Enteropathogenic Bacteria in Water Sources Associated with Faecal Waste from Open Defecation and Animals in Rural Communities of Vhembe District, South Africa

Abstract

The lack of improved sanitation in rural areas of developing countries, including South Africa, exacerbates open defecation, leading to the significant contamination of water sources by human and animal waste. This study aimed to establish the association of Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Shigella flexneri, and Yersinia enterocolitica in open defecation sites and animal waste with the contamination of water sources in Vhembe District, South Africa. A total of 1032 water samples and 111 faecal samples from the Collins Chabane and Thulamela municipalities were analysed using qPCR. Regression models were used to assess associations, with S. Typhimurium (19–60%) and S. flexneri (11–44%) being the most prevalent bacteria in faecal matter and water, showing detection rates of 4–100% and 5–100%, respectively. Strong associations were found during the wet season between faecal waste and water contamination for S. flexneri (R² = 0.7, p = 0.005) and S. Typhimurium (R² = 0.619, p = 0.091). Urgent measures are needed to address the contamination of rural water sources due to open defecation and livestock waste.

1. Introduction

The availability and accessibility of clean water is a fundamental human right; however, its quality is determined by biological, physical, and chemical properties. While many communities may have access to water, the crucial question remains of whether this water is safe for human consumption. As of 2020, approximately 5.8 billion people had gained access to improved water sources, whereas 368 million people relied on unprotected wells and springs, and an additional 122 million sourced untreated surface water from lakes, ponds, rivers, and streams [1]. Global reports also highlight that around two million people utilise water contaminated with faeces [1]. Faecal contamination of drinking water sources, especially in low-income regions such as sub-Saharan Africa, is prevalent, particularly in rural areas. Factors such as open defecation, animal waste, and economic activities are cited as primary causes of water source pollution [2]. In the Vhembe District of Limpopo Province, diarrhoeal diseases remain a significant public health concern, especially among children under five years old. According to the South African National Department of Health, Limpopo has reported higher rates of diarrhoea-related illness compared to the national average, with Vhembe District identified as one of the hotspots due to inadequate water and sanitation infrastructure [3]. Furthermore, a study by Murei et al. [4] reported the frequent detection of faecal indicators and pathogens in drinking water sources across the district, correlating with high incidence rates of diarrhoea. Additionally, local clinics have documented seasonal spikes in diarrhoeal cases during the rainy season, likely due to increased faecal contamination of surface water [4]. These data underscore the need for comprehensive investigations into the links between faecal contamination and waterborne disease risks in rural Vhembe communities. Worldwide, about 494 million people still engage in open defecation, 92% of whom reside in rural areas of developing nations [5,6]. Furthermore, in most parts of the world, particularly in developing regions, livestock commonly grazes around water sources such as rivers, ponds, and lakes [1]. In sub-Saharan Africa, around 70% of rural water sources are contaminated by livestock waste, because of the prevalence of free-ranging livestock in areas without fenced grazing lands [7]. In South Asia, studies conducted in India and Bangladesh have reported that 50 to 60% of surface water is contaminated by livestock faecal matter [8]. In South Africa, open defecation remains a persistent issue in certain rural and informal settlements, where access to adequate sanitation facilities is limited. In 2021, approximately 2.2% of households in the country still practiced open defecation, with higher prevalence in rural provinces such as Limpopo, Eastern Cape, and KwaZulu-Natal [9].

For many rural communities, safe drinking water remains a luxury, leading them to depend on unsafe water sources that are inadequate to meet their daily needs [10]. The reliance on contaminated water sources has far-reaching consequences, particularly for sustainable development in these regions [11]. In South Africa, the safeguarding of water resources is paramount, especially considering that the demand for water is projected to increase by 2025. The nation’s limited water resources, coupled with its semi-arid conditions and deteriorating water quality, have hindered economic growth, particularly in rural regions, where potable water is scarce [12]. In Vhembe District Municipality (VDM) in Limpopo Province, many rural communities face intermittent water supplies. This scarcity forces residents to store water obtained from various sources, including community standpipes, yard taps, rivers, springs, hand-dug wells, or dams [4,13,14]. In addition, many of VDM’s residents engage in subsistence farming, relying heavily on water sources for both irrigation and drinking purposes.

Water sources contaminated with faecal matter are at a high risk of hosting pathogenic bacteria, which have been linked to waterborne diseases, such as infectious diarrhoea, in many developing countries, including South Africa [15]. Previous studies have noted that agricultural activities, urban runoff, and urbanisation have introduced pathogens into the Luvuvhu River and its basin. Despite this, the river remains a critical water source for agricultural and domestic use in Vhembe District. The deteriorating condition of the water sources, including the Nandoni Dam, which supplies water to several rural communities, presents an environmental challenge [16]. The effective management of water sources is, therefore, crucial to ensure the protection of drinking water supplies. This calls for collaboration between stakeholders such as water suppliers, environmental protection agencies, public health authorities, and communities to manage water resources effectively [17].

Diarrhoeal diseases, due to bacterial pathogens such as Escherichia coli, Shigella, and Salmonella, are a leading cause of illness and mortality among children under five. The prevalence of these diseases is attributable to poor sanitation infrastructure and the contamination of water sources due to inadequate waste management [3,18]. This study focused on rural communities in Thulamela Local Municipality (TLM) and Collins Chabane Local Municipality (CLM) and aimed at tracking the prevalence of diarrhoeagenic bacterial pathogens such as Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Shigella flexneri, and Yersinia enterocolitica from open defecation sites to water sources used in these areas in VDM. These pathogens are known as major causative agents of bacterial gastroenteritis, particularly in settings with poor sanitation and unsafe water.

2. Materials and Methods

2.1. Study Design

This study focuses on the diverse water sources used by households in the VDM region. Based on daily water usage patterns, four primary scenarios were considered: (1) households relying on treated surface water from rivers and dams, distributed following treatment at water plants; (2) households exclusively using untreated river water; (3) households depending on untreated groundwater from springs; and (4) households using untreated groundwater from hand-dug wells. This approach is adapted from a similar study design by Mogane et al. [19], which informed the categorisation of the water sources and treatment scenarios. A survey was conducted using structured questions to identify the open defecation sites surrounding the water sources used by the local municipalities of the VDM and to track the target enteropathogenic bacteria from these sites to water sources and to dwellings.

2.2. Ethical Clearance

This study was conducted with ethical approval from the Faculty Committee for Research Ethics (FCRE) at Tshwane University of Technology (TUT) (ref no.: FCRE 2019/10/008 (FCPS 03) (SCI)), following the ethical framework established by Mogane et al. [19]. Permission to conduct research in Thulamela Local Municipality (TLM) and Collins Chabane Local Municipality (CLM) was granted by Vhembe District Municipality. Informed consent was obtained from household owners prior to collecting water samples, with confidentiality maintained for all data. The study objectives were fully explained to participants, and only defecation sites around water sources accessed by rural communities in TLM and CLM were included in the scope.

2.3. Study Area

The study was conducted in Thulamela and Collins Chabane Local Municipalities within Vhembe District, Limpopo Province, South Africa (Figure 1). Thulamela comprises 38 wards and 163 villages, while Collins Chabane consists of 36 wards and 198 villages. The local terrain is a mix of elevated (Thulamela) and low-lying (Collins Chabane) areas, with differing rainfall and temperature profiles that influence bacterial persistence and runoff patterns. Communities in both municipalities rely on multiple water sources, including treated surface water (e.g., from Nandoni Dam), untreated river water (e.g., from the Luvuvhu, Mutshindudi, and Mvudi Rivers), springs, and hand-dug wells. Sampling sites were selected based on proximity to open defecation sites and household water use patterns. The map was generated using ArcGIS Desktop version 10.8 (Esri, Redlands, CA, USA). The software was employed to digitize shapefiles, overlay village boundaries, and visualize spatial relationships between water sources and open defecation sites.

2.4. Collection of Samples

2.4.1. Faecal Sample Collection

Figure 2 shows some of the open defecation sites identified within the target municipalities. Following the methods outlined by Mogane et al. [19], a total of 111 human and animal composite faecal samples were aseptically collected from surface and groundwater source areas in selected villages during both the wet (March–April 2021) and dry (June–July 2021) seasons to assess the influence of seasonal variation on the faecal contamination of water sources. The wet season in the study area was characterised by heavy rainfall, increased surface runoff, and elevated water table levels, which may have facilitated the transport of faecal matter from open defecation sites and livestock grazing areas into nearby surface and groundwater sources. In contrast, the dry season typically featured reduced precipitation and hence lower runoff of faecal material into adjacent waterbodies. The collected faecal samples in this study included human (n = 49), cow (n = 49), pig (n = 3), chicken (n = 3), and dog (n = 7) faeces and were promptly transported in a cooler box to the microbiology laboratory at the University of Venda for preliminary analysis within 24 h.

The attribution of faecal samples to specific host sources was guided by direct observation and community knowledge, as outlined in Mogane et al. [19]. Human faecal samples were collected from open defecation sites identified through household surveys. Animal faecal samples were collected from areas where livestock were observed grazing or roaming near water sources, with species identification confirmed visually and through consultation with local residents and livestock owners. Composite sampling involved pooling multiple samples of faecal matter from the same species within a defined area to ensure representative sampling and reduce variability. Each composite sample was collected using sterile stool collection tubes and gloves to prevent cross-contamination. Samples were immediately placed in cooler boxes and transported to the microbiology laboratory at the University of Venda, where they were processed within 24 h of collection.

2.4.2. Drinking Water Samples

Following the procedures outlined in Mogane et al. [19], a total of 1032 water samples were collected from village water sources, water treatment facilities, and residences (for detailed numbers of samples and sampling sites, see Supplementary Materials, Table S1) during the wet season (March–April 2021) and dry season (June–July 2021). Each 2 L water sample was aseptically collected in a labelled sterile bottle as per the APHA guidelines [20] and immediately transported on ice, within 6–8 h of collection, to the Tshwane University of Technology laboratory for further analysis. Upon arrival, samples were processed immediately or stored at 4 °C for a maximum of 12 h before processing to preserve microbial integrity.

2.5. Processing of Samples

2.5.1. Faecal Samples

Faecal samples (150 mg for each sample) were transferred into test tubes containing 9 mL of Bolton Selective Enrichment Broth, supplemented with Bolton Selective Supplement and Laked Horse Blood (Thermo Scientific™, Oxoid™, Johannesburg, South Africa), for the enrichment of Campylobacter jejuni (C. jejuni). These tubes were incubated at 42 °C for 48 h in anaerobic jars containing anaerobic sachets (Thermo Scientific™, Oxoid™). For the selective enrichment of Salmonella enterica serovar Typhimurium (S. Typhimurium) and Shigella flexneri (S. flexneri), 150 mg faecal samples were transferred into test tubes containing 9 mL of GN Broth (BioLab Inc., Budapest, Hungary). These tubes were incubated at 37 °C for 24 h. For the enrichment of Yersinia enterocolitica (Y. enterocolitica), 150 mg faecal samples were transferred into test tubes containing 9 mL of Peptone Sorbitol Bile Broth (BioLab Inc., Budapest, Hungary), and the tubes were incubated at 29 °C for 48 h.

2.5.2. Water Samples

To concentrate and enrich the target bacteria, a modified membrane filtration technique described by Saingam et al. [21], with some adjustments for specific pathogens, was employed. The technique utilised 0.45 µm and 47 mm filters (Merck Millipore, Darmstadt, Germany) for the concentration and enrichment of the bacteria. To minimise the risk of cross-contamination during membrane filtration, strict aseptic techniques were followed throughout the procedure. All filtration units, forceps, and workspace surfaces were thoroughly disinfected with 70% ethanol before and after each filtration. Sterile filter membranes were handled with sterile gloves and forceps, and a new membrane was used for each water sample. Between each sample, the filtration apparatus was rinsed with sterile distilled water and wiped down. Briefly, 1.5 L of water was filtered to concentrate Campylobacter jejuni (C. jejuni). The filters were then placed into 50 mL sterile Falcon tubes containing 15 mL of Bolton Selective Enrichment Broth, supplemented with Bolton Selective Supplement and Laked Horse Blood (Thermo Fisher Scientific, Oxoid™), and incubated at 42 °C for 48 h in anaerobic jars with anaerobic sachets. For Salmonella enterica serovar Typhimurium (S. Typhimurium) and Shigella flexneri (S. flexneri), 100 mL of water was filtered. The filters were submerged in 50 mL tubes containing 15 mL of GN Broth (BioLab Inc., Budapest, Hungary) and incubated at 37 °C for 24 h. For Yersinia enterocolitica (Y. enterocolitica), 100 mL of water was filtered, and the filters were placed into 50 mL tubes containing 15 mL of Peptone Sorbitol Bile Broth (BioLab Inc., Budapest, Hungary), followed by incubation at 29 °C for 48 h.

2.6. Molecular Identification of Target Enteropathogenic Bacteria

2.6.1. DNA Extraction

Prior to DNA extraction, the processed faecal and water samples, along with positive controls for each target enteropathogenic bacterium, were vigorously vortexed for approximately 5 min to dislodge bacterial cells. About 200 µL of the concentrated bacterial sample was then transferred into a ZR BashingBead™ lysis tube containing BashingBead™ buffer (Zymo Research, Irvine, CA, USA). The samples were subjected to bead beating, followed by DNA extraction using the Quick-DNA Fecal/Soil Microbe Microprep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. The quality and quantity of the extracted DNA were assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Johannesburg, South Africa).

2.6.2. Determination of qPCR Efficiency and Lower Limit of Quantification of Primers

The extracted DNA from positive controls of the enteropathogenic bacteria Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Yersinia enterocolitica, and Shigella flexneri (Table S1, Supplementary Materials) was serially diluted in 10-fold increments to construct standard curves. These curves were then used to assess the quantitative PCR (qPCR) efficiency and determine the limit of quantification (LOQ) for each pathogen. This approach ensured the accurate calibration of the qPCR assay, allowing for the reliable quantification of the bacterial DNA in the water samples.

2.6.3. Enteropathogenic Bacteria Identification

Target enteropathogenic bacteria were identified using primers, probes, and cycling conditions previously described by the investigators (Table S2, Supplementary Materials). The extracted DNA was subjected to qPCR assays using a CFX96™ Real-Time PCR thermal cycler (Bio-Rad, Hercules, CA, USA), which was optimised prior to use. Each 25 µL PCR mixture contained 2 µL of template DNA, 12.5 µL of GoTaq^® Probe RT-PCR Master Mix (1X), 0.5 µL of probe (0.2 µM), 1 µL of each primer (0.4 µM), and 8 µL of nuclease-free water. Four controls were included in the reactions: a negative control (nuclease-free water), a no-template control (only PCR Master Mix), an extraction blank (from the DNA extraction process), and a positive control.

2.7. Statistical Analysis

Microsoft Excel 365 (version 2205) was used to create a spreadsheet documenting the frequencies of the target enteropathogenic bacteria in faecal samples and various water sources. Prevalence data were represented in tables. A regression model was then applied using the Statistical Package for the Social Sciences (SPSS) software (version 27) to assess associations between the presence of enteropathogenic bacteria in faecal samples (independent variable) and water sources (dependent variable). The model was also used to evaluate the relationship between bacteria detected in water sources (independent variable) and household-stored water (dependent variable). The strength of these associations was measured using the coefficient of determination (R²).

3. Results

3.1. Parameters Used for Detection and Quantification of Enteropathogenic Bacteria

The efficiency of the qPCR and limit of detection (LLOQ) for each assay were determined using the standard curves. Overall, the highest efficiency values were noted for Y. enterocolitica and S. flexneri at 100%, followed by C. jejuni (96%) and S. Typhimurium (95%). The LLOQ calculated from the average Ct values obtained using the standard curve for each assay showed C. jejuni with the highest value (37.9) compared to other target enteropathogenic bacteria, which had values ranging between 19 and 23. The y-intercept parameter, used as the cut-off value for each assay, ranged between −3.24 and −3.49 (Table S3 and Figure S1, Supplementary Materials).

3.2. Prevalence of Enteropathogenic Bacteria in Faecal Samples Collected in the Vicinities of Various Water Sources

Surface water sources—During the wet and dry seasons, faecal waste from humans and/or cows was found in the vicinities of all rivers and the dam used by the communities of the local municipalities under investigation. Despite the small sizes of the faecal samples in CLM, all target enteropathogenic bacteria were detected in these wastes during the wet season, except for Y. enterocolitica. Out of the nine (9) faecal samples collected, the highest prevalence was found for S. Typhimurium (44%), followed by S. flexneri (33%), while C. jejuni was detected at the lowest rate (22%). It is also important to mention that S. flexneri was not detected in four cow stool specimens collected during the wet season in CLM. Additionally, during the dry season, only human faecal samples harboured S. Typhimurium at a frequency of 30% and S. flexneri at a prevalence rate of 20% (

Table 1. Prevalence of enteropathogenic bacteria in faecal waste collected around surface water sources.

CLM
Wet Season
Frequency (No. of Positive Samples/No. of Tested Samples)
Enteropathogenic Bacteria
Sampling Site	Faecal Samples	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
Luvuvhu River downstream	Cow	4	50%	25%	0%	0%
Luvuvhu River upstream	Human	5	40%	20%	0%	60%
Total		9	44%	22%	0%	33%
Dry season
Luvuvhu River downstream	Cow	2	0%	0%	0%	0%
	Human	4	50%	0%	0%	25%
Luvuvhu River upstream	Human	4	25%	0%	0%	25%
Total		10	30%	0%	0%	20%
TLM
Wet season
Mvudi River downstream	Cow	3	33%	0%	0%	67%
	Human	3	0%	0%	0%	33%
Mvudi River upstream	Cow	2	50%	0%	0%	0%
Luvuvhu River downstream	Human	4	25%	0%	0%	0%
Luvuvhu River upstream	Cow	2	0%	0%	0%	50%
	Human	3	33%	0%	0%	33%
Mutshindudi River downstream	Human	2	50%	0%	0%	0%
Mutshindudi River upstream	Human	3	67%	0%	0%	67%
Nandoni Dam	Cow	3	0%	0%	0%	33%
	Human	2	50%	0%	0%	0%
Total		27	27%	0%	0%	27%
Dry season
Mvudi River downstream	Human	4	0%	0%	0%	0%
Mvudi River upstream	Cow	4	25%	0%	0%	25%
	Human	2	0%	0%	0%	0%
Luvuvhu River downstream	Cow	2	0%	0%	0%	0%
Luvuvhu River upstream	Human	3	67%	0%	0%	0%
Mutshindudi River downstream	Cow	3	0%	0%	0%	0%
Mutshindudi River upstream	Dog Human Cow	3 2 2	33% 50% 0%	0% 0% 0%	0% 0% 0%	0% 0% 50%
Nandoni Dam	Cow	2	0%	0%	0%	50%
Total		27	19%	0%	0%	11%

).

In TLM, the community relies on three (3) main rivers and one dam (Table 1). Human and cow faecal matter was found in the vicinities of the surface water sources during the wet season, while human, cow, and dog faecal matter was found in the vicinities of the surface water sources during the dry season. During the wet season, S. Typhimurium and S. flexneri were detected in faecal matter, but C. jejuni and Y. enterocolitica were not detected. Out of the 27 faecal waste samples collected, S. Typhimurium and S. flexneri had the same detection frequency (both 27%). However, during the dry season, the highest frequency was found for S. Typhimurium (19%), while a detection frequency of 11% was recorded for S. flexneri.

Spring water—The spring water sources used by the communities in both municipalities during the wet and dry seasons exhibited faecal matter from humans and animals (dogs, cows, and pigs) (

Table 2. Prevalence of enteropathogenic bacteria in faecal samples collected around spring water sources.

CLM
Wet Season
Frequency (No. of Positive Samples/No. of Tested Samples) Enteropathogenic Bacteria
Sampling Site	Faecal Samples	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
Dididi spring	Pig	3	67%	0%	33%	67%
	Dog	2	50%	0%	50%	0%
Total		5	60%	0%	40%	40%
Dry season
Dididi spring	Cow	3	67%	0%	0%	0%
	Pig	2	0%	0%	50%	33%
Total		5	40%	0	20%	20%
TLM
Wet season
Tshivulani spring	Cow	3	33%	0%	0%	0%
Tshilapfene spring	Cow	4	0%	0%	0%	75%
	Human	2	50%	0%	0%	50%
Total		9	22%	0%	0%	44%
Dry season
Tshivulani spring	Cow	3	0%	0%	0%	0%
	Human	2	50%	0%	0%	0%
Tshilapfene spring	Cow	4	0%	0%	0%	0%
	Human	2	50%	0%	0%	50%
Total		11	18%	0%	0%	9%

). In CLM, all target enteropathogenic bacteria were detected in faecal matter during the wet season, except for C. jejuni. Out of the five (5) faecal samples collected, the highest prevalence was recorded for S. Typhimurium (60%), followed by S. flexneri and Y. enterocolitica (both 40%). During the dry season, out of the five faecal samples collected, the highest prevalence was also recorded for S. Typhimurium (40%), followed by Y. enterocolitica and S. flexneri (both 20%). Moreover, C. jejuni was also not detected in any of the faecal samples collected during this season.

In TLM, only cow and human faecal samples were collected in the vicinity of the spring water source during both seasons. During the wet season, only S. Typhimurium and S. flexneri were detected in the faecal matter. Out of the nine faecal samples collected during the wet season, the highest prevalence was recorded for S. flexneri (44%), followed by S. Typhimurium (22%). However, the situation was different during the dry season, where the highest frequency was recorded for S. Typhimurium (18%), followed by S. flexneri (9%) (Table 2).

Hand-dug well water source—In TLM, faecal samples from chickens and dogs were collected during the wet season, while only cow faecal samples were collected during the dry season in the vicinities of the hand-dug wells. All target enteropathogenic bacteria were detected in the faecal samples during the wet season, except for C. jejuni. Out of the five faecal samples collected, the highest frequency was recorded for S. flexneri (40%), followed by S. Typhimurium and Y. enterocolitica (both 20%). During the dry season, S. Typhimurium and S. flexneri were detected in cow faecal samples, but not C. jejuni or Y. enterocolitica; however, both S. flexneri and S. Typhimurium exhibited a detection frequency of 33.33% in the vicinities of the hand-dug wells (

Table 3. Prevalence of enteropathogenic bacteria in faecal samples collected around hand-dug well water sources.

TLM+
Wet Season
Frequency (No. of Positive Samples/No. of Tested Samples)
Enteropathogenic Bacteria
Sampling Site	Faecal Samples	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
Tshivulani hand-dug wells	Chicken	3	0%	0%	0%	67%
	Dog	2	50%	0%	50%	0%
Total		5	20%	0%	20%	40%
Dry season
Tshivulani hand-dug wells	Cow	3	33%	0%	0	33%
Total		3	33	0%	0%	33%

).

3.3. Prevalence of Enteropathogenic Bacteria in Surface Water Sources and Household Water (End Users)

3.3.1. Households Using Treated Surface Water (Rivers and Dams)

Table 4. Prevalences of enteropathogenic bacteria in surface water sources prior to treatment, after treatment, and at point of use (household-stored water).

CLM
		Wet Season				Dry Season
Frequency (No. of Positive Samples/No. of Tested Samples
Enteropathogenic Bacteria
Sampling Site	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
	n = 176					n = 176
Luvuvhu River downstream	4	100%	0%	0%	100%	75%	0%	0%	100%
Luvuvhu River upstream	4	100%	0%	0%	100%	50%	0%	0%	100%
WTP abstraction point	4	100%	0%	0%	50%	100%	0%	0%	50%
WTP—treated water at point of treatment	4	0%	0%	0%	0%	0%	0%	0%	0%
HH *—standpipe	80	15%	0%	0%	5%	6%	0%	0%	3%
HH—container-stored water	80	9%	0%	0%	20%	6%	0%	0%	5%
				TLM
	n = 188					n = 188
Mvudi River downstream	4	100%	0%	0%	100%	75%	0%	0%	75%
							0%
Mvudi River upstream	4	100%	0%	100%	50%	0%	0%	75%	100%
Luvuvhu River downstream	4	100%	0%	0%	100%	75%	0%	0%	100%
Luvuvhu River upstream	4	100%	0%	0%	100%	75%	100%	0%	0%
Nandoni Dam	4	100%	0%	0%	75%	75%	0%	0%	100%
WTP abstraction point	4	100%	0%	0%	100%	100%	100%	0%	0%
WTP—treated water at point of treatment	4	0%	0%	0%	0%	0%	0%	0%	0%
HH *—standpipe	80	9%	0%	0%	5%	4%	0%	0%	3%
HH—container-stored water	80	14%	0%	0%	5%	6%	0%	0%	4%

* HH = household.

illustrates the prevalence of enteropathogenic bacteria in CLM. The water samples collected upstream and downstream of open defecation sites along the Luvuvhu River tested positive only for S. Typhimurium and S. Flexneri during both seasons. These two enteropathogenic bacteria were detected in all samples collected during the wet season (100%) at both sampling points, while, during the dry season, the detection rates were 50–75% upstream and 100% downstream, respectively. At the point of treatment, the assessed enteropathogenic bacteria were also present in all samples (75% for S. Typhimurium, 100% for S. flexneri) during the wet season. The detection rates were 75% for S. Typhimurium and 50% for S. flexneri during the dry season. None of the target enteropathogenic bacteria were detected in the treated water. At the point of use (household level), S. Typhimurium was detected at a frequency of 15%, while S. flexneri was detected at a frequency of 5% during the wet season (during the dry season, these values were 6% for S. Typhimurium and 4% for S. flexneri) in treated water prior to storage. As for the treated water stored in household containers, the same enteropathogenic bacteria were detected during both seasons at higher frequencies compared to household water prior to storage. During the wet season, S. Typhimurium was detected at a rate of 9% and S. flexneri was recorded at a frequency of 20%, while the detection frequencies were 6% for S. Typhimurium and 5% for S. flexneri during the dry season.

In TLM, all water samples collected upstream and downstream of open defecation sites, along the Mvudi River, Luvuvhu River, and Nandoni Dam, tested positive for S. Typhimurium and S. Flexneri during both seasons. The detection frequencies for the samples collected at all sampling points during the wet season were 100% for S. Typhimurium and 75–100% for S. flexneri; meanwhile, during the dry season, the detection rates were 50–75% for S. Typhimurium and 75–100% for S. flexneri.

At the WTP abstraction point, the same enteropathogenic bacteria were detected within all samples (100%) during both seasons. None of the target enteropathogenic bacteria were detected in the treated water. However, at the point of use, S. Typhimurium and S. flexneri were detected at low frequencies during the wet season (9% and 5%, respectively), and, during the dry season, the same enteropathogenic bacteria were detected (4% and 3%, respectively) in treated water prior to storage. Regarding the treated water stored in household containers, S. Typhimurium was detected at a frequency of 14% and S. flexneri at 5% during the wet season, and, during the dry season, the detection rates for these enteropathogenic bacteria were 6% for S. Typhimurium and 4% for S. flexneri.

3.3.2. Households only Depending on Untreated River Water Sources

Water samples collected from the surface water source tested positive for S. Typhimurium and S. flexneri during both seasons, but not C. jejuni and Y. enterocolitica (

Table 5. Prevalences of enteropathogenic bacteria in surface water sources and in household container-stored water.

TLM
	Wet Season					Dry Season
	Frequency (No. of Positive Samples/No. of Tested Samples)
	Enteropathogenic Bacteria
	n = 44					n = 44
Sampling Site	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
Mutshindudi River downstream	4	100%	0%	0%	100%	50%	0%	0%	100%
Mutshindudi River upstream	4	100%	0%	0%	100%	75%	0%	0%	50%
HH *—container-stored water	36	33%	0%	0%	22%	25%	0%	0%	14%

* HH = household.

). All water samples collected from the downstream and upstream regions of open defecation sites along the Mutshindudi River harboured S. Typhimurium and S. flexneri during the wet season, while, during the dry season, S. Typhimurium was detected at frequency of 50–70% and S. flexneri at a frequency of 50–100%. At the point of use, the prevalences of S. Typhimurium and S. flexneri decreased to 33% and 22% during the wet season, respectively, and to 25% and 14%, respectively, during the dry season.

3.3.3. Only Depending on Untreated Spring Water and Household Container-Stored Water

Water samples collected from the spring in CLM tested positive for S. Typhimurium, S. flexneri, and Y. enterocolitica, although variations occurred during the seasons. During the wet season, these enteropathogenic bacteria (S. Typhimurium, S. flexneri, and Y. enterocolitica) were detected in all water samples collected from the target sampling points. However, during the dry season, the detection rates were 75% for both S. Typhimurium and Y. enterocolitica and 50% for S. flexneri. At the point of use, the water samples exhibited S. Typhimurium at a frequency as high as 56%, followed by S. flexneri (38%) and Y. enterocolitica (28%), during the wet season. During the dry season, S. Typhimurium still predominated in water samples (34%) compared to S. flexneri (31%) and Y. enterocolitica (16%). Water samples collected from the spring water in TLM tested positive for S. Typhimurium and S. flexneri during both seasons, with detection rates of 100% during the wet season and 75% during the dry season. At the point of use, S. flexneri predominated (33%) compared to S. Typhimurium (28%) during the wet season. A similar observation was also noted during the dry season (22% for S. Typhimurium and 8% for S. flexneri) (

Table 6. Prevalences of enteropathogenic bacteria in spring water sources and in household container-stored water.

CLM
		Wet Season				Dry Season
Sampling Site	Number of Samples	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
			n = 36			n = 36
Spring	4	100%	0%	100%	100%	75%	0%	75%	50%
HH *—container-stored water	32	56%	0%	28%	38%	34%	0%	16%	31%
TLM
n = 40						n = 40
Spring	4	100%	0%	0%	100%	75%	0%	0%	75%
HH *—container-stored water	36	28%	0%	0%	33%	8%	0%	0%	22%

* HH = household.

).

3.3.4. Households only Depending on Untreated Groundwater such as Hand-Dug Wells and Household Container-Stored Water

The hand-dug well water samples in TLM tested positive for all target pathogenic bacteria except C. jejuni, although seasonal variations were observed in their detection frequencies (

Table 7. Prevalences of enteropathogenic bacteria in hand-dug wells and in household container-stored water.

TLM
	Wet Season					Dry Season
Sampling Site	Number of Samples (n)	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri	S. Typhimurium	C. jejuni	Y. enterocolitica	S. flexneri
	n = 32					n = 32
Hand-dug well	4	100%	0%	100%	100%	50%	0%	50%	75%
HH *—container-stored water	28	18%	0%	7%	43%	14%	0%	7%	32%

* HH = Household.

). All samples collected at both sampling points during the wet season (100%) contained all assessed enteropathogenic bacteria, while, during the dry season, the detection rate fell to 50% for S. Typhimurium and Y. enterocolitica and 75% for S. flexneri. At the point of use, S. flexneri was detected at a high frequency (43%), followed by S. Typhimurium (18%) and Y. enterocolitica (7%). Similar results were observed during the dry season, where S. flexneri still predominated in water sources (32%) compared to S. Typhimurium (14%) and Y. enterocolitica (7%).

3.4. Associations Between Target Enteropathogenic Bacteria in Faecal Samples and in Water Sources

The regression model (Table S4, Supplementary Materials) established the associations among the target enteropathogenic bacteria in faecal matter and their presence in the water sources used by the selected villages in both municipalities. During the wet season, statistical evidence showed a strong association for S. flexneri (R² = 0.72; p = 0.005) and a moderate association for S. Typhimurium (R² = 0.62; p = 0.091) originating from faecal matter and their presence in water sources. Furthermore, a very weak association was found for Y. enterocolitica (R² = 0.15; p = 0.40). During the dry season, a weak association for S. Typhimurium (R² = 0.313; p = 0.032), a moderate one for S. flexneri (R² = 0.4; p = 0.001), and a very weak one for Y. enterocolitica (R² = 0.04; p = 0.281) were established. The association between C. jejuni detected in faecal matter and in water sources was not determined during both seasons due to the lack of detection of this bacterium in these matrices.

3.5. Associations Between Target Enteropathogenic Bacteria in Water Sources and in Household Container-Stored Water

The associations between the enteropathogenic bacteria detected in water sources and those in household (HH) container-stored water ranged from strong to very weak (strong (R² > 0.6) to very weak (R² < 0.2), with weak associations falling in the range of R² = 0.2–0.39) in the two (2) local municipalities (

Table 8. Associations between enteropathogenic bacteria detected in water sources and enteropathogenic bacteria detected in household container-stored water.

Wet Season
S. Typhimurium					S. flexneri				Y. enterocolitica
Water Sources and HH * Container-Stored Water	Coefficient	Standard Error	R2	p Value	Coefficient	Standard Error	R2	p Value	Coefficient	Standard Error	R2	p Value
River water	0.334	0.141	0.567	0.092	0.073	0.114	0.344	0.014	3.86	1.021	0.190	0.169
Dam water	0.282	0.064	0.184	0.066	1.265	0.172	0.227	0.028	1.285	0.015	0.147	0.187
Treated water	0.358	0.258	0.246	0.261	3.216	0.135	0.164	0.225	2.284	0.731	0.101	0.125
Spring water	0.418	0.361	0.346	0.041	0.447	0.095	0.358	0.057	1.135	0.074	0.141	0.211
Hand-dug well water	0.364	0.017	0.651	0.003	2.319	0.103	0.424	0.022	0.354	0.059	0.046	0.141
Dry Season
River water	0.352	0.135	0.319	0.041	1.117	0.026	0.211	0.147	1.715	0.255	0.024	0.251
Dam water	0.213	0.510	0.140	0.242	2.465	0.324	0.105	0.028	0.386	0.024	0.165	0.138
Treated water	0.485	0.143	0.194	0.191	0.317	0.069	0.121	0.242	1.081	0.312	0.067	0.111
Spring water	0.418	0.263	0.117	0.032	1.693	0.096	0.138	0.001	0.187	0.151	0.125	0.313
Hand-dug well water	0.494	0.412	0.292	0.024	2.754	0.010	0.291	0.038	0.165	0.021	0.103	0.276

* HH = household.

). During the wet season, S. Typhimurium detected in hand-dug wells and in HH container-stored water had a strong association (R² = 0.65), followed by S. Typhimurium detected in river and HH container-stored water, which had a moderate association (R² = 0.5). Weak to very weak associations were established between S. Typhimurium detected in household container-stored water (R² = 0.184), dam water (R² = 0.246), and treated spring water (R² = 0.36). Furthermore, the associations were shown to be statistically significant (p ≤ 0.05), with the exception of the association between the treated water and the HH container-stored water (p ≥ 0.05). S. flexneri detected in water sources and HH container-stored water showed associations ranging from moderate to very weak. S. flexneri detected in hand-dug well water, spring water, and HH container-stored water displayed a moderate association (R² = 0.42 and 0.358, respectively), followed by the association between S. Typhimurium detected in river and HH container-stored water, which had a weak association (R² = 0.344), and between S. flexneri detected in dam water, treated water, and HH container-stored water, which exhibited a very weak association (R² = 0.22 and R² = 0.164, respectively). These associations were also shown to be statistically significant (p ≤ 0.05), with the exception of the association between the treated water and the HH container-stored water (p ≥ 0.05). Y. enterocolitica detected in all water sources and in HH container-stored water displayed very weak associations (R² = 0.190 to 0.046), with statistical significance (p ≤ 0.05).

During the dry season, S. Typhimurium detected in water sources and in HH container-stored water showed associations ranging from weak to very weak. S. Typhimurium detected in river water, hand-dug well water, and HH container-stored water exhibited a weak association (R² = 0.319 and R² = 0.292, respectively), followed by S. Typhimurium detected in treated water, spring water, dam water, and HH container-stored water, which had a very weak association (R² = 0.194, R² = 0.117, and R² = 0.140, respectively). These associations were shown to be statistically significant (p ≤ 0.05), with the exception of the association between treated water, dam water, and HH container-stored water (p ≥ 0.05). Similar results were observed with S. flexneri, where this enteropathogenic bacterium detected in river water, hand-dug wells, and HH container-stored water had a weak association (R² = 0.211 and R² = 0.291, respectively), followed by S. flexneri detected in spring water, treated water, dam water, and HH container-stored water, which had a very weak association (R² of 0.138, R² of 0.121, and R² of 0.105, respectively). However, these associations were shown to be statistically significant (p ≤ 0.05), with the exception of the association between treated water, river water, and HH container-stored water (p ≥ 0.05). Furthermore, Y. enterocolitica detected in all water sources and in HH container-stored water had a very weak association (R² of 0.165 to R² of 0.024), and this association did not show statistical significance (p ≥ 0.05).

4. Discussion

The pollution of water sources by human and animal faecal matter carrying potential pathogens poses an environmental threat, hence affecting the microbiological quality of water sources. Reports have illustrated the impact of water sources polluted by human faecal matter on public health [22]. Furthermore, animals have also been regarded as hotspot reservoirs for several enteric pathogens [23,24]. In rural communities, most households are reliant on water sources where the microbial quality is unknown, and Vhembe District Municipality is no exception. In this district municipality, communities are still dependent on untreated water sources that are microbiologically unfit for human consumption [25,26,27]. Despite various national sanitation programs, such as the Bucket Eradication Programme and the Rural Household Infrastructure Programme (RHIP), access to improved sanitation remains limited in parts of Vhembe District. According to Statistics South Africa [9], a significant proportion of households in rural Limpopo still rely on unimproved sanitation facilities, with open defecation practiced by over 2% of the national population—higher rates are observed in municipalities such as Thulamela and Collins Chabane. While municipal interventions, including pit latrine construction and conventional flush toilet installations, have been implemented, their effectiveness has been constrained by limited resources, poor maintenance, and insufficient community involvement.

Moreover, livestock waste management in these areas is largely informal, as animals frequently roam and graze near water sources, further exacerbating environmental contamination.

However, very few studies have focused on tracking the sources of the contamination impacting these water sources. Consequently, the aim of the present study was to establish the association between target enteropathogenic bacteria in faecal waste originating from open defecation sites and their presence in water sources used for multiple purposes by selected rural communities of Vhembe District Municipality in South Africa. The qPCR technique used in this study allows for the detection of a single enteropathogenic bacterium through targeting a specific DNA sequence [28]. Furthermore, this technology is able detect and quantify low amounts of target DNA, thereby increasing the sensitivity of detection of microorganisms at low concentrations, especially in environmental samples [29]. The results of this study demonstrated the efficiency of the primers, which ranged from 95% to 100%. In other words, the desired qPCR efficiency normally ranged from 95% to 100%, within a slope ranging between −3.6 and −3.3. The slopes in this study were within the desired slope range, which resulted in the qPCR efficiency of the primers [30].

Although C. jejuni and Y. enterocolitica were detected in human, cow, dog (as shown in Table 1 and Table 3), and pig faecal samples (Table 2) collected in the vicinities of some water sources within the two (2) municipalities, S. Typhimurium and S. flexneri were found to be the predominant pathogens in both animal and human faecal samples, despite the low number of open defecation sites sampled. However, the situation was found to be different in a study conducted in Burkina Faso, where Salmonella was detected only in animal faecal waste—specifically, in swine (16%), cow (52%), and chicken (55%) waste [31] Despite the predominance of S. flexneri in both human and animal faecal samples, the results also differed from those reported in a study conducted in Pantnaga, India, where S. flexneri was detected only in human faecal waste (3%), whilst cow and chicken faecal samples tested negative for this enteropathogenic bacterium [32]. The low prevalence of C. jejuni in human and animal faecal samples recorded during the present study corroborates the findings of a study conducted in the Dodoma Rural and Bagamoyo districts in Tanzania, where the prevalence of this bacterium in human and cow faeces was also reported to be low [33]. Moreover, C. jejuni was not the only pathogen that displayed a low prevalence during the present study; Y. enterocolitica was also detected at a low prevalence, mostly in pig faecal samples (Table 3). Pigs have been reported to be a major reservoir of enteropathogenic Y. enterocolitica in most countries across the globe [34]. Additionally, the prevalence of Y. enterocolitica has been reported to be low, with rare foodborne outbreaks, and it has been isolated from animals, raw food, the environment, water, and humans [35,36,37].

Water sources generally contain a variety of enteropathogenic bacteria, originating from municipal sewage discharge, rainfall runoff from agricultural land, and direct human and animal faecal deposition [38]. To establish the sources of faecal pollution in water sources used by the rural communities of VDM, water source samples and human and animal faecal waste samples collected in the vicinities of these water sources were concurrently tested for similar enteropathogenic bacteria. The advanced technique qPCR revealed that the target populations within the local municipalities of VDM are exposed to S. Typhimurium, S. flexneri, C. jejuni, and Y. enterocolitica. Human and faecal waste could, therefore, be the sources of faecal contamination of water sources, which may pose a health risk to these communities. It has been reported that S. Typhimurium and S. flexneri are common in the environment when sanitation is inadequate [39]. This observation aligns with the findings of the current study, which revealed the predominance of these two enteropathogenic bacteria in faeces collected from open defecation sites and in water sources (surface water, treated surface water, spring water, hand-dug well water, or household container-stored water) commonly used by the communities of CLM and TLM (Table 4), regardless of the seasonal variations in terms of their prevalence. While S. Typhimurium and S. flexneri were detected in the river water source (Mutshindudi River) during this study (Table 5), previous studies have reported the prevalence of Salmonella spp. in the same water source [26,40]. Salmonella species in water are known to not only multiply significantly but also survive for long periods, especially for many months in dried animal faeces [41]. At the household level, S. Typhimurium was detected at a high frequency during the wet season (33%), in contrast to the dry season (25%), in HH container-stored water. The results obtained in this study show that human faeces (due to open defecation) and animal faeces could be considered as the sources of faecal pollution of this water source, and they corroborate the findings of a study by Schriewer et al. [42], who also detected humans, cattle, and dogs as sources of faecal pollution in surface water (rivers).

Groundwater sources are perceived as clean in most rural communities and not usually subjected to any treatment. This study has demonstrated the presence of enteropathogenic bacteria in groundwater sources and in the subsequent storage of these water types at the household level (Table 6 and Table 7). The prevalence of S. Typhimurium and S. flexneri in groundwater sources (springs, boreholes, and hand-dug wells) is a matter of concern as people who use these contaminated water sources may be prone to gastrointestinal diseases. In studies conducted in rural communities of China, spring water sources also harboured S. Typhimurium [43,44]. Once again, these findings confirm the ubiquity of this enteropathogenic bacterium in water sources used by rural communities globally. Not only were S. Typhimurium and S. flexneri detected in groundwater sources; Y. enterocolitica was also detected, although at low frequencies, in HH container-stored water and only during the wet season (16%) (Table 6). The hand-dug well water sources in TLM were found to be microbiologically unsafe, as they harboured S. Typhimurium and S. flexneri during both seasons (Table 7). Furthermore, at the household level, S. flexneri was detected at the highest frequency during the wet season (43%) and during the dry season (32%) in HH container-stored water (Table 7).

The overall results of this study show higher detection frequencies of S. Typhimurium and S. flexneri during the wet season compared to the dry season (Table 4, Table 5, Table 6 and Table 7). The high prevalence during the wet season could be a result of surface runoff due to frequently heavy rainfall events in warmer months [45]. The regional temperature and precipitation lead to the persistence and proliferation of waterborne pathogens. Furthermore, according to Pandey et al. [46], the detection frequency of pathogens is lower in the dry season. Similar observations occurred at the household level, where the detection frequency of these enteropathogenic bacteria was lower in treated water prior to storage in households, in contrast to an increased detection frequency in HH container-stored water. Although, in South Africa, approximately 88% of households have gained access to an improved drinking water source [47], most households in rural communities still store water for domestic purposes [48]. It has been demonstrated that, during storage, the microbial quality of drinking water deteriorates as a result of various processes. These include a lack of or inadequate cleaning of storage containers, poor hygiene practices in dwellings, and/or biofilm formation on the inner surfaces of household storage containers, which enhances the growth of pathogenic bacteria inside the containers [49,50,51].

Regardless of the types of water sources used by the communities of the target local municipalities, this study observed an association between enteropathogenic bacteria in faecal matter and in water sources (Table S3, Supplementary Materials). During the wet season, a strong association was identified between Shigella flexneri and S. Typhimurium (R2 of 0.7 and R2 of 0.619, respectively) in faecal matter and in water sources. Moreover, the association was statistically significant for both enteropathogenic bacteria, with p-values below 0.05 (Table S3, Supplementary Materials). During the dry season, S. flexneri had a moderate association (R2 of 0.4), while S. Typhimurium had a weak association (R2 of 0.313) during the wet season, and the association was shown to be statistically significant for both enteropathogenic bacteria (p-value < 0.05). Only Y. enterocolitica had a very weak association during both seasons (R2 of 0.151 and p-value of 0.401 during the wet season and R2 of 0.044 with a p > 0.05 during the dry season). The strong association observed for S. flexneri (R² = 0.7) reinforces the conclusion that faecal contamination is a primary driver of its presence in water sources, particularly in areas practicing open defecation near water bodies. In contrast, the weak association observed for Y. enterocolitica (R² = 0.15) may point to alternative transmission pathways or environmental reservoirs beyond direct faecal input. Y. enterocolitica is known to persist in soil, water, and aquatic environments, particularly under cooler temperatures, and has been isolated from biofilms, vegetables, and surface waters, where faecal contamination is not always evident. Its ability to survive in low-nutrient environments may allow it to persist independently of recent contamination events. These findings highlight the need to consider non-faecal environmental niches and food-related exposure pathways for certain pathogens, especially those like Y. enterocolitica, which exhibit weak detection correlations with traditional faecal markers.

An association between enteropathogenic bacteria detected in water sources and HH container-stored water was also identified (Table 8), and a strong association was established between S. Typhimurium detected in hand-dug well water sources and in HH container-stored water during the wet season (R2 of 0.651), with a p-value of 0.003, which indicated statistical significance. Field observations confirmed that most of the hand-dug wells included in this study were unprotected and shallow, lacking structural covers, proper linings, or drainage features. These wells were often located in low-lying areas prone to surface runoff, especially during the wet season, making them highly susceptible to contamination from nearby open defecation sites and livestock activity. The absence of fencing or barriers also allowed direct access by animals, further increasing the risk of faecal contamination. These conditions likely contributed to the strong statistical association between hand-dug well water and the presence of enteropathogenic bacteria in household-stored water. However, during the dry season, a weak association was established between S. Typhimurium detected in river water sources and in HH container-stored water (R2 of 0.319), with a p-value of 0.041, which was also significant. Open defecation might be linked to the pollution of water sources in CLM and TLM, as associations have been observed between the target pathogens and water sources, especially for S. flexneri and S. Typhimurium. These findings highlight the urgent need for integrated interventions to reduce the microbial contamination of water sources and improve public health outcomes. The most important strategies include (i) improving sanitation infrastructure, particularly by eliminating open defecation through the construction of lined pit latrines at safe distances from water sources; (ii) protecting water sources, especially hand-dug wells and springs, with fencing, proper drainage, and concrete linings to prevent surface runoff contamination; (iii) enhancing household water hygiene, including the use of covered, narrow-mouthed containers and regular cleaning practices; (iv) community education and awareness programs on hygiene, safe water handling, and the health risks of waterborne pathogens; and (v) monitoring of residual disinfectant levels in treated water to ensure continued microbiological safety through the distribution system. These strategies should be implemented through a community-based, multisectoral approach, supported by local authorities, health departments, and water management agencies.

5. Conclusions

This study highlights the significant role of enteropathogenic bacteria in the faecal contamination of water sources used by rural communities in Vhembe District. The predominance of S. Typhimurium and S. flexneri in both animal (pig, cow, chicken, and dog) and human faecal matter, in contrast to the low prevalences of C. jejuni and Y. enterocolitica, underscores the critical public health risks posed by these bacteria. The findings from the regression model demonstrate a statistically significant association between the presence of S. Typhimurium and S. flexneri in faecal matter and their detection in water sources and household-stored water. This evidence confirms that both human and animal faecal matter are key sources of these pathogens, which are transported into community water supplies regardless of their origin. This study provides compelling evidence that faecal contamination, particularly from open defecation and unmanaged animal waste, has a direct impact on water quality. These findings emphasise the urgent need to control such practices near water sources to mitigate the transmission of enteropathogenic bacteria, which threaten the health of rural communities in Vhembe District. Policymakers and local stakeholders should prioritise the development and implementation of locally adapted water, sanitation, and hygiene (WASH) strategies.

6. Recommendations

Future research can build on the current findings by assessing the impacts of seasonal rainfall patterns and hydrological conditions through the integration of environmental and meteorological data; evaluating the effectiveness of household-level interventions, such as container disinfection and point-of-use water treatment technologies; and investigating behavioural and socioeconomic factors that influence sanitation and hygiene practices. Moreover, the incorporation of residual disinfectant monitoring, molecular typing, and pathogen viability assays would provide deeper insights into contamination risks and health implications. Collaborative research involving local communities, health departments, and environmental scientists will be crucial for developing evidence-based, scalable interventions.