Antimicrobial-Resistance Pathogen Load and Public Health Risk of Drinking Water in South Gondar, Ethiopia


 Objectives: This study aims to determine the contamination Antimicrobial-Resistance Pathogen load and Public Health Risk of Drinking Water in the case of Debre Tabor Town, Northwest Ethiopia.Study design and period: A laboratory-based cross-sectional study design was employed from March to May 2020.Methods: A total of 60 water samples were collected from the household tap and household drinking water storage container by following the standard microbial analysis method. Besides Sanitary survey was conducted for the municipal water supply system. Water samples were analyzed for differences in bacteriological parameters and drug-susceptibility patterns. Descriptive statistics, independent sample t-test, and multiple linear regression models were used to analyze the data.Results: The drinking water was mostly contaminated with multiple antibiotics-resistance waterborne Escherichia coli 35% (95% CI: 31.2%, 46.9%), Salmonella 22.7% (95% CI: 23.2%, 28.7%), and Shigella 15% (95% CI: 11.2%, 20.9%). Approximately 52.78% and 36.11% of the tap and an equal 23.33% of the household storage container water samples were categorized under low and intermediate risks respectively, and the overall health risk index of the water samples showed that 45.83%, 41.67%, and 12.5%, of them, are categorized under low, intermediate and high-risk classes respectively.Conclusion: The contamination of drinking water with antimicrobial-resistant waterborne bacteria in the community could indicate the likelihood of the occurrence of treatment failure and increased mortality. Hence, proper drinking water treatment and strict supervision are needed to prevent the contamination of the water and related consequences.


Introduction
Globally, antimicrobial resistance (AMR) pathogens posed the most signi cant public health and economic threats where ten million humans at risk, with 700,000 deaths per year and 100 trillion USD of economic loss by 2050. Hence, these gures are assumed to increase signi cantly if preventive measures could not be done (1).
In Africa, most bacteria are 50 percent to 100 percent resistant to widely used antibiotics and results in more common treatment failure, increased morbidity, and mortality, chronic infection, increased infant and child death, and other worsening conditions (2). These threats may occur due to weak antibiotics regulatory capacity, circulation of substandard/counterfeit antibiotics, lack of antimicrobial surveillance strategies, weak laboratory capacity on AMR testing, lack of essential laboratory reagents for monitoring and evaluation, and limited quality assurance (3,4).
The increasing occurrence of AMR pathogens and their threats was a concern of the high and low-income countries (5).
The widespread emergence of AMR bacteria has become one of the most serious challenges in Ethiopia due to antibiotic-drug misuse, drug prescription without susceptibility test, self-medication, and a long stay in the hospital environment (6). Some experimental investigation and surveillance in Ethiopia, E. coli, Shigella, and Salmonella species showed a high level of resistance to frequently recommended antibiotics (7).
Assessment of qualities of urban water source and tap water distribution systems in Arba-Minch town showed that the distribution lines are the most contaminated with Waterborne Bacteria (WBB) such as Salmonella and Shigella (8).
On the other hand, a study conducted in North Gondar showed that 50% of water samples collected from water lines were contaminated with indicator WBB E.coli (9). Assessment of the level of AMR contamination and contamination source identi cation is highly relevant for policy intervention.
Therefore, this study aimed to assess the AMR Pathogen load and Public Health Risk of Drinking Water in the case of Debre Tabor Town, Northwest Ethiopia.

Study design and period
A laboratory-based cross-sectional study was carried out during a month from March-May 2020 at Debre Tabor town.

Sample size determination and sampling technique
A total of 60 water samples was taken by using equations derived from the World Health Organization( WHO) and United Nations International Children's Emergency Fund (UNICEF) (10).

Water sample collection
For bacteriological analysis, the samples were collected aseptically in an autoclaved bottle containing two drops of sodium thiosulfate (10% Na 2 S 2 O 3 ) for complete neutralization of any residual chlorine if present and for preserving microbial contamination. Before taking a sample from the tap, the mouth of the tap was cleaned with a clean cloth to remove any dirt if present (11). Then, water taken from the tap was ushed for 5 min and; then sterilization of the mouth of the tap was done with a spirit of ame followed by cooling it with the water to run for 1-3 min at a medium ow (12). Then the sterilized bottle was opened and lled with water by leaving a small air space to make shaking before analysis easier.
The collected water samples from each source were labeled and kept in a cold box containing ice freezer packs (< 4ºC) and were transported to Felege Hiwot Referal Hospital.
2.4 Sample processing techniques of WBB isolation and susceptibility testing 2.4.1 Sample preparation Every 10 ml of water samples from different sources were aseptically homogenized into 90 ml of sterile peptone water in a clean 250 ml sterile ask, shaken and 1:10 dilution was subsequently made (13). For the samples collected from tap and household storage container sources, further dilution was made by diluting 1 ml of each homogenized sample and 9 ml of sterile 0.85% physiological saline solution (NaCl) to make 10 − 5 dilution by using a vortex mixer. For water samples collected from the household tap and household storage container, the rst homogenized sample was plated.

WBB isolation and identi cation technique
A 0.1 ml of the prepared diluted sample was directly inoculated on differential and selective agar media after primary and secondary enrichment and incubated at 37 °C for 18-24 hours. After incubation, the isolates were determined by Lactose fermentation and precipitated bile salts with the appearance of dry, donut-shaped, with the surrounded dark pink area were used for presumptive identi cation of E. coli on the selective medium MacConkey agar and Thiosulfate and Ferric Citrate were used for observing hydrogen sul de production with black centers for presumptive identi cation of Salmonella and Shigella on the selective medium SS agar as shown the Fig. 1.

Multiple antibiotic-resistant pro le testing
Antimicrobial susceptibility testing was done for all the isolated WBB species. The slanted cultures were subcultured and puri ed. The pure colonies were inoculated into Nutrient Broth and incubated at 37 O C for 18-24 hours. After incubation, the turbidity of the culture was adjusted to 0.5 McFarland Standard to bring the cell density to approximately 107-108 cfu/ml. The 0.5 McFarland turbidity standard was prepared by mixing 0.05 ml BaCl2 (1%) with 9.95 ml H2 SO4(1%). Muller-Hinton (MH) (Oxoid) plates were prepared and warmed to ambient temperature for plating. A sterile cotton swab was dipped into the standardized suspension. The culture was spread evenly over the entire surface of the Muller-Hinton agar plates by swabbing in three directions at 90 0 of each spreading. The plates were allowed to dry before applying antimicrobial discs. The following standard and Oxoid drug discs were used: Vancomycin (VA) of 30 µg; Cotrimoxazole (SXT) of 25 µg; Cipro axicillin (Cip) of 5 µg; Doxycycline (DC) of 30 µg and Amoxicillin (Amo) of 2 µg, that were commonly used antibiotics in Ethiopian healthcare facilities. multiple antibiotic-resistance indexes (MAR) were found out by the formula: MAR index of isolate = No. of antibiotics to which an isolate is resistant/Total no. of antibiotics to which the isolate was exposed, based on the guidelines developed from Clinical and Laboratory Standards Institute of US (14).

Health Risk analysis
To assess the sanitary condition of the water samples and to analyze the risk to health matrix, the World Health Organization (WHO) standards de ned and recommended to determine the degree of contamination were used (15). Besides, Data on sanitary inspection of water sources were collected using the standard format described by WHO and UNICEF (16).
Observation checklist containing 10 item observation checklists (sanitary inspection form). These forms consist of a set of questions with "yes" or "no" answers to help in nding the most important factors that contribute to the contamination of water. To each item, a risk factor was assigned with a score of 1 for "yes" while anyone that does not constitutes a risk factor was assigned a score of 0 for "no". A combination of the scores for each item gave the sanitary risk scores which were grouped into four categories: 0-2, 3-5, 6-8, and 9-10 for low, moderate, high, and very high risk of contamination respectively.

Quality control
To ensure reliability, con dentiality, and validity during eld and experiment, different strategies were employed. Triplicate and duplicate samples were collected for physicochemical and bacteriological analysis of both municipal water supply and hand-dug well water and the average of each result were taken for the analysis. Observational checklists were also used to make sure the handling of water sources. Information on each sampling site and identi cation of the sampling locations were done by GPS. To assure and control the quality of data during sample analysis, series of events like cleaning of laboratory glassware and calibration of eld equipment were performed just before and just after eld measurements to ensure the validity and accuracy of acquired data. Bacteriological quality of drinking water was processed according to standard operating procedures and laboratory safety rule was followed. Sample collecting bottles were serialized before sample collection. To check the sterility of prepared media, 5% of the prepared batch of media was incubated overnight and checked for microbial growth in the media. Field blank, positive control negative control and also internal quality control was used to assure the quality of species identi cation and the antimicrobial susceptibility test.

Data analysis
The data were coded and entered using Epi info 7 and exported to STATA version 16.then the Mean prevalence, variability, and linear regression were executed by using STATA statistical software version 16. The variances between groups were handled by analysis of variance (ANOVA). A multiple linear regression model was conducted to determine the relationship between AMR WBB of drinking water with associated factors.

Household hygienic practices for handling the drinking water
The hygienic practice, observation of household tap water, and household storage containers concerning the sanitary inspection checklists are presented in Table 1. Accordingly, in the study area, most (63.9%) of the respondents treat their water, which is fetched from the tap before consumption. Out of the total respondents treat their drinking water, most of them (55.88%) uses ceramic water lter while the remaining 23.53% and 20.58% add chorine and boiling method, respectively. From the total tap waters assessed about 30.56% of the area around the tap was a bad hygienic environment. Besides, 36.11% of the drinking water storage containers had poor hygiene (Table 1). In this study, about 23% (95% CI: 10-100 CFU/100 ml) and 3% (95% CI: 1-10 CFU/100 ml) water samples were collected from taps and household containers that had Shigella counts respectively.

SD-standard deviation
The three major public health concern and common WBB isolate obtained from different combinations of enrichment, selective and differential media were tested for an array of ve different antibiotics. Most of the isolated WBB are resistant to SXT (25 µg); Amo (2 µg); VA (30 µg), none was resistant to cipro oxacin. Most isolated WBB showed MDR (Table 3).

Health Risks of Drinking Water of Debre Tabor Town
In this study, analysis of household container water samples revealed that half 10 (50%), of the household's container water samples, was free from Fecal contamination (FC) (low risk of contamination) ( Table 4). The Chi-square test analysis revealed that the sanitary inspection (SI) level of risk score had a strong association with the FC quality of both tap and household storage container water samples (p < 0.05). However, the SI level of risk score had no signi cant association with the TC quality of both tap and household storage container water samples (p > 0.05).

Associated factors of AMR WBB on drinking water
Correlation analysis of the WBB, their growth parameters, and the level of sanitary risk is shown in Table 5. Most of the bacteriological parameters showed a signi cant positive correlation with the SI level of risk and a signi cant negative correlation with residual chlorine concentration. From all the bacteriological parameters assessed, TC in the drinking water storage container showed a strong positive correlation (r = 0.856, p < 0.01) with the SI and a strong negative correlation with residual chlorine concentration (r= -0.622, p < 0.01) ( Table 5).

Discussion
WBB of drinking water determination is a good representative of public health risk since it can be a medium for the transmission of pathogenic disease, particularly deriving from fecal contamination.
A similar study conducted in Nekemte town has shown that 37% of the drinking water were contaminated with FC which is above the present study (17). But, the present study E.coli nding is higher than the studies conducted in Addis Ababa City (2.4%) (18) and Dharan, Nepal town (21.1%) (11). This difference may be due to the difference in the safety and quality control of water through the evaluation of water sources and manage contamination of water supply. Also, it may associate with the frequent pipe breakage, leakage, and the passing of pipelines through the ditches and drainage systems.
The E.coli load was higher than the study conducted in Kolladiba town of Ethiopia (32.5%) (19) and lower than Babati town, Tanzania (86%) of drinking water samples were contaminated with E.coli (20). The difference might be treatment of the drinking water and variation in climatic conditions.
According to  drinking water can be contaminated with AMR WBB due to improper treatment of water and the existence of poor sanitation (22). Assessment of qualities of urban water source and tap water distribution systems in Arba-Minch town revealed that the distribution lines are the most contaminated with AMR WBB such as Salmonella and Shigella (8).
The relationship between sanitary inspection scores and the bacteriological risk category is used to identify the level of risk of contamination due to AMR WBB. A study conducted by Tsega et al. (2013), showed that the total sanitary risk score has a signi cant relationship with the level of fecal contamination (23). And also A similar study conducted in Bahir Dar town showed that 45.7% and 11.4% of drinking water samples had low and very high-risk scores respectively (24 The nding of this study has shown possible health hazards related to the consumption of drinking water. Identi cation of these hazards would help health o cials to pay attention to safety and quality issues regarding drinking water. It will also contribute to awareness of consumers and water sector o cials about safety and quality issues related to consumption of drinking water. It may encourage water sector o cials to follow proper water treatment procedures during distribution up to consumption. The nding suggests the importance of water quality training for human working in water sectors, implementation of proper water treatment and strict follow up of the implementation of acceptable hygienic practices might improve water quality. Besides minimizing irrational drug use could also help to reduce AMR in water and the environment.
6. Declarations 6.1 Ethics Approval: Ethical clearance was obtained from the Institutional Review Board of the Jimma University and an o cial letter was submitted to the concerned bodies. The concerned bodies were informed to get the assurance of the study and con dentiality was maintained at all levels of the study. Informed consent was obtained from all participants and the Institutional Review Board of the Jimma University approved it with Ethical approval of Research protocol letter with its reference number IRB00010/2020.

Consent to Publish:
Not applicable.
6.3 Availability of data and materials: All data generated and analyzed during this study are included in the manuscript.