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

Abstract

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 h^-1 versus 0.15 h^-1) led to higher conjugation frequencies (10^-4 versus 10^-6 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. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.

Keywords: E. coli; chemostat; conjugation; growth rate; nutrient level; tetracycline resistance.

FIG 1

Experimental setup of the chemostat reactor system.

FIG 2

Conjugation frequency under different growth rates (0.45 h⁻¹ and 0.15 h⁻¹) and different nutrient levels (1/10 MHB and 1/3 MHB) at 16 h. Asterisk represents statistically significant differences (* and *** indicate P < 0.05 and P < 0.001, respectively [Tukey’s post doc test]). Error bars represent standard deviations (n = 6).

FIG 3

Concentrations of transconjugants (n = 6) at 0.45 h⁻¹ and 0.15 h⁻¹ growth rates with nutrient levels of 1/10 MHB (a) and 1/3 MHB (b). Asterisks represents statistically significant differences (i.e., *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively [Tukey’s post doc test]).

FIG 4

Change in tetracycline MIC relative to the J53 control strain after 4 days of the conjugation experiment at 0.45 h⁻¹ and 0.15 h⁻¹ growth rates with nutrient levels of 1/10 MHB (a) and 1/3 MHB (b). The results are averages from six replicate chemostats, and error bars are too small to be visible.