Abstract: Background and Objective: Intrusion of wide range of antibiotics through the pollutants in river water makes the bacteria resistance to some specific antibiotics over a short period of time. Thus, the objective of this study was to determine antibiotic resistant pattern of Escherichia coli and Vibrio cholerae in water and sediment of Padma river to commonly used antibiotics. Materials and Methods: Multiple tube fermentation technique (MPN) and spread plate technique were used to the enumeration of E. coli and V. cholerae from water and sediment samples. Antimicrobial susceptibility testing was done by using Kirby-Bauer disc-diffusion method according to Clinical and Laboratory Standards Institute. Results: About 47 Escherichia coli and 38 Vibrio cholerae isolates were collected from water and sediment. The E. coli isolates were found highly resistant to ampicillin in water and to ciprofloxacin in sediment samples. The V. cholerae isolates showed the highest resistance to tetracycline and ciprofloxacin in water and sediment samples, respectively during summer season. During winter season, the highest resistance was obtained for ampicillin and for ciprofloxacin in water and sediment samples, respectively. The value of multiple antibiotic resistances (MAR) index crossed the high risk line at Site-2 (Padma garden) for both E. coli and V. choleare. Conclusion: It was concluded that the presence of multiple antibiotic resistant bacteria in Padma river is a serious concern for health status of the people inhabiting along the river.
INTRODUCTION
No doubt that the problem of antimicrobial resistance of micro-organisms has become a significant public health concern worldwide1,2 and especially in countries like Bangladesh where the antibiotic resistance pattern of its hundreds of river water is remain unrevealed. The river Padma (one of the largest river of Bangladesh) is situated along the Rajshahi city of Bangladesh, considered a very vital source of drinking water and irrigation, as well as for recreation along the region of Rajshahi city. It also serve as a reservoir for many micro-organisms from municipal waste as well as illegal dumping from industries, hospitals, households and from agricultural pollutants3. Therefore, use of this water for recreational activities together with fishing, drinking, bathing and irrigation of crops sometimes causes serious infectious disease. The indiscriminate use of antibacterial agent in fish feed and other agricultural products, household products and their uses in hospitals increase the level of antimicrobial resistant bacteria in surface water. Therefore, water pollution with multi drug resistant (MDR) bacteria requires evaluation4. Escherichia coli, a well-known resident of vertebrate’s digestive tract, sometimes used as an indicator of fecal contamination in water5. The E. coli are found as normal flora in the human intestine. The E. coli and related bacteria constitute about 0.1% of gut flora and fecal-oral transmission is the major route for causing disease in human6. On the other hands, Vibrio cholerae is well known as the relevant agent for the human disease cholera that sometimes causes significant mortality. Generally, V. cholerae is diffused through contaminated food and water to the people who do not have proper sewage and water treatment systems. However, little is known about the level of microbial contamination and antimicrobial susceptibility patterns from Padma river. Therefore, the aim of this study was to determine antimicrobial susceptibility profiles among E. coli and V. cholerae isolated from this river.
MATERIALS AND METHODS
Selection of study locations: The present study was conducted along the Padma river from T-dam to Talaimari covering most of the part of Rajshshi city Corporation along the bank of the Padma river. Sampling was done on two, respective seasons namely summer and winter in the years 2016. Location and description of sampling sites is shown in Fig. 1 and Table 1.
Sampling: For the bacteriological analysis, 500 mL of water samples and required 500 g of sediment samples were collected from the selected sampling points. The materials used in sampling were sterile glass bottle, ice box and marker7. The samples were labeled and transported to the laboratory. Ice during transportation was used to prevent the reproduction of bacteria as well to ensure better survivability at low temperature. The time gap between sampling and analysis was maintained below 3 h.
Isolation, identification and confirmation of E. coli and V. cholera isolates: Multiple tube fermentation technique was used to enumerate the fecal coliforms from water sample, while spread plate method was used for the enumeration of fecal and V. cholerae from sediment samples. For isolation of E. coli from the confirmed fecal coliform collected from water and sediment samples, Eosine methylene blue agar (EMB) was used. Individual colonies showing a green metallic sheen on EMB agar were further confirmed using biochemical tests. The biochemical tests that were used for further identification of E. coli from other fecal coliform bacteria were indole, methyl-red, voges-proskauer and citrate test8. Antibiotic sensitivity testing was done for the isolates that showed positive results for biochemical tests. For the isolation of V. cholerae, 25 g of the sample was added with approximately 225 mL of alkaline peptone water in a warring blender flask and blended for 1 min. The sample was than incubated at 37°C for 6-8 h. After the incubation, a loopful of the alkaline peptone water was streaked on Thiosulphate Citrate Bile Salts (TCBS) agar plates. At the end of the incubation period TCBS agar plate was checked for growth of typical yellow or blue-green colonies. Confirmation of isolates of V. cholerae was done by Voges-Proskauer test and motility test.
Antibiotic sensitivity testing: Antimicrobial susceptibility testing was done by using Kirby-Bauer disc-diffusion method and using Mueller-Hinton agar (Difco, MI, USA) according to Clinical and Laboratory Standards Institute9. The standardized antibiotic discs of ampicillin (10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), streptomycin (10 μg), tetracycline (30 μg) and azithromycin (15 μg) (Hi media) were used for antibiotic sensitivity testing. The disc was distributed evenly at a distance of 24 mm from center to center of each disc. The 3 discs were used for a 100 mm plate. After an overnight incubation, the diameters of the zones of complete inhibition were measured to the nearest mm using slide calipers. The sizes of the zones of inhibition were interpreted according to protocols standardized for the assay of antibiotic compounds as guided by Clinical Laboratory Standards Institute10.
| Fig. 1: | Location of the study area (indicated with yellow stars). Modified from Google Earth-2017 |
| Table 1: | Sampling sites, sampling code and observation |
Multiple antibiotic resistance (MAR) indexing: The MAR index was performed to evaluate the health risk of the environments. Multiple antibiotic resistance index (MAR) for each test isolate was calculated as recommended by Krumperman11. The equation is as follows:
Statistical analysis: Values were represented as the mean (without standard deviation) of the replicates by using SPSS (Statistical Package for Social Science) version 20.0 (IBM Corporation, Armonk, NY, USA). Microsoft Excel (version 10) was used to represent the data in figures.
RESULTS
Antibiotic sensitivity testing of E. coli isolates: Antibiotic susceptibility test was carried out for 47 E. coli and 38 V. cholerae isolates collected from water and sediment samples. Antibiotic resistance profile of E. coli isolates of water samples is shown in Table 2. About 75% of the isolates were found to show resistance to ampicillin, 50% to ciprofloxacin and 25% to streptomycin during summer season at Site-2. During winter season, 16.67% isolates showed resistance to both gentamicin and streptomycin. At Site-3, 33.33% of the isolates showed resistance to streptomycin and azithromycin during summer, while 50% of the isolates showed resistance to ampicillin and streptomycin (Table 2). In sediment samples, isolates of E. coli did not show resistant to the studied antibiotics at Site-1 during summer and at Site-4 during both summer and winter season, respectively (Table 3). Only 50% isolates of E. coli showed resistance to ampicillin during winter season in Site-1. At Site-2, 40% isolates showed resistance to tetracycline and ciprofloxacin and 20% showed resistance to gentamicin in summer season. At Site-3, 16.67% isolates of E. coli showed resistance to ampicillin, gentamicin and ciprofloxacin, while 33.33% isolates showed resistance against azithromycin during summer season. During winter season, 50% of isolates showed resistance to ciprofloxacin and 25% to gentamicin at Site-3, respectively (Table 3).
Antibiotic sensitivity testing of V. cholerae isolates: Isolates of V. cholerae from water sample did not show any resistance to studied antibiotics at Site-1 and Site-4 during both summer and winter season (Table 4). While at Site-2, 16.67% of isolates showed resistance to tetracycline, streptomycin and ciprofloxacin and 33.33% isolates to azithromycin during summer season. During winter season, 33.33% isolates showed resistance to ampicillin, tetracycline and ciprofloxacin and 66.67% to azithromycin at Site-2. At Site-3, 66.67% isolates of V. cholerae showed resistance to tetracycline and 33.33% isolates to gentamicin and streptomycin during summer season and in winter season only 50% of isolates of V. cholerae showed resistance to ampicillin (Table 4). Antibiotic resistance profile of V. cholerae isolates in sediment samples, collected from the studied locations of Padma river, is shown in Table 5. Here isolates from Site-1 during both summer and winter season and from Site-4 during summer season did not show resistance to any types of antibiotics used. While at Site-2, 25% of the isolates showed resistance to tetracycline, gentamicin, ciprofloxacin and azithromycin during summer season and 50% of isolates to ampicillin and ciprofloxacin during winter season, respectively. At Site-3, 66.67% isolates were resistant to only azithromycin during summer season and 33.33% isolates showed resistance to ampicillin, gentamicin and ciprofloxacin during winter season. However, at Site-4, 50% of isolates of V. cholera from sediment sample showed resistance to only azithromycin during winter season (Table 5).
| Table 2: | Antibiotic resistance (%) profile of E. coli isolates in water samples collected from the studied locations of Padma river |
| Su: Summer, Wi: Winter | |
| Table 3: | Antibiotic resistance (%) profile of E. coli isolates in sediment samples collected from the studied locations of Padma river |
| Su: Summer, Wi: Winter | |
| Table 4: | Antibiotic resistance (%) profile of V. cholerae isolates in water samples collected from the studied locations of Padma river |
| Su: Summer, Wi: Winter | |
| Table 5: | Antibiotic resistance (%) profile of V. cholerae isolates in sediment samples collected from the studied locations of Padma river |
| Su: Summer, Wi : Winter | |
| Fig. 2(a-b): | Calculated MAR index for the study locations during summer and winter seasons based on E. coli in (a) Water and (b) Sediment samples of Padma river |
Multiple antibiotic resistance (MAR) indexing: The MAR indexing of different study locations is shown in Fig. 2. The MAR index value of maximum 0.25 was considered high risk level in terms of resistance of multiple antibiotics. Resistance of E. coli isolates, collected from water samples, to antibiotics crossed the high risk value of MAR index during summer season at Site-2 (Fig. 2a). However, resistance of V. cholerae isolates collected from water samples crossed the high risk value of MAR index during winter season at Site-2 (Fig. 2a). In case of sediment sample, isolates of E. coli and V. cholerae showed multiple resistances to selected antibiotics and crossed the high risk value of MAR index at Site-2 except for E. coli isolates of winter season. However, isolates of V. cholerae during winter season crossed the high risk value of MAR index at Site-3 (Fig. 2b).
DISCUSSION
Contamination of river water through the microbes of vertebrate’s digestive tract is a major threat to the people drinks river water because it may be responsible for numerous waterborne disease outbreaks. The presence of drug resistant bacteria in surface water contributed to the spread and persistence of antibiotic resistance bacteria in environment12. Antibiotic sensitivity testing was done during summer and winter season, as because isolates of E. coli and V. cholerae were not found during monsoon season. Although Kistemann et al.13 stated that microbial contamination is increased following rainfall and runoff events, but a different result was observed in the present study. However, as the major factors affecting the microbial quality of surface water and underground waters are sewage runoff water, seepage from nearby sewage or septic tank14, the presence of studied pathogenic bacteria during summer and winter might be due to the low level of water in the river that might be easily contaminated by sewage and septic tank disposal and other domestic waste, which is in agreement with an earlier study by Kenyon et al.15. The E. coli isolates exhibited highly resistant to ampicillin in water and to ciprofloxacin in sediment samples. Ampicillin is a broad spectrum penicillin and Ciprofloxacin is a broad spectrum fluoroquinolone antibiotic which are frequently added to the poultry feed. Both of these antibiotics are effective against gram negative bacteria. Therefore, Padma river was considered a natural reservoir of these Gram-negative bacteria as because 12.5% of the isolates that were resistant could represent a natural reservoir of antibiotic resistance16. Resistance to Ampicillin is worrying because this antibiotic is in World Health Organization list of agents that are frequently used for treatment of many infections. On the other hand, V. cholerae isolates showed their highest resistance to tetracycline in water and ciprofloxacin in sediment samples during summer season. While during winter season, the highest resistance was obtained for ampicillin in water and for ciprofloxacin in sediment samples, respectively. The relatively high level of resistance to the studied antibiotics was an indicative of adverse use of these agents in the environment. Similar observation was also reported by Lateef17, who stated that high resistance of antimicrobial agents represents misuse and abuse of these agents in environment. The value of MAR index crossed the high risk line during both summer and winter season, respectively for E. coli and V. choleare Site-2. This indicated that the phenomenon of multiple antibiotic resistant bacteria in aquatic environment was a serious concern for health status of the people inhabiting at Site-2 along the Padma river. The increasing resistance and multiple resistances of the microbial strains in this area might be due to the inefficiency of the water treatment plant. The fecal residues discharged from house-holding activities and hospital wastes sometimes also overflowed during monsoon and caused severe pollution in the Padma river at Site-2. Similar to the findings of the present study, a related study conducted by Armstrong et al.18 showed that high multiple drug resistance (MDR) strains are more common from water contaminated with runoffs from land occupied by animal and human and those with urban and industrial sewerage. Presence of MDR in enteric bacteria isolates from aquatic environment has also been reported by Olaniran et al.19, Abdo et al.20 and Emmanuel et al.21. Findings of the study indicated that the E. coli and V. cholera recovered in this study most often expressed resistance to the antibiotics that are commonly used in clinical medicine it may limit the availability of antimicrobials for clinical management of waterborne outbreaks of the people living in this locality in future. This could contribute to the spread and persistence of antimicrobial resistant bacteria.
CONCLUSION
It was concluded that the presence of E. coli and V. cholera pathogens and the multiple antibiotic resistance tendencies among the isolates in the surface water and sediment of Padma river highlighted the human health risk associated with exposure to fishing, recreational and other household activities. Therefore, adequate risk prevention strategies need to be undertaken to protect the water and sediment and consequently to protect public health from these infectious pathogens.
SIGNIFICANCE STATEMENTS
The present study has established understanding on the antibiotic resistance of isolates of E. coli and V. cholerae. To detect any changing patterns in bacterial contamination, there is a need to monitor antibiotic sensitivity at regular intervals and knowledge of local presence of bacterial organisms and antibiotic sensitivities of the present study will provide the direction of antibiotic selection. These results would assist the authority to manage further bacterial contamination of water through proper guidelines.