala, Mt.Lavinia, Ambalangoda, Ginthota, Unawatuna, Hikkaduwa, Galle, Rathgama, Koggala, Weligama, and Mirissa. Therefore, swimmers should be warned against entering the water in these areas following rain events. Due to the pathogenic nature of the E. coli O157 and its association with severe illnesses, locations where E. coli is detected in abundance are deemed unsuitable for recreational activities such as swimming and surfing due to health concerns. Further monitoring and assessment are strongly recommended to identify sources and manage options.

The authors thank the University of Sri Jayewardenepura in Sri Lanka for financially supporting the study (Centre for Water Quality and Algae Research).

This study was funded by the Center for Water Quality and Algae Research, University of Sri Jayewardenepura, Sri Lanka.

S.B. contributed to sample collection, formal analysis, writing the original draft. P.W. contributed to conceptualization, formal analysis, review & editing. G.L. contributed to formal analysis, review & editing. S.P. contributed to conceptualization. P.M. contributed to conceptualization, sample collection, review & editing.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.