Antibiotic resistance associated with bacteria in irrigation water : a case study of irrigation ponds in Southern Sweden

Abstract: The focus of this project was to characterize the occurrence of ESBL-­‐producing bacteria in two irrigation water ponds in Southern Sweden. Samples were taken from two ponds nearby each other, from which analyses were made based on levels of community and individual isolates. Community samples were used for attempts for characterization of resistance patterns in irrigation water using the Omnilog PM-­‐plate system. The growth of community microbiota in the presence of the following antibiotic substances was assessed; amikacin, gentamicin, kanamycin, penicillin G, ampicillin, piperacillin, trimethoprim, potassium tellurite and ciprofloxacin. Isolates were pure cultured using semi-­‐selective media: LB, Rainbow Agar, mEnterococcus, 0.1 TSA and VRBD. A total of 177 isolates were isolated randomly. Each isolate was identified using the Omnilog GENIII-­‐system of biochemical characterization. Further, the isolates were tested on Brilliance ESBL-­‐agar for their ability to grow. Out of the total number of isolates, 30 were selected, preferably Enterobacteriaceae or Pseudomonas spp., based on their high similarity to library strain during the identification in the Omnilog-­‐system. These were further characterized using Etest-­‐sticks. Three selected strains were picked for further analyses using exogenous isolation for attempts to simulate horizontal gene transfer, and Omnilog PM-­‐panels containing various antibiotics of interest. Selections of samples were chosen for molecular tests targeting plasmid-­‐borne resistance genes CTX-­‐M1, CTX-­‐M2 and SHV. The method for assessing community-­‐resistance patterns showed ambiguous data that was difficult to interpret; further optimization is recommended. For all tested PM-­‐plates, generally greatest differences between substances were found among wells with the strongest concentration of antibiotic substance. A total of 175 isolates displayed ability to grow on ESBL-­‐agar. Etests confirmed ability of isolates to resist many β-­‐lactam antibiotics and many were clinically resistant to certain substances. No plasmids were transferred during exogenous isolation. During molecular tests, a few matches were found for the CTX-­‐M1 gene. Isolates analyzed with Omnilog PM-­‐plates showed resistance to most compounds tested. The isolates showed greatest resistance toward penicillin G, ampicillin, amikacin, kanamycin and trimethoprim. In the study, it was established that resistance among bacteria was frequently occurring in the irrigation water, and β-­‐lactamase producing bacteria was very common. Extended studies are needed to assess the frequency-­‐ and occurrence of horizontal gene transfer (HGF) in this environment. Although some potential corresponding data is found, it is at this point not possible to determine whether the resistances are due to antibiotics used in veterinary medicine, human medicine or one additional reason is travel and trade. in irrigation water using the Omnilog PM-­‐plate system. The growth of community microbiota in the presence of the following antibiotic substances was assessed; amikacin, gentamicin, kanamycin, penicillin G, ampicillin, piperacillin, trimethoprim, potassium tellurite and ciprofloxacin. Isolates were pure cultured using semi-­‐selective media: LB, Rainbow Agar, mEnterococcus, 0.1 TSA and VRBD. A total of 177 isolates were isolated randomly. Each isolate was identified using the Omnilog GENIII-­‐system of biochemical characterization. Further, the isolates were tested on Brilliance ESBL-­‐agar for their ability to grow. Out of the total number of isolates, 30 were selected, preferably Enterobacteriaceae or Pseudomonas spp., based on their high similarity to library strain during the identification in the Omnilog-­‐system. These were further characterized using Etest-­‐sticks. Three selected strains were picked for further analyses using exogenous isolation for attempts to simulate horizontal gene transfer, and Omnilog PM-­‐panels containing various antibiotics of interest. Selections of samples were chosen for molecular tests targeting plasmid-­‐borne resistance genes CTX-­‐M1, CTX-­‐M2 and SHV. The method for assessing community-­‐resistance patterns showed ambiguous data that was difficult to interpret; further optimization is recommended. For all tested PM-­‐plates, generally greatest differences between substances were found among wells with the strongest concentration of antibiotic substance. A total of 175 isolates displayed ability to grow on ESBL-­‐agar. Etests confirmed ability of isolates to resist many β-­‐lactam antibiotics and many were clinically resistant to certain substances. No plasmids were transferred during exogenous isolation. During molecular tests, a few matches were found for the CTX-­‐M1 gene. Isolates analyzed with Omnilog PM-­‐plates showed resistance to most compounds tested. The isolates showed greatest resistance toward penicillin G, ampicillin, amikacin, kanamycin and trimethoprim. In the study, it was established that resistance among bacteria was frequently occurring in the irrigation water, and β-­‐lactamase producing bacteria was very common. Extended studies are needed to assess the frequency-­‐ and occurrence of horizontal gene transfer (HGF) in this environment. Although some potential corresponding data is found, it is at this point not possible to determine whether the resistances are due to antibiotics used in veterinary medicine, human medicine or one additional reason is travel and trade.