Effect of Urban Wastewater Discharge on the Abundance of Antibiotic Resistance Genes and Antibiotic-Resistant Escherichia coli in Two Italian Rivers

Abstract

Background: Wastewater treatment plants (WWTPs) are microbial factories aimed to reduce the amount of nutrients and pathogenic microorganisms in the treated wastewater before its discharge into the environment. We studied the impact of urban WWTP effluents on the abundance of antibiotic resistance genes (ARGs) and antibiotic-resistant Escherichia coli (AR-E. coli) in the last stretch of two rivers (Arrone and Tiber) in Central Italy that differ in size and flow volume.

Methods: Water samples were collected in three seasons upstream and downstream of the WWTP, at the WWTP outlet, and at sea sites near the river mouth, and analyzed for the abundance of ARGs by qPCR and AR-E. coli using cultivation followed by disk diffusion assays.

Results: For all studied genes (16S rRNA, intI1, sul1, ermB, blaTEM, tetW and qnrS), absolute concentrations were significantly higher in the Tiber than in the Arrone at all sampling sites, despite their collection date, but the prevalence of target ARGs within bacterial communities in both rivers was similar. The absolute concentrations of most ARGs were also generally higher in the WWTP effluent with median levels between log 4 and log 6 copies per ml but did not show differences along the studied stretches of rivers. Statistically significant site effect was found for E. coli phenotypic resistance to tetracycline and ciprofloxacin in the Arrone but not in the Tiber.

Conclusions: In both rivers, diffuse or point pollution sources other than the studied WWTP effluents may account for the observed resistance pattern, although the Arrone appears as more sensitive to the wastewater impact considering its lower flow volume.

Keywords: Arrone River; Escherichia coli; Tiber River; antibiotic resistance; antibiotic resistance genes.

Figure 1

Comparison of the absolute abundance of target genes between studied rivers at the different sampling sites. The lower and upper edges of each boxplot are the first and third quartiles, the midline shows the median and the whiskers extend from the minimal to the maximal values. Outliers are indicated by gray dots. Asterisks denote statistical significance as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Absolute abundance of target genes in sampled sites from Arrone (left) and Tiber (right) rivers. Different letters above boxplots indicate significant differences (two-way ANOVA and post hoc Tukey test after correction of p-values for multiple comparisons). The lower and upper edges of each boxplot are the first and third quartiles, the midline shows the median and the whiskers extend from the minimal to the maximal values. Outliers are indicated by black dots. WWTP effluent sample is highlighted in gray.