Effects of Nutrient Level and Growth Rate on Mutation and Conjugation Processes That Confer Antibiotic Resistance to E. coli

Authors

Mohammadreza Shafieifini, University of Nebraska - LincolnFollow

First Advisor

Xu Li

Date of this Version

Summer 8-5-2019

Citation

Water research

Comments

A THESIS Presented to the Faculty of the Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Environmental Engineering, Under the Supervision of Professors Xu Li. Lincoln, Nebraska: July 2019

Copyright (c) 2019 Mohammadreza Shafieifini

Abstract

Antibiotic resistance genes (ARGs) may enter surface water with the discharge of treated wastewater. Conjugation is a main mechanism for the horizontal gene transfer of ARGs from wastewater microbiome to the microbiome indigenous to surface water. However, little is known about how environmental factors affect the conjugation process of ARGs.

In this thesis, an extensive research related to spreading antibiotic resistance genes in the environment is reported. The project investigated how environmental parameters may affect the rate of dissemination of ARGs and in what extent the resistance level of the population is changed after mutation and conjugation event. The chemostat reactors were built to simulate surface water receiving wastewater effluent. Two E. coli strains, CV601 and J53, were used as donor and recipient cells, respectively. First, CV601 cells received plasmids from an activated sludge sample through filter mating and became resistant to tetracycline. Then, J53 cells (i.e., to simulate river microbes) were established in chemostats before CV601 cells (i.e., to simulate wastewater microbes) were introduced. Two nutrient levels (3× and 10× diluted Muller Hinton broth) and two growth rates (0.15 and 0.45 hr^-1) were included. Results show that regardless the nutrient level, the conjugation frequency was 10^-2 and 10^-6 for the high and low growth rate, respectively. The minimum inhibitory concentration (MIC) of the recipient cells increased from 2 to 64-128 mg/L after the conjugation test, which was 2 to 4 fold lower than donor for the same plasmid. In addition, recipient cells grown under 0.45 hr^-1 and 1/10 MHB showed MIC increased from 2 to 8 mg/L in mutation experiment. Whole genome sequencing was conducted to 1) prove the presence of tetracycline resistant plasmid in donor; 2) Transferability of the plasmid to recipient cells; 3) and the emergence of mutants in recipient cells.

The results will allow us to better model and predict the proliferation of ARGs in the environment.

Advisor: Xu Li