Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h21 versus 0.15 h21) led to higher conjugation frequencies (1024 versus 1026 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water.