Water Resources Research Act Program

Details for Project ID 2014AR350B

Is persistence of plasmids in antibiotic resistant E. coli isolated from stream water impacted by integrons and conjugation or mobilization genes?

Institute: Arkansas
Year Established: 2014 Start Date: 2014-03-01 End Date: 2015-02-28
Total Federal Funds: $6,000 Total Non-Federal Funds: $6,410

Principal Investigators: Mary Savin, FNU Suhartono

Abstract: Antibiotic resistant bacteria (ARB) are a major public problem, with concern increasing about their dissemination and persistence throughout the environment. Previous local investigations signified that ARB might emerge in Northwest Arkansas streams, indicated by a number of E. coli possessing antibiotic resistance and plasmids that were recovered in the effluent of wastewater treatment plants (WWTP) and receiving stream water in Northwest Arkansas. Many antibiotic resistance genes (ARG) are located on plasmids and, furthermore, are associated with genetic elements that assist in the transfer and integration of antibiotic resistance into other organisms, providing mechanisms which can serve to maintain and spread antibiotic resistance throughout the environment. Thus, the persistence of ARG can be facilitated by the presence of integrase (intI1 and intI2 genes) and mobilization (mob) genes associated with bacterial plasmids. The objectives of the research are to determine the presence of integrase and mobilization genes in plasmids and the relationship with multiple antibiotic resistance (MAR) number in antibiotic resistant E. coli as well as to determine the influence of the integrase and mobilization genes towards the persistence of antibiotic resistant plasmids recovered from E. coli that were isolated from WWTP effluent and receiving stream water. Antibiotic resistant E. coli strains (140) will be regrown on selective media and plasmid DNA will be extracted. The size of the plasmid DNA will be determined using restriction endonuclease digestions with EcoRI and HindIll. Class I and II integrase and mob genes will be detected and characterized using PCR amplification. The influence of integrons and mobilization apparatus (mob+int+, mob-int+, mob+int-, or mob-int-) towards plasmid persistence will be tested on E. coli isolates for which the determinants for trimethoprim and sulfamethoxazole antibiotic resistance can be confirmed by PCR. Persistence of plasmids in the four mob/int categories will be determined using mesocosms built in flasks containing synthetic wastewater with and without addition of antibiotics of trimethoprim and sulfamethoxazole. Plasmid persistence will be evaluated by quantitative PCR (qPCR).