Antibiotic Resistance Index of Heterotrophic and Coliform Bacteria in Water and Sediment of Goharrood River

 Overuse of antibiotics and their entry into the environment is a global concern today. Much of the antibiotics taken by animals and humans are excreted unchanged and found in their feces which enter the environment through livestock waste and municipal wastewater disposal. Uncontrolled release and continuous introduction of antibiotics to the environment induced antibiotic resistance in microorganisms living in other habitats which pose a potential hazard to existing aquatic ecosystems and animals. Rivers act as the main sink for the effluents that distribute antibiotics and antibiotic resistant microorganisms in the environment. This study aimed to investigate the antibiotic resistant index (ARI) in Goharrood River. Seasonal variations and source of contamination, focusing on urban surface wastewater of Rasht, were investigated through sampling of river water and sediment in different points along the river during four seasons of a year.

The water and sediment of the river were sampled at three points along the river (focused on river course in Rasht city), in autumn, 2016 and in winter, spring and summer, 2017. The number of antibiotic resistant heterotrophic and coliform bacteria were counted via colony count method in the antibiotic supplemented 100 µg/mL Nutrient Agar and Eosine-Methylene Blue agar media respectively. Cephalexin, gentamicin, doxycycline, ciprofloxacin, and trimethoprim antibiotics were tested in this study. ARI was calculated by dividing number of bacteria colonies (heterotrophic and coliform bacteria) in plates supplemented with antibiotics to the number of colonies in control plate (without antibiotic). Escherichia coli as an indicator coliform bacterium was isolated from water and sediment samples (12 strains; 4 season and 3 sampling points) and their resistant pattern to these antibiotic was also tested by disk diffusion (Kirby-Bauer) method in Mueller-Hinton agar medium. The inhibition zone (ZOI) of E. coli growth was measured and its sensitivity/resistant was assessed based on CLSI standard protocol. The calculated ARI of heterotrophic and coliform bacteria of water and sediment of the River and the determined ZOI of E. coli isolated from water and sediment were analyzed by repeated measures of factorial arrangement in a completely randomized design format by SAS software package. Factors included sampling point at 3 levels (before entering river to the Rasht city; A, in the Rasht city; B, and after river exit from the Rasht city; C), and antibiotics at 5 levels (cephalexin, gentamicin, doxycycline, ciprofloxacin, and trimethoprim) as main plot and sampling time at 4 levels (autumn, winter, spring, and summer) as sub-plot.

 The highest ARI value of water heterotrophic bacteria was obtained to cephalexin at the sampling point C. Mean ARI of water heterotrophic bacteria to all antibiotics (regardless of type of antibiotic) at three points of A, B and C was 3.77, 4.54 and 7.53%, respectively. The highest ARI levels of heterotrophic and coliform bacteria in water were obtained in the summer season. In fact, the change of seasons and clearly the summer season controlled the ARI in water bacteria rather than the type of antibiotics. So that in this season 30.78% of water heterotrophic bacteria were resistant to ciprofloxacin antibiotic and about half (50.78) of the river water coliforms were resistant to cephalexin. Although ARI for heterotrophic and coliform bacteria was lower in sediment rather than that in water, the highest ARI levels of heterotrophic and coliform bacteria in sediment were obtained against cephalexin in autumn and winter, respectively. In general, the mean ARI in water and sediment bacteria was as follows: cephalexin > gentamicin > ciprofloxacine > doxycycline > trimethoprim. The lowest ZOI value for E. coli was obtained against cephalexin. Therefore, in all three sampling points, isolated E. coli bacteria from water and sediment were resistant to cephalexin. In the study of the sampling time and sampling point interaction, it was also seen that the lowest ZOI of E. coli was in autumn and at sampling point C. Therefore, it seems that E. coli has become resistant to antibiotics when river crosses the city.

 According to the results of this study, Goharrood river is contaminated with antibiotic-resistant, especially cephalexin resistant bacteria and it may distribute pollution downstream. If the river water is used in aquaculture and irrigation of downstream agriculture fields, the antibiotic resistant bacteria may be spread in the other ecosystems and finally may enter the human food chain.