**2. Methods**

*Pathogenic Bacteria*

age tanks before distribution.

the rise [7], and many unregulated producers exist.

contribute to contamination.

to self-help by using water from boreholes as the only source of water supply for drinking and general use. Use of borehole is a simple way of obtaining potable water from the aquifer below the ground, after which the water can be pumped into stor-

Many people that went into borehole drilling business, which reduced the price of new boreholes, aided the proliferation of boreholes in Nigeria, and many citizens were ready to pay more money in rent for houses, which had boreholes. Furthermore, the dependence on groundwater, which is believed to be purified, is on the increase due to the increasing contamination of the surface water [3]. It is known that properly designed and constructed borehole both ensures the success of the borehole as an adequate supply of water and minimizes the risk of local pollution affecting the source [4]. If a borehole facility is not properly managed, contamination may occur in the process through the accumulation of physical, chemical, and biological agents in the pipelines and storage tanks of a distribution system or water packaging company. One direct use of boreholes is in the production and packaging of drinking water in sachets made from low-density polyethylene sheets. These products are popularly known as "pure water" in Nigeria. From the early 1990s, the production of sachet water increased exponentially and provided jobs for producers and sellers of the product. There is hardly any community in Nigeria without a sachet water facility. It is possibly the most widely consumed commercial liquid in Nigeria, and no sophistication is required for production. The quest for a cheap, readily available, and inexpensive source of potable water contributed to the emergence of sachet water [5], and it is far better and safer than the hand-filled, hand-tied packaged water in polyethylene bag [6] sold in Nigeria in the past. In developing countries, production and consumption of sachet water are rapidly on

Packaged drinking water like the sachet water could be water from any potable source such as tap, well, and rain, which may be subjected to further treatments like decantation, filtration, demineralization, remineralization, and other methods to meet established drinking standards [8, 9]. Packaged water is susceptible to microbial and chemical contamination regardless of their source [10]. Researchers have previously performed microbial analysis of sachet water in Nigeria using different laboratory techniques and found different bacteria and fungi. Occurrence of bacteria could lead to different disease conditions such as gastroenteritis, typhoid fever, cholera, bacillary dysentery, and hepatitis [11]. It has been reported [12] that waterborne diseases account for 80% of illnesses and diseases in developing countries, which leads to the death of several children every 8 seconds. In Nigeria, like most developing countries, various factors predispose packaged sachet water to contamination, and these include poor sanitation and source of raw material for food or water production [13]. Long storage of sachet under unfavorable environmental conditions and lack of good manufacturing practices (GMP) in general also

It has been found that the microbiome dynamically changes during different stages of water treatment distribution and the main important group in the past and present are fecal-associated bacterial pathogens like *Escherichia coli* [14]. However, opportunistic bacteria like *Legionella* and process-related bacteria, which form biofilms, are also a cause for concern [15, 16]. A review [17] elucidated that drinking water comprises a complex microbiota that is influenced by disinfection and that members of the phylum *Proteobacteria* represent the most frequent bacteria in drinking water. It was also pointed out that their ubiquity has serious implications for human health and that the first step to address the persistent nature of bacteria in water would be to identify and characterize ubiquitous bacteria. The manifestation of bacterial contamination in drinking water can become known when

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Water samples from boreholes were collected on different days using Whirl-Pak sampling bags (Nasco, Wisconsin, USA) and analyzed within 2 hours after collection. Twenty private boreholes and 20 different brands of commercial sachet water sold in four areas of a community were analyzed on different days. Sachet water was purchased (five each) from the different areas and were inspected for the inscription of an approved product registration code from the National Agency for Food and Drug Administration and Control (NAFDAC), the Nigerian national regulatory body. It was ensured that the same brand was not purchased twice from one area. The human population of the community (all 4 areas) was estimated to be over 5000 but less than 100,000.

Heterotrophic plate and total coliform count of bacteria were carried out using standard membrane filtration performed previously [21]. A slight modification of the method was introduced. Instead of using factory-made ready to use nutrient media sets, plate count agar (Oxoid, United Kingdom, CM0325) and violet red bile lactose agar (Oxoid, CM0107) for coliforms were prepared and used according to manufacturer's instructions. Briefly, the filtration process involved placing of 100 ml of water sample in a sterile multibranched stainless steel manifold and filter holder system. A 0.45 μm membrane filter was fitted into the filter system after which water was drawn through to retain bacteria on the membrane. The membrane filter was placed on the media prepared and then incubated at 32°C over 48 h for membrane filters placed on plate count agar, whereas incubation at 30°C for 48 h was used for filters grown on violet red bile lactose agar. The heterotrophic count was noted, and estimated coliform results obtained for boreholes and sachet water were compared to WHO quality guidelines for drinking water [22].

#### **3. Results**

#### **3.1 Heterotrophic and total coliform count of borehole samples**

This survey was carried out to have an overview of the bacterial load in water quality of some boreholes in the community surveyed. The borehole owners were apprehensive and thought they were being investigated for possible closure. To allow sample collection, it was agreed that the name of borehole owners and their location should remain anonymous when the findings were published. Results showed that borehole samples from area "C2" had the highest heterotrophic aerobic count. Two boreholes had counts of over 500 Cfu/mL, which is above the

recommended heterotrophic limit [21]. All the other samples were below 500 Cfu/mL. Seven boreholes indicated the presence of coliforms because purple-pink colonies, which were 1–2 mm in diameter surrounded by a purple zone, were formed on the plates after incubation. Samples C2a, C2b, C2c, C2d, and C2e had coliform count of 17, 15, 9, 6, and 5 Cfu/mL, respectively, whereas samples C3b and C4b had coliform count of 4 and 2 Cfu/mL. The rest of the samples had no coliform on the plate used after incubation. A definitive trend was that samples with the highest heterotrophic count had the most coliform count (**Figure 1**).
