Assessment of bacterial diversity in hand rinse, stored and drinking water in low-income neighbourhoods of Ibule-Soro, Nigeria

Water is an important medium through which many illnesses are spread in the human population. The essential role it plays in human survival cannot be over-emphasized, especially in the sustenance of life and promotion of health. Water, sanitation and hygiene (WASH) has a significant impact on human health and general well-being (Orimoloye et al. 2015). Inadequate WASH remains a critical challenge in many parts of the world. For instance, over 2 billion people lack access to readily available safe water at home, approximately 263 million people spend over 30 min per trip collecting water from external sources and about 159 million people drink untreated water from sources such as streams or lakes (WHO 2017).

Hand hygiene may be described as the act of removing or killing microorganisms on the hands. It is an effective method of preventing the spread of communicable diseases and infections (Martin 2023). Globally, about 2.3 billion people do not have handwashing facilities with water and soap available at home. Low-income countries have numerous households without access to basic hand hygiene (Gizaw et al. 2023). In addition, those living in rural areas are generally more disadvantaged than people living in urban areas in terms of access to basic handwashing facilities (UNICEF 2020). In rural households, where resources and infrastructure may be limited, the microbial contamination of water sources and inadequate hand hygiene practices may lead to the spread of waterborne diseases as hands may serve as vehicles for disease transmission if not properly cleaned (Prüss-Üstün et al. 2008; Dreibelbis et al. 2014). The high prevalence of enteric bacteria on hands indicates the potential for the contamination and transmission of pathogenic microorganisms. Poor hand hygiene practices, including inadequate handwashing, can contribute to the spread of enteric diseases. Regular handwashing with soap and water is essential to reduce the risk of infection. Chinakwe et al. (2019) reported that pathogenic organisms were present in hand-wash water samples and that bacteria isolated from the samples were Escherichia coli, Pseudomonas sp., Shigella sp. and Enterobacter sp.

Household water storage remains a necessity in many communities worldwide, especially in developing countries. Water collected from sources with good microbial quality may become contaminated during storage in households (Tadesse et al. 2010; Adane et al. 2017). The tanks or vessels used for storage and related user practices may be a source of water contamination (Manga et al. 2021). Water storage containers when not properly cleaned and maintained may serve as a reservoir and play a significant role in the contamination of water. Other sources of contamination may include the source water, the air and the hands of people who handle the containers. Safe drinking water is a major challenge for rural communities across the globe. Clean water is crucial to human health and well-being (Wu et al. 2023). Drinking unsafe water impairs human health through illnesses such as diarrhoea, dysentery, and typhoid (Olalemi et al. 2020, 2021; Udoh et al. 2021).

Rural communities, including Ibule-Soro, often have challenges in terms of access to safe water. Microbial contamination of stored and drinking water may pose significant health risks to humans, and proper hand hygiene practices play a major role in controlling the spread of infectious diseases. The study aimed to determine the bacterial communities and levels of enteric bacteria in hand rinse, stored and drinking water from selected households in rural neighbourhoods of Ibule-Soro in Akure, Ondo State, Nigeria. Understanding the levels and diversity of enteric bacteria on hands, an important vehicle for pathogen transmission, stored and drinking water is important in identifying potential health risks, and this may be used to develop effective strategies for reducing the risk of contamination in order to prevent the occurrence of waterborne diseases in rural areas of Ibule-Soro.

The study was conducted in the community of Ibule-Soro, a town in the Ifedore Local Government Area of Ondo State, South-Western Nigeria, with a population of approximately 30,000 people. It has an area of 1.318 km² and is located between latitude 7.3173°N and 5.1172°E. Most of the residents of Ibule-Soro engage in subsistence farming, trading and civil service. The community has a primary school and a health centre, and five households were selected based on the presence of children of less than five years of age, the presence of pit latrines, animals and pets and limited access to safe and clean water. Hand-dug wells are the major sources of drinking water in the households and are situated between 50 and 100 m away from the households. The water is neither heated nor treated in any way before drinking. The residents also fetch the water into large plastic storage containers within the households for domestic use.

Hand rinse, stored and drinking water samples were collected biweekly over six months (i.e., July to December 2023). July represents the peak period of heavy rainfall and September is typically the end of wet months, whereas October to December mark the beginning of dry months to the middle of the dry period when the amount of rainfall is relatively low. Informed consent was obtained before sample collection, and hand rinse, stored and drinking water samples were collected from residents in the selected households following standard procedures. Briefly, hand rinse water samples were collected by asking the residents to wash their hands into a sterile 500 mL container. Stored water samples were collected by dipping a sterile screw-capped bottle into the household storage containers, and drinking water samples were collected directly from the wells. A total of 180 water samples (i.e., 60 hand rinse, 60 stored and 60 drinking water samples) were collected from five households in Ibule-Soro. All water samples were labelled appropriately and transported within 1 h to the laboratory for analysis.

Hand rinse water samples were serially diluted in sterile distilled water to obtain dilutions (3rd and 4th) suitable for bacterial growth, and stored and drinking water samples were examined using the pour plate method. Selective agar, such as Eosin Methylene Blue agar, Salmonella Shigella agar and MacConkey agar were prepared according to the manufacturer's instructions for the enumeration of E. coli, Salmonella, Shigella, Enterobacter, Proteus and Klebsiella. The water samples were filtered through 0.45 μm membrane filters, and the filters were placed on freshly prepared media. Thereafter, the plates were incubated at 37 °C for 24 h, and colonies were counted, calculated and expressed as colony-forming units per 100 millilitres (cfu/100 mL). E. coli had blue-black colonies with a green metallic sheen, Salmonella had colourless colonies with a dark centre, Shigella had translucent colonies, Enterobacter had small pink colonies, Proteus had pale-coloured colonies and Klebsiella had large, mucoid and pink colonies (Dhengesu et al. 2022).

The ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Catalogue No. D4300) was used for genomic DNA extraction. The water samples (250 μl) were vortexed in the ZR BashingBead™ Lysis Tube (0.1 and 0.5 mm) with 750 μl of ZymoBIOMICS™ Lysis Solution for 30 s. A bead beater fitted with a 2 mL tube holder assembly was secured and processed using optimized beat-beating conditions. The ZR BashingBead™ Lysis Tubes (0.1 and 0.5 mm) were centrifuged in a microcentrifuge at ≥ 10,000 × g for 1 min. About 400 μl of the supernatant was transferred to the Zymo-Spin™ III-F Filter in a collection tube and centrifuged at 8,000 × g for 1 min. The Zymo-Spin™ III-F Filter was then discarded, and 1,200 μl of ZymoBIOMICS™ DNA binding buffer was added to the filtrate in the collection tube and mixed thoroughly. About 800 μl of the mixture was transferred to a Zymo-Spin™ IICR column in a collection tube and centrifuged at 10,000 × g for 1 min. The flow-through was discarded from the collection tube, and 400 μl of ZymoBIOMICS™ DNA wash buffer 1 was added to the Zymo-Spin™ IICR column in a new collection tube and centrifuged at 10,000 × g for 1 min, then the flow-through was discarded. Afterwards, 700 μl of ZymoBIOMICS™ DNA wash buffer 2 was added to the Zymo-Spin™ IICR column in a collection tube and centrifuged at 10,000 × g for 1 min and the flow-through was discarded. Approximately 200 μl of ZymoBIOMICS™ DNA wash buffer 2 was added to the Zymo-Spin™ IICR column in a collection tube and centrifuged at 10,000 × g for 1 min. The Zymo-Spin™ IICR column was transferred to a clean 1.5 mL microcentrifuge tube, and 100 μl of ZymoBIOMICS™ DNase/RNase free water was added and incubated for 1 min and centrifuged at 10,000 × g for 1 min to elute the DNA. A Zymo-Spin™ III-HRC filter was placed in a new collection tube and 600 μl of ZymoBIOMICS™ HRC prep solution was added and centrifuged at 8,000 × g for 3 min. The eluted DNA was transferred to a prepared Zymo-Spin™ III-HRC filter in a clean 1.5 mL microcentrifuge tube and centrifuged at 16,000 × g for 3 min. The filtered DNA was then suitable for polymerase chain reaction (PCR).

The extracted genomic DNA samples were PCR amplified using a universal primer pair, 27F and 1492R, targeting the V1–V9 region of the bacterial 16S rRNA gene. The resulting amplicons were barcoded with PacBio M13 barcodes for multiplexing through limited cycle PCR with M13 tailed forward primer/5AmMC6/GTAAAACGACGGCCAGT(N)n and M13 tailed reverse primer/5AmMC6/CAGGAAACAGCTATGAC(N)n. The resulting barcoded amplicons were quantified and pooled in equimolar amounts, and the AMPure PB bead-based purification step was performed. The PacBio SMRTbell library was prepared from the pooled amplicons following the manufacturer's protocol (PacBio 2022).

Sequencing primer annealing and polymerase binding were done following the SMRTlink Link software protocol to prepare the library for sequencing on the PacBio Sequel IIe system. Raw sub-reads were processed through the SMRTlink (v11.0) Circular Consensus Sequences algorithm to produce highly accurate reads (>QV40). These highly accurate reads were then processed through vsearch (https://github.com/torognes/vsearch), and taxonomic information was determined based on QIMME2, DADA2 and MEGAN.

Data obtained were inputted into the Excel Workbook using Microsoft Excel 2019, and descriptive statistics were carried out. One-way analysis of variance (ANOVA) was carried out, and means were separated by Duncan's new multiple range test using SPSS version 23.0.

All cases of values below the detection limit of 1 log₁₀ cfu/100 mL were treated as the detection limit. The load of Salmonella was the highest (2.88 log₁₀ cfu/100 mL) in the stored water and the least (0.1 log₁₀ cfu/100 mL) in the hand rinse water, whereas the load of Shigella was the highest (2.27 log₁₀ cfu/100 mL) in the drinking water and the least (0.1 log₁₀ cfu/100 mL) in the hand rinse water. The load of E. coli was the highest (3.62 log₁₀ cfu/100 mL) in the stored water and the least (0.1 log₁₀ cfu/100 mL) in the hand rinse water. Similarly, the load of Enterobacter was the highest (2.60 log₁₀ cfu/100 mL) in the drinking water and the least (0.1 log₁₀ cfu/100 mL) in the stored water. The load of Klebsiella was the highest (3.60 log₁₀ cfu/100 mL) and the least (0.4 log₁₀ cfu/100 mL) in the hand rinse water. The load of Proteus was the highest (2.95 log₁₀ cfu/100 mL) in the stored water and the least (0.1 log₁₀ cfu/100 mL) in the hand rinse water. Overall, the stored and drinking water samples had higher loads of enteric bacteria than the hand rinse water samples (Table 1).

Furthermore, Klebsiella (29%) had the highest percentage frequency of occurrence in hand rinse water samples, while Salmonella (7.3%) and Proteus (7.3%) had the least. Similarly, in stored water samples, Klebsiella (23.6%) had the highest percentage frequency of occurrence, whereas Enterobacter (7.4%) had the least. In drinking water samples, E. coli (19.9%) had the highest percentage frequency of occurrence, whereas Salmonella (9.2%) had the least (Table 1).

Water is vital for sustaining life and health, making it imperative to ensure its uninterrupted availability in households, as any disruption in the supply of clean and safe water may have negative consequences (Rai et al. 2021). The overall well-being of individuals inhabiting the households may be influenced by the prevalence and distribution of pathogenic bacteria in household water supply. This study investigated the levels of enteric bacteria and bacterial diversities in hand rinse, stored and drinking water obtained from selected households in low-income neighbourhoods of Ibule-Soro, Nigeria. The results revealed that all the stored and drinking water samples had interesting patterns of bacterial loads exceeding acceptable standard limits and guidelines (WHO 2022). The source of bacteria in the drinking water may likely be due to contamination of the water sources (i.e. wells) from point (use of unsterile containers as fetchers) and non-point sources (run-off after rainfall events, animal rearing in the households, etc.). The contamination of the stored water may also be due to the use of unsterile plastic storage containers, inadequate regular cleaning and long storage of water in the containers within the households, whereas inadequate hand hygiene may be responsible for the observed levels of bacteria in hand rinse water samples obtained from selected households.

Globally, E. coli is the preferred microbial water quality indicator and its presence in water suggests faecal contamination. E. coli was detected in all the water samples from households, suggesting faecal contamination and poor hygiene in the household settings. The high number of diarrheal disease cases in low- and middle-income countries is related to sanitation conditions, consumption of untreated water and inadequate hygiene (Salamandane et al. 2021). Bacteria, such as Salmonella, Shigella, Klebsiella and Proteus, in the water samples obtained from the selected household raise significant concerns about the water quality. These bacteria are commonly associated with faecal contamination and may pose associated health risks to consumers (Noor 2023; Olalemi et al. 2023). The load of the bacteria in the water samples that increased in the wet months (July and August) with a gradual reduction in the dry months (October to December) may likely be due to an increased rate of contamination that is often associated with increased precipitation. Salmonella was detected in all hand rinse water samples obtained from households, indicating a ubiquitous presence and inadequate hand hygiene practices (Ikede et al. 2022). This observation is in agreement with Tagar & Ahmed (2022), where the authors isolated faecal indicator bacteria and multiple antibiotic-resistant Salmonella from a butcher's hand. Pickering et al. (2018) also reported that hands were the most strongly associated with increased subsequent risk of diarrheal illness among children under five years old.

Bioinformatic analysis performed on the 16S metagenomics data from the water sample obtained from the most contaminated household, using a culture-based identification method, provided insights into the number of bacterial genomes associated with the samples. A total of 11 different bacterial phyla were detected in the water samples in the selected household. The most abundant phyla were Proteobacteria, Actinobacteria, Bacteriodetes and Firmicutes, of which Proteobacteria had the highest percentage abundance. This is in agreement with Bautista-de los Santos et al. (2016) where the authors reported Proteobacteria as the most abundant phylum in full-scale drinking water distribution systems. The dominance of these four phyla of bacteria may be due to their ability to thrive in water matrices. Studies have shown that phyla such as Proteobacteria, Actinobacteria and Bacteriodetes are commonly found in water samples (Pinto et al. 2012; Prest et al. 2014; Wu et al. 2023). The analysis performed at the genus level revealed the common presence of well-known aquatic bacterial genera such as Novosphingobium, Sphingomonas and Flavobacterium (Allen et al. 2004). Acetinobacter, one of the most abundant taxa encountered in this study, is an aerobic, non-motile, Gram-negative bacterium that is ubiquitous in the environment and has been identified in stored waters, rivers and swamps (Álvarez-Pérez et al. 2013). They exhibit resistance to multiple antibiotics; therefore, their presence in stored and drinking water may pose a significant risk to human health (Zhang et al. 2016). Bacterial species identified from the water samples included Flectobacillus, a Gram-negative, aerobic, rod-shaped, non-motile bacterium that is often isolated in streams (Lu et al. 2023). H. stevensii is a Gram-negative bacterium associated with contaminated environments. It is a human pathogen involved in nosocomial infections possessing virulence factors that may affect immunocompromised individuals. Similarly, H. hydrothermalis has been isolated from hydrothermal fluids and is capable of exhibiting resistance to multiple antibiotics. Ideonella is capable of degrading polyethylene terephthalate plastics (Palm et al. 2019). Their presence in the water samples may likely be connected to the household water storage facility that is made of plastics. Polynucleobacter is abundant in lakes, ponds and streams and has also been detected in groundwater systems (Pitt et al. 2018). Overall, this study demonstrates the need for public health interventions to reduce the contamination of household water supply, especially in low-resource settings. Residents of the households should be encouraged to practice hand washing with soap under running water, use adequate covering and protection of water in household storage containers, regularly clean the storage containers and use the point-of-use water treatment systems to improve the quality of the stored and drinking water.

The presence of E. coli, Salmonella, Shigella, Klebsiella, Proteus and Enterobacter in the hand rinse water samples suggests poor hand hygiene practices in households. The stored and drinking water samples had high loads of bacterial loads exceeding acceptable standard limits and guidelines. The metagenomic data revealed that Proteobacteria, Sphingomonadales and Flectobacillus were the most abundant phyla, orders and species in the water samples. The identification of these organisms emphasizes the urgent need for improved water quality management, sanitation and hygiene practices in low-income rural households.

The authors are grateful to the Department of Microbiology, School of Life Sciences, The Federal University of Technology, Akure, Ondo State, Nigeria for providing appropriate support in terms of equipment and laboratory used for the study.

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

The authors declare there is no conflict.