Potential Risk Assessment of Drinking Water Source Exposed to Contamination Using Microbial Indicators and Multiple Antibiotic Resistance Index

In this study, drinking water sources including drilled wells (DW), water tankers (WT), stabilized water tanks (SWT), and plastic bottles (PB) as sequential sample were assessed at Dhamar City, Yemen to investigate the microbial contamination and the potential risk of contamination using microbial indicators and multiple antibiotic resistance index. The sequential sample involving, 5 drilled wells, 10 water tankers, 20 water tanks, and 100 plastic bottles. The study encompassed five sequential samples. Each water sample was collected in triplicate and analyzed for Escherichia coli as a microbial indicator and antibiotic susceptibility testing. Results indicated that all the water samples were contaminated by E. coli and total coliform exceeded the acceptable levels recommended by WHO of microbial quality of drinking water. E. coli isolates (240) showed high resistance to the tested antibiotics comprising 79.82% to ampicillin, 78.32% to gentamicin, 67.5% to ceftriaxone, 19.98% to ciprofloxacin, 18.32% to amoxiclav, and 33.34% were resistant to tetracycline. The multiple antibiotic resistance index of E. coli that showed resistant to three antibiotics ranged from 0.19 to 0.24 for all the tested samples and exceeded the threshold value of 0.2 for all samples except sequential sample 4, indicating a high risk of contamination for drinking water effected by anthropogenic activities related to urbanization, accumulation of microbial contamination during the various transferring processes of water from the source into households, as well as misuse and greater exposure to antibiotics in humans and poultry farms, which may pose a high ecological risk to the waters. doi: 10.5829/ijee.2021.12.01.10


INTRODUCTION 1
Water is essential to mankind's life, so an adequate, safe, and accessible water supply must be available to all [1][2][3]. Access to safe drinking water has been recognized as a basic human right; nevertheless, many people particularly in developing countries struggle to get access to safe water [4,5]. In developing countries, the residents of rural and urban areas depend on both surface water and groundwater as drinking water sources without any treatment. Therefore, the use of these sources has been associated with outbreaks of lethal diarrheal diseases [6][7][8]. Frequently, the ability to make comprehensive decisions regarding water and sanitation is limited by data scarcity, particularly concerning the effects of environmental conditions on water contamination.

*Corresponding
Author Email: Fawaz.albadai@tu.edu.ye (F. Al-Badaii) Therefore, it is imperative to investigate the microbial contamination of water sources in terms of occurrence and causes [8]. Also, many rural and urban communities have forced to use untreated water sources as alternative supplies such as springs, dams, and rainwater or water tankers due to the failure to meet the increasing demand for drinking water services [9][10][11]. Usually, the microbial water quality of these sources is significantly influenced by a microbial load of atmosphere washing, surface runoff, and adjacent agricultural activities [4,[12][13][14][15].
Water sources contaminated by human and animal excreta is a vehicle for the transmission of microorganisms responsible for several diseases, particularly in rural and urban areas where inhabitants entirely depend on untreated drinking water [2,7,[16][17][18]. From the pathogens present in water sources, enteric pathogens such as E. coli, Salmonella spp., Shigella spp. and Vibrio cholerae that are the most spreading and frequently transmitted through the ingestion of contaminated water and food to human [14,19,20]. The risks associated with these pathogens increase if were resistant to the antibiotic. Commonly, acute diarrheal diseases remain a major public health problem associated with the consumption of untreated water, poor hygiene facilities, and low financial resources [3,21]. The mortality of diseases associated with water exceeded five million people per year, 50% related to microbial intestinal infection such as Vibrio cholerae [22].
Indeed, the microbial quality of drinking water was the major focus of the water treatment plants; therefore, the occurrence of antibiotic resistant bacteria in drinking water may significantly affect public health and cause an emerging problem of the industry of drinking water [1,23,24]. Prevalently, microbial indicators particularly E. coli has been used worldwide as a tool to assess the microbial quality of drinking water, the occurrence of E. coli in drinking water indicates fecal contamination that may be accompanied by waterborne diseases which cause harms to human health [25][26][27]. Currently, the concern is growing respecting the water quality influenced by antimicrobials agents due to they may accelerate the selection for antibiotic-resistant bacteria [28,29]. In recent decades, the antibiotic resistance among enteric bacteria has increased since the escalating usage of antibiotics; therefore, bacteria with antibiotic resistance are isolated from various water sources such as surface water and groundwater [25,30,31]. Additionally, the microbiological contamination of water is the primary cause of waterborne diseases [32], and the ingestion of water contaminated with excreta causes most of the hazardous health risks [18,27].
The number of suspected cholera cases reported fro m October 2016 to December 2018 were 1417156 including 2870 associated deaths with a case fatality rate of 0.20%. Besides, in 2019, the suspected cholera cases started increasing and reached more than 29500 cases during the first three months. The trend of suspected cases then fluctuated over the following months until June when it stabilized. Only in January 2019, 4429 samples of 90552 samples have been confirmed as cholera-positive by the culture at the central public health laboratories [33].
Dhamar city is one of the Yemeni cities that lack access to clean water and sanitation, as reported by the world health organization [33]. Dhamar has recently confronted rapid population growth associated mainly with the conflict in Yemen that lead to the arrival of large numbers of national refugees from struggle areas to the city. The WHO stated that most of the population in Dhamar city relies essentially on drilled wells water transported by water tankers as sources of drinking water [33]. The deterioration of the water distribution system due to the war events make the water tankers are the alternative source of drinking water, although its consideration one of contamination source associated with the spreading of various diseases by various microorganisms such as cholera which become a widespread epidemic in Dhamar city. The microbial contamination of drinking water at Dhamar city is a public health problem. Therefore, for the best understanding of the potential risks posed by the microbial contamination of this water, this study aimed to precisely determine the microbial indicators as evidence on the fecal contamination as well as its focus on the occurrence of several antibiotic-resistant E. coli in drinking water sources to assess the potential risk using multiple antibiotic resistance index (MARindex).

Study area
Dhamar city is located at 14°33'08" N. and 44 023'50" E., in the middle of the northern portion of Yemen and far from the capital of Yemen (Sana'a city) with around 100 km to south direction ( Figure 1). In Dhamar city, the unique source of drinking water is groundwater. According to the Census of 2004, the population of Dhamar city is 175159 residents [34]. The annual average of rainfall at Dhamar city ranges from 200 to 400 mm/ y [34][35][36]. The highest rainfall occurs in July and August as well as lower rainfall amounts in March, April, and May with an annual average of temperature around 24°C which was measured during 2019 [34,35].

Sampling sites and samples collection
In this study, sampling was selected during the period of cholera outbreaks (July 2019) as well as sampling sites were selected to include entirely Dhamar city, the selection of sampling sites was depended on study area survey to pick out the sites considered as main sources of drinking water at Thamar city, including drilled wells , water tankers, water tanks (stabilized tanks distributed in the city) and domestic plastic bottles. Each sample was comprised of consecutive sample collection (Sequential sample), started with drilled wells and completed with domestic plastic bottles ( Figure 2). Permanently drilled wells are utilized to fill water tankers by which water transport into the water tanks distributed in the city squares to be utilized by a human. From water tanks, people use plastic bottles (20 L) to obtain water for drinking and other domestic purposes. Five drilled wells , 10 water tankers, 20 water tanks, and more than 100 plastic bottles were including in sampling. Each water sample was collected in triplicate in sterile glass (500ml) according to literature [37,38].

Counting, isolation, and identification of coliform bacteria
Coliforms bacteria were counted and isolated from water samples using the procedure of membrane filtration  [34] technique recommended by the Standard Methods for the Examination of Water and Wastewater [37]. Six water samples from each collection point were filtered by a sterile membrane with pore size 0.45±0.02 μm and 47mm-diameter [37]. After filtration, Petri dishes with eosin methylene blue agar (EMB) (Merck) and chromocult coliform agar (CCA) (Oxoid) were utilized as selective and differential media. Three filtrated membranes comprising bacteria placed on EMB, while the three remaining filters placed on CCA. Then, Petri dishes containing the filtrated membranes were incubated at 37°C for 24 h [37,38]. After incubation and based on colonial morphology, coliform bacteria colonies were distinctly counted into E. coli and total coliform [12,37]. Then, three E. coli strains from each plate were transferred into the nutrient broth (Oxoid) to confirm the identification of E. coli and incubated at 35°C for 24 h. Bacteria from the broth were identified by biochemical tests (Biomerieux, Marcy l'Etoile-France). Based on the biochemical results, the bacterial isolates confirmed as E. coli were chosen for the antibiotic susceptibility testing. Finally, the confirmed bacterial isolates were resuspended in normal saline until the turbidity of suspension equaled 0.5 of McFarland standards.

Antibiotic susceptibility testing
The bacterial isolates of E. coli from plastic water bottles (20 L) were used in the antibiotic resistance test due to people uses bottles to obtain water from the water tank for drinking and other domestic purposes. The antibiotic resistance pattern of E. coli isolates was performed using the Kirby-Bauer method [38,39]. The pure colonies that transferred from nutrient broth into normal saline until the turbidity of solution equaled McFarland 0.5 standards were utilized in the antibiotic resistance test. The bacterial suspensions were entirely streaked onto Muller Hinton agar (MHA) (Oxoid) using a sterile cotton swab. Then, the antibiotic discs were placed on MHA 30 mm apart and 10 mm away from the plate edge and incubated at 37°C for 18-24 hours under aerobic conditions. After incubation, the inhibition zone of each antibiotic disk was measured and recorded. Based on the measured inhibition diameter, the bacterial isolates were categorized into resistant and sensitive as described in the guidelines of the National Committee for Clinical and Laboratory Standards [40]. The tested antibiotics were including, Ampicillin (10 µg), Gentamicin (10 µg), Ceftriaxone (30 µ g), Amoxiclav (20/10 µg ), Ciprofloxacin (5 µg), and Tetracycline (30 µ g). The antibiotic discs were obtained from Oxoid, UK. In addition, the common antibiotic in the treatment of bacterial infections of humans was selected in this study to provide data about the multiple antibiotic resistance by which the potential risk can be assessed [12,21,30,32].

Multiple Antibiotic Resistance index (MAR index)
Multiple antibiotic resistance index is executed to evaluate the potential risk of the environments contaminated by the antibiotic-resistant bacteria [29,32,41]. The bacteria are termed multiple antibiotic resistant if it is found to be resistant to three or more antibiotic [42]. Multiple antibiotic resistance index of 0.2 is used as threshold limit for differentiating between low and highrisk contamination with multiple antibiotic resistant bacteria, the values greater than 0.2 are indicating of a high risk of contamination, while the values less than 0.2 are indicating to low risk of contamination [25,26,32]. Generally, the multiple antibiotic resistance index of the water samples is calculated by the Equation (1) [43,44]: where, y= Total number of resistance scored; n = number of isolates; x = Total number of antibiotics tested.

Statistical analysis
Statistical software (SPSS, v 20) was used to count and calculate the mean values of microbial from triplicat e samples obtained from each sequential sample. In addition, regression analysis was carried out to determine the impact of microbial indicators on the prevalence of antibiotic resistance of E. coli.

Microbial indicators (E. coli and total coliform)
The detection of microbial indicators in water used for drinking is of particular concern since they have been related to gastrointestinal infections such as diarrhea and dysentery, shigellosis, and other diseases [2,21]. These diseases remain a heavy burden in developing countries and have been particularly complicated due to the occurrence of antibiotic resistance, particularly used in the treatment of these bacterial infections [26]. In this study, the E .coli values of water samples ranged between a minimum 0.00 cfu/100ml at drilled wells of sequential samples 4 and 5 and a maximu m of 375 cfu/100ml in bottle 3 of sequential sample 1 (Table 1). For total coliform, the values ranged from 0.00 to 3135 TC/ 100ml. The highest value was recorded at bottle 6 of sequential sample 2, whereas the lowest value was at the drilled well of sequential sample 3 ( Table 2). The highest values of E. coli and total coliform were recorded in plastic bottles of all sequential samples by which water transport fro m stabilized water tanks to households. Water samples of bottles recorded high levels of microbial contamination compared to the other water samples. The values of E. coli and total coliform of bottles for all samples exceeded the acceptable level for WHO [45]. According to the WHO [45], E. coli and total coliform must not be detectable in any 100-ml sample, also U.S. Environmental Protection Agency [47] reported that E. coli and total coliform must be zero/100ml in drinking water.
For samples collected from water tankers, the highest contamination by E. coli was recorded at water tanker 2 of sequential sample 1 (185 cfu/100ml), while the lowest contamination was recorded at water tanker 1 of sequential sample 5 (55 cfu/100ml). Furthermore, the total coliform showed high contamination at water tanker 1 of sequential sample 2 (735 cfu/100ml), while showed low contamination at water tanker 2 of sequential sample 4 (125 cfu/100ml). The contamination of water tankers was attributed to the lacking of cleanliness and chlorination of the water tankers, also the lacking of good coverage of tanks that result in the entry of dust loaded by bacteria. In samples collected from stabilized water tanks in the city, total coliforms ranged from 120 cfu/100 ml at stabilized water tanks 3 of sequential sample 4 to 2450 cfu/100 ml at stabilized water tanks 1 of sequential sample 2. E. coli population ranged from 35 to 375 cfu/100ml, the lowest value was at stabilized water tanks 1 of sequential sample 4, whereas the highest value was at stabilized water tanks 3 of sequential sample 1. For the samples from plastic bottles utilized to transport water to households, the maximum value of E. coli was recorded in plastic bottle 3 (1375 cfu/100 ml) of sequential sample 1, whereas the minimum value was recorded in plastic bottle 7 (40 cfu/100 ml) of sequential sample 4. Conversely, the maximu m value of total coliform was recorded in plastic bottle 6 (3135 cfu/100 ml) of sequential sample 2, whereas the minimum value was recorded in plastic bottles 3 and 8 (110 cfu/100 ml) of sequential sample 4. Samples from plastic bottles recorded the highest levels of contamination by colifo rm bacteria compared to the other samples. The contamination of plastic bottles is affected by the poor personal hygiene of persons (generally children) who fill bottles with water from stabilized water tanks as well as the contamination during the transportation of water bottles for long-distances from stabilized water tanks to households that make plastic bottle exposed to contamination due to pollutants such as small landfills that are virtually dispersed around the stabilized water tanks.
The Occurrence of coliform in drinking water is adequate evidence of the fecal contamination associated with microbial pathogens. Among the coliform bacteria, E. coli is of importance as a microbial indicator of drinking water so its presence is usually correlated to gastrointestinal pathogens [18]. In this study, the results of E. coli and total coliform showed that the drilled wells of sequential samples 3, 4, and 5 were free of E. coli, therefore, these results were consistent with those obtained by Johnson et al. [48]. In the study of drinking water quality in Lalu commune ( Benin ), they found that the drinking water was uncontaminated by E. coli due to the lack of human and animal excreta. On the other hand, E. coli and total coliform population recorded high concentrations at all sequential samples. Consequently, these results were higher than the results obtained by Gwimbi [49], in the study of the microbial quality of drinking water in Manonyane community: Maseru District (Lesotho), due to the high contamination by coliform bacteria in this study is largely attributed to several contamination sources, including the existence of the small landfills close to the stabilized water tanks utilized to distribute water to citizens, the suspended particles in the air that contain bacteria, the wind carrying microbes from these landfills and insects that may carry microbes to water sources. Besides, the utilization of deep holes to collect wastewater at each house due to the lacking of good sanitation is also a major cause of contamination of wells water in the sequential samples 1 and 2 because these wells are located in urban frequently is an indication of fecal contamination and can represent a risk of water-borne diseases [3,7,45,50].

Antibiotic resistance of E. coli bacteria
In developing countries such as Yemen, where the people is exposed to various diseases (cholera epidemic, malaria, malnutrition, and other diseases), people only use contaminated water sources as a source of drinking water, the exposure to antibiotic-resistant bacteria can increase exacerbation of health risk, mostly to the children, elderly, immunocompromised [26,44,[51][52][53]. In this study, a total of 240 isolates of E. coli were obtained from drinking water in plastic bottles used to transport water to households. From eight plastic bottles of each sequential sample, six bacterial isolates were tested against the antibiotic. The antibiotic resistance pattern (Table 3) of E. coli strains for sequential sample 1 (bottles) was ampicillin (100%), gentamycin (79.2%), ceftriaxon e  (45.8%), ciprofloxacin (33.3%), amoxiclav (33.3%) and tetracycline (45.8%). In addition, E. coli strains for sequential sample 2 (bottles) were resistant to ampicillin (91.7%), gentamycin (83.3%), ceftriaxone (66.7%), ciprofloxacin (25%), amoxiclav (16.7%) and tetracycline (41.7%). The extreme prevalence of E. coli resistant to antibiotics at sequential samples 1 and 2 was ascribed to small rubbish dumps and untreated sewage holes located beside the stabilized water tanks. According to Kümmerer [30], the resistance of several antibiotics can be preserved in bacteria populations over time, despite selection pressure, which can cause a comprehensive increase in antibiotic resistance over time. Therefore, the bacterial population exposed to one or more antibiotics may induce resistance to other antibiotics without any previous exposure. Furthermore, the resistance of E. coli isolated from sequential sample 3 (bottles) was recorded as follows: ampicillin 70.8%, gentamycin 83.3%, ceftriaxone 62.5%, ciprofloxacin 8.3%, amoxiclav 8.3% and tetracycline 16.5%, while the E. coli isolates of sequential sample 4 (bottles) were 58.3 % resistant to ampicillin, 58.3% to gentamycin, 70.8 % to ceftriaxon e, 8.3 to ciprofloxacin, 8.3 to amoxiclav and 16.7 % to tetracycline. Lastly, E. coli isolates of sequential sample 5 (bottles) were resistance to ampicillin(83. 3%) , gentamycin (87.5%), ceftriaxone (66.7%), ciprofloxacin (25%), amoxiclav (25%) and tetracycline (45.8%). Generally, E. coli bacteria isolated from sequential samples 3 and 4 (bottles) showed low resistance to antibiotics than other samples mainly regarding amoxiclav, tetracycline, ampicillin, and ciprofloxacin due to these samples exposed to small amounts of contaminants compared to the other samples. Berto et al. [54] stated that bacteria resistant to antibiotics were found in drinking water affected by contaminants around the primary water source such as wells water. Vanneste et al. [55] also found that pathogenic bacteria isolated fro m various watercourses were resistant to antibiotics even in regions where no apparent pollutants, the results indicated that natural watercourse could be sources of antibiotic resistance. E. coli isolated from the water samples in plastic bottles showed high levels of resistance to several antibiotics used. Ordinarily, antibiotics resistance among bacteria will not only complicate future antibiotics therapy but can also potentially stimulate the transmission of resistance genes to other bacteria [1,43].
Previously, several studies emphasized the prevalence of antibiotic-resistant bacteria in drinking water, a potential risk to human and animals [1,21,24,26,32,52,[56][57][58][59]. Frequently, the direct infections of human by antibiotic-resistant pathogens may provoke the transmission of antibiotic resistance genes to opportunist bacteria and decrease the efficiency of the antibiotic therapy, drinking water considers an important source of antibiotic resistance particularly in developing countries where access to drinking water is limited to contaminated environmental sources which are utilized without any treatment [24,26,31,44].

Regression analysis
The impact of microbial indicators on the antibioticresistant E. coli occurrence by the linear regression analyses have been carried out for the microbial indicators as independent variables and the E. coli bacteria resistant to the different antibiotics as dependent variables. The different dependent characteristics of antibiotic-resistant E. coli bacteria were calculated using the regression equation and by substituting the values for the independent variables in the equations. From the regression analysis (Table 4), the P values between the microbial indicators and antibiotic-resistant E. coli bacteria including ampicillin, gentamycin, and ceftriaxone showed statistical significance (P≤0.05). This provides evidence of the existence of a linear relationship between the predictor (microbial indicators) and response (antibiotic-resistant E. coli bacteria). This means that, the regression analysis we have executed is well determined by the factors.
The regression analysis also revealed that colifo rm bacteria (E. coli and total coliform) showed statistical significance with antibiotic-resistant E. coli bacteria (gentamycin, tetracycline, ciprofloxacin, and chloramphenicol) (P < 0.05) because antibiotic-resistant E. coli bacteria is part of coliform bacteria. Generally, the   environmental conditions which are suitable for colifo rm bacteria growth are also suitable for antibiotic-resistant E. coli bacteria; hence the linear relationship between those bacteria was nearly perfect. Commonly, the antibioticresistant bacteria from various sources such as sewage, animal farms, domestic wastewater, and agricultural runoff (contain non-antibiotic resistant and antibioticresistant bacteria) can transmit their resistance to another bacteria through lateral transfer contributed to the spread of antibiotic-resistant bacteria, increasing selective pressure on the bacteria, and thus favoring resistance [60]. This was observed in this study in plastic bottles that recorded high concentrations of coliform bacteria related to high concentrations of antibiotic-resistant E. coli bacteria due to the accumulation of pollutants from the different sources that contain them together. Generally , ampicillin, gentamycin, and ceftriaxone were from the antibiotic used in the treatment of humans and animals as well as their use in agricultural activity since the last century, therefore, throughout the years, the bacteria acquired resistance against these antibiotics [61].
On the other hand, the regression analysis (Table 4), between the microbial indicators as independent variables and the E .coli bacteria resistant to ciprofloxacin , amoxiclav, and tetracycline were insignificant at P ≥ 0.05. In this study, the coliform bacteria showed high resistance to ampicillin affected by the preservation of resistance due to the selection pressure, which caus es an increase in antibiotic resistance over time. Thus, this verified the statistical significance of regression between the microbial indicators and ampicillin-resistant E. coli bacteria. According to Eregno [60], the use of antibiotics for a long time, even when used appropriately, creates selective pressure for resistant microorganisms. This result was similar to those reported by many research around the world in their studies regions [62]. From these relationships, it is concluded that the regression analysis has led to the formulation of equations of the linear regression for each antibiotic-resistant E. coli bacteria in the drinking water (Table 5). Based on this table, the regression equation can be used to predict the dependent variables. For example, from a Table 5, it is seen from the equation that for every unit increase in total coliform, a 73.41 unit increase in the gentamycin resistant E. coli bacteria is predicted.

MAR index in drinking water
In this study, The multiple antibiotic resistance index of E. coli bacteria resistant to three antibiotics of plastic bottles samples of sequential sample 4 (0.19) was found in a low range indicating a low risk of contamination, whereas the high values of multiple antibiotic resistance index were observed to be exceeded the threshold limit of plastic bottles samples of sequential sample 1, 2, 3 and 5 with value exceeded 0.21 at these stations ( Table 6). The main source of water of all sequential samples particularly 1, 2, 3, and 5 were situated in the urban area impacted by various anthropogenic activities. Moreover, plastic bottle samples of sequential samples 1, 2, 3, and 5 showed multiple antibiotic resistance index above 0.2, indicating high-risk contamination of drinking water. The difference in multiple antibiotic resistance index among samples indicated the effect of anthropogenic activities related to urbanization on antibiotic resistance levels. Commonly, the high values of multiple antibiotic resistance index reveal misuse and greater exposure to antibiotics in humans and poultry farms, which may pose a high ecological risk to the waters [41]. This was observed at sequential sample 1, which is considered a high-risk sample of contamination. The high value of this sample was attributed to the small landfills and domestic wastewater (situated nearby the source) that cause contamination of the main source of water (drilled well) due to the high concentration of detergents containing antibacterial components as well as the landfills containing poultry dumps, which may previously this poultry use the antibiotic for disease treatment and growth promotion, all these factors resulted in the high-risk contamination sample with antibiotic-resistant bacteria. According to Bohm and Gozalan [63], the observation on the high multiple antibiotic resistance index points to that isolates originated from high risks sources of contamination where antibiotics are frequently used and high levels of antibiotics usage and resistance are related to poultry farms and domestic wastewater. In addition, in the plastic bottles samples of sequential sample 2, 3, and 5, the multiple antibiotic resistance index was exceeded the high-risk level (0.2), signifying these samples were considered as high-risk sources of contamination, affected by the accumulated microbial contamination during the various transferring processes of water from the water source into households. According to Berendonk et al. [64], antibiotic resistance hotspots are present not only in medical settings but also in environmental systems, which are subjected to precipitation of polluted air and anthropogenic activities, including municipal wastewater, agricultural activities, pharmaceutical manufacturing, and animal husbandry farms. These locations are characterized by tremendously high bacterial loads joined with subtherapeutic concentrations of antibiotics, and they contribute to the release of antibiotic-resistant bacteria into the aquatic environment. The findings of this research are in agreement with those reported by Odonkor and Addo [26], for the drinking water sources in Accra, Ghana. They found that the multiple antibiotic resistance index was above 0.2 associated with the act that the water sources may be extremely contaminated with antibiotics caused by the massive usage of these chemicals in the adjacent areas of the various sources of water. Besides, the results of this study are consistent with the results reported by Chen et al. [32] for the drinking water sources in Hangzhou City, China, which showed a high risk of contamination with multiple antibiotic resistance index exceeded 0.2 for 25% of deliberated samples, influenced by the several sources of domestic and industrial sewage that discharge into the drinking water sources. Additionally, the multiple antibiotic resistance index is lower than the range of 0.11 to 0.55, reported by Varghese and Roymon [31], for the E .coli bacteria from different water sources in India, they showed that water sources were contaminated with antibiotic-resistant E. coli arising from high-risk sources of contamination due to human and non-human fecal contamination of surface water and groundwater. Furthermore, the results were lower than those observed by Titilawo et al. [41] for ten rivers utilized for drinking and domestic purposes in Osun State, South-western Nigeria, in which multiple antibiotic resistance index in E.c oli bacteria was significantly greater than 0.2 resulted in high-risk contamination in the area associated with the misuse and overuse of antibiotic in animal husbandry farms. A similar study was carried out by Chitanand et al. [25] who concluded that Godavari River in India is a high risk of the contaminated environment due to high values of multiple antibiotic resistance index (ranged from 0.15-0.48) related to the domestic waste and urban runoff, which impacted on the antibiotic resistance levels in the river. This indicates that the phenomenon of multiple antibiotic-resistant bacteria in the aquatic environment used as drinking water is of worldwide concern since it is an international rather than national problem [4,28,32]. Generally, bacteria that have resistance to antibiotics can cause direct or indirect risks to human health due to the use and exposure to contaminated water by these bacteria. The direct risks are related to exposure to harmful bacteria, which are resistant to antibiotics relevant for the treatment of infection caused by these harmful bacteria, as this can result in diseases that are difficult to treat [61,65,66]. The indirect risks are related to exposure to harmless bacteria, which carry antibiotic resistance. These bacteria can colonize skin or intestines without resulting in disease, and transfer their resistance to other bacteria that inhabit these tissues [61,67]. The resistance gene transfer occurs through a horizontal gene transfer process between the same or different species of bacteria [68,69]. Thus, the resistance genes can be transferred to pathogenic bacteria (non-resistant) to become resistant, resulting in difficulty to treat the Infection caused as well as most bacteria such as E. coli are called opportunistic bacteria, which can result in disease in people who are more vulnerable to infection.

CONCLUSION
The study concluded that the contamination level of drinking water at Dhamar city by E. coli and total coliform of all the five sequential samples, particularly related to plastic bottles samples, exceeded the acceptable level for WHO concerning the quality of drinking water affected by the accumulated contaminants of sequential samples, the lacking chlorination of the water, the poor personal hygiene of persons (generally children) who fill bottles with water from stabilized water tanks as well as the contamination during the transportation of water bottles for long-distances from stabilized water tanks to households that make plastic bottles exposed to contamination by bacteria in the air and small landfills that are virtually dispersed around the stabilized water. Also, the antibiotic resistance of E. coli bacteria isolated from plastic bottle samples was widespread principally for the sequential samples 1, 2, and 5. The E. coli isolates demonstrated high resistance to ampicillin and gentamicin as well as moderate resistance to ceftriaxon e and tetracycline, whereas the lowest resistance was recorded against ciprofloxacin and amoxiclav. Moreover, the multiple antibiotic resistance index of E. coli bacteria resistant to antibiotics of plastic bottles samples of sequential samples were found in a high range indicating a high risk of contamination impacted by various anthropogenic activities associated with urbanization. The risks are related to the exposure to harmful bacteria that are resistant to antibiotics relevant for the treatment of infection caused by these harmful bacteria, as this can result in diseases that are difficult to treat, as well as the exposure to harmless bacteria that carry antibiotic resistance and colonize skin or intestines without resulting in disease can transfer their resistance to other bacteria that inhabit these tissues .