Isolation and characterization of Escherichia coli pathotypes and factors associated with well and boreholes water contamination in Mombasa County

Introduction Safe water for human consumption is important, but there is a limited supply. Mombasa County has water shortages making residences rely on other sources of water including boreholes and wells. Microbiological evaluation of drinking water is important to reduce exposure to water borne enteric diseases. This cross sectional study aimed at determining the frequency and characterization of Escherichia coli (E. coli) pathotypes from water samples collected from wells and boreholes in Mombasa County. Methods One hundred and fifty seven (157) water samples were collected from four divisions of the county and a questionnaire administered. The samples were inoculated to double strength MacConkey broth and incubated at 370C for up to 48 hours. Positive results were compared to the 3 tube McCrady MPN table. The E. coli were confirmed by Eijkman's test and antibiotic susceptibility carried out. Using polymerase chain reaction (PCR), the E. coli were characterized to establish pathotypes. Results One hundred and thirty one (n = 131; 83.4%) samples had coliform bacteria with only 79 (60.3%) samples having E. coli. Significant values (<0.05) were noted when coliforms were compared to variables with E. Coli showing no significance when compared to similar variables. E. coli (n = 77; 100%) tested were sensitive to Gentamicin, while all (n = 77; 100%) isolates were resistant to Ampicillin. PCR typed isolates as enteroinvasive E. Coli (EIEC). Conclusion Findings suggest that coliforms and E. coli are major contaminants of wells and boreholes in Mombasa County. The isolates have a variety of resistant and sensitivity patterns to commonly used antibiotics.


Introduction
Water is an important resource that is prone to bacterial contamination from a variety of hosts including mammals and avian species [1]. The rapid expansion of Mombasa County has led to the residents relying on groundwater to supply them with portable water [2]. Drinking water comes from surface water and ground water. Surface water includes rivers, lakes, and reservoirs while Ground water is pumped from wells or boreholes that are drilled into aquifers. However due to dwindling resources and faulty sanitation especially in developing world makes the availability of safe water almost unattainable, this is due to bacterial and chemical contamination [2]. Water-borne diseases are one of the major public health problems in developing countries. It is estimated that contaminated water has caused more than 20 million deaths [1], of which more than 80% were among children under age five [3]. In the developing world, more than one billion people have no safe drinking water, or water for washing their food, hands and utensils before eating, while 2.4 billion also have no adequate sanitation [4].
This leads to; water-borne diseases (e.g. cholera, typhoid), waterrelated diseases (e.g. malaria, yellow fever, river blindness, sleeping sickness), water-based diseases (e.g. guinea worm and bilharzias), water-scarce diseases (trachoma and scabies), diarrhea. Mombasa and the Coast province experience perennial water shortages. There is no sewerage system except within Mombasa Island. Shallow wells are dug near toilets or septic pits. Outbreaks of cholera and dysentery occur during raining seasons or shortly after the rains [2,5]. Exchange of microbes between wells and toilets/septic pits has been documented [6]. Mombasa gets most of its water from Mzima springs, Marere and Baricho water works. However ground water forms an important source of water for Mombasa and its environs. This study intends to isolate and characterize Escherichia coli pathotypes and possible factors associated with wells and boreholes water contamination in Mombasa County [2]. After getting the number of wells/bore holes to be sampled per division, wells and borehole were assigned numbers and simple random sampling was used to identify the well and boreholes to be sampled.Water samples (200mls) were collected from bore holes and wells using sterile water collection labeled bottles. For water that had been treated with chlorine, 5% sodium thiosulfate was added to the sterile bottles to neutralize the chlorine. The bottles were placed in a cool box and transported to the laboratory for processing. At the site of collection, a questionnaire was administered to assess and determine the risk factors.  Antibiotic sensitivity testing: this was determined by picking1-2 colonies of the isolated organism to obtain 0.5 MacFarland standard, then was spread/streaked onto the Muller Hinton agar and the disc for drugs (Table ) were be placed on the media. The plates were incubated at 35°C for 18-24 hours and the zones of inhibition

Association between coliform and Escherichia coli against variables tested
The Table 1, Table 2 compared the association between different variables with detection of coliform and E. coli contaminants. The Page number not for citation purposes 4 results of coliform detection determined that sampling site location (X 2 value = 13.308, p value = 0.004), recent pump overhaul/repair (X 2 value 13.308, p value = 0.003) and distance to pit latrine from water source (X 2 value 9.113, p value = 0.021) had significant association. No association could be determined when detection of E. coli was compared to the variables tested.

Molecular characterization of E. coli pathotypes
To further characterize the Escherichia coli samples isolated in this study from the contaminated water samples, two molecular assays were carried out using type specific primers were used. The first test was a conventional multiplex PCR to detect three common pathotypes of Escherichia coli in developing countries that included ETEC, EPEC and EAEC. The primers (Table 3)  The isolated E. coli from this study were EIEC as shown in Figure   2 and representative real time PCR CT values (Table 4).

Discussion
The development and standardization of bacteriological indicator organisms as indicators for faecal contamination of water has developed over time with coliform bacteria and Escherichia coli being finally accepted as indicators for faecal contamination of water samples [7,8]. The presence of coliform bacteria andE. coli in water is an indicator of recent fecal contamination indicating the possible presence of disease-causing pathogens, such as bacteria, viruses, and parasites [9,10]. This also led to the development of the standard, most probable number (MPN) that requires that less than one tube in all the dilutions used to detect contamination of water should show contamination. Anything above this value would indicate contamination of a water sample [7,11]. Studies carried out in the same area show that there is the presence of both coliforms and E. coli [2]. This could be due to the lack of sanitation due to expansion of towns and or counties [2,11]. The results of this study have indicated that majority of the samples collected by the study were contaminated by coliforms and also E. coli thus indicating recent faecal contamination. In this study, many variables were tested against coliform and E. coli detection, but only location of well samples collected, recent overhaul of sampling site and distance of water source and pit latrine indicated significant relationships when tested against the presence of coliforms but not E. coli detection. These similar significant relationships could not be established when compared to a previous study in the same area by Munga, and others in 2005. Studies in Zimbabwe have showed decreased contamination in distances that are more than 5 meters between the water source and source of contamination [12]. Even though this study did not look at practices of water storage, other previous studies have indicated that when people have collected water from protected water sources, they may have a tendency to consume the water without treatment because of belief chlorine will protect them [13], leading to outbreaks. Also other studies have showed that contamination of water sources can be linked to contaminated hands and collection containers such as cups [14].
Unprotected water sources have been implicated as sources of water contamination [14]. Also important to note is that, this study detected the E. coli subtype EIEC, which is associated with diarrhea [15]. This strain of E. coli causes watery, dysentery-like diarrhea, associated with cramps and fever [16]. It leads to the inflammation of the large intestines and occurs commonly in developing countries [16]. These bacteria in Kenya, have been isolated from stool samples [17], but have never been isolated from water sources before. A limitation to this study includes thenone detection of other bacterial species associated with water contamination from the samples. None determination of physical and chemical characteristics such as chlorine, calcium carbonate, ammonium, phosphates, nitrates and sulphates levels is also another limitation of this study is compared to other similar studies from the same County and in the country [2,18]. Surveillance is an important tool in that it contributes to protection of health. According to WHO, in populations having more than 100,000 people, it is expected that the quality of drinking water samples should have proportions of between 85-99% clean/without E. coli [7]. What is known about this topic  The detection of E. coli in water has been ongoing for a long time. This microorganism is associated with fresh fecal contamination of water sources. This is because the microorganism resides in the guts of animals, birds and humans and will only be found in water when open defecation occurs or when pit latrines are close to water sources such as boreholes and springs.

What this study adds
 This study has gone further by not only detecting E. coli in portable water from boreholes and springs, but also subtyping the E. coli to determine which pathotypes are contaminating these water sources. This is the first time that this has been done in Kenya. Sub typing of E. coli is mainly done from diarrheal samples.
 The study has also looked at other factors distance between pit latrine and water sources and its effects on contamination of these sources. Again this study has highlighted that treatment of water with chlorine will not affect contamination with E. coli of these water sources.
Probably the contamination is occurring from other sources.