Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant <i>Escherichia coli</i>

<div><p>Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of antimicrobial-resistant bacteria in wastewater.</p></div

The number of drugs to which <i>E</i>. <i>coli</i> isolates from four different sewage treatment plants (STPs) in South India showed antimicrobial resistance.

<p>The X-axis indicates the number of resistant-antimicrobials of <i>E</i>. <i>coli</i> isolates. The Y-axis represents the percentage of the resistant <i>E</i>. <i>coli</i> isolates. Data for STP-1, -2 and -3 were retrieved from Akiba et al., 2015.</p

The estimated associations between gene expression potential and sewage treatment facility, season and treatment process in STP wastewater.

<p>The explanatory variables, STP-2 (facility: relatively newly build and receives mixed domestic and hospital wastewater), pre-monsoon (season) and step 1 (process) were used as the reference levels. The asterisks shown to the P values shows the levels of statistical significance (* <0.05, **<0.01, ***<0.001) difference.</p><p>The estimated associations between gene expression potential and sewage treatment facility, season and treatment process in STP wastewater.</p

Comparison of CYP2B15 and CYP 3A gene expressions in rat hepatocytes exposed to wastewater extracts from STPs.

<p>A: Pre-monsoon; B: Monsoon. Data are reported as means ± SD (standard deviation). STPx-y: x is the type of STP; y is the treatment step [equalization (step 1), aeration (step 2), settling (step 3), and outlet (step 4)]. The data for STP3-1 were not available.</p

Comparison of CYP gene expression by the prototypical inducers in rat hepatocytes.

<p>Data are reported as means ± SD (standard deviation).</p

The estimated associations between resistance of <i>E</i>. <i>coli</i> isolates to an antimicrobial agent and gene expression potential with other confounding variables in STP wastewater.¹

<p>¹Data shows if only significant association between an antimicrobial agent and gene expression was found. All other data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138391#pone.0138391.s002" target="_blank">S2 Table</a>. STP-2, pre-monsoon and Step 1, were assigned as the reference level for the variables of the sewage treatment facility, season and processing step, respectively. The asterisks shown to the P values shows the levels of statistical significance (* <0.05, **<0.01).</p><p>The estimated associations between resistance of <i>E</i>. <i>coli</i> isolates to an antimicrobial agent and gene expression potential with other confounding variables in STP wastewater.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138391#t003fn001" target="_blank">¹</a></p

Comparison of CYP1 gene expression in rat hepatocytes exposed to wastewater extracts from STPs.

<p>A: Pre-monsoon, B: Monsoon. Data are reported as means ± SD (standard deviation). STPx-y: x is the type of STP, y is the treatment step [equalization (step 1), aeration (step 2), settling (step 3), and outlet (step 4)]. The data for STP3-1 were not available.</p