The research team set out to answer a deceptively simple question: how does temperature affect the abundance and composition of antibiotic resistance genes in activated sludge systems, when everything else is held constant? This question matters because real-world treatment plants experience seasonal swings, and previous field studies have struggled to disentangle the effects of temperature from the effects of changing influent composition. Winter brings not only colder basins but also different patterns of water use, different chemical loads, and different microbial communities arriving at the plant. To isolate temperature as a single variable, the researchers designed an elegant controlled experiment conducted directly at an urban wastewater treatment plant in Portugal, ensuring that the wastewater feeding their experimental systems reflected genuine, real-world conditions rather than synthetic laboratory mixtures.

Two laboratory-scale conventional activated sludge systems were operated side by side for 147 days under identical conditions, with one crucial exception. The control system was maintained at approximately 20 degrees Celsius, a temperature representative of moderate operating conditions, while the test system was run sequentially at 10, 5, 15, and 28 degrees Celsius. This sequential design allowed the researchers to observe how the same microbial community responded as the thermal regime shifted up and down over time. Throughout the experiment, the team sampled the feed, which consisted of primary effluent from the host treatment plant, as well as the treated effluent and the sludge from each system. These samples were analyzed for physicochemical parameters, fecal indicator bacteria, the abundance of specific antibiotic resistance genes, and the composition of the bacterial communities using molecular profiling techniques.

The results revealed a striking interplay between what enters the plant and what happens inside it. The patterns of antibiotic resistance genes in the treated effluent were clearly influenced by the composition of the incoming feed, confirming that influent characteristics drive much of the resistance dynamics in activated sludge systems. However, temperature exerted an additional, independent influence on top of this influent-driven variability. The most dramatic effect emerged when the test system was operated at 5 degrees Celsius. Under this cold regime, the abundance of intI1, a gene widely used as a proxy for anthropogenic antibiotic resistance pollution and a marker of mobile genetic elements, dropped significantly compared with the control system held at 20 degrees Celsius.

Even more importantly from a clinical standpoint, the cold treatment significantly reduced genes of direct medical relevance, including blaIMP, which confers resistance to carbapenem antibiotics, a last-resort class of drugs used against multidrug-resistant infections, and qnrS, which is associated with reduced susceptibility to fluoroquinolones. These are not obscure genetic markers; carbapenem resistance in particular represents one of the most alarming developments in infectious disease medicine. The observation that a simple operational parameter like temperature could measurably suppress these genes in treated effluent carries substantial implications for how treatment plants might be managed to limit the environmental dissemination of resistance.

To understand why cold temperatures had this effect, the researchers turned to the bacterial community composition data. The shifts in gene abundance were associated with changes in the microbial community itself, particularly a decrease in genera such as Acinetobacter and Escherichia-Shigella, both of which are considered potential hosts for antibiotic resistance genes and both of which include clinically significant pathogens. In other words, the cold did not simply disable the genes while leaving their carriers intact; it appears to have reduced the fitness of the key bacterial hosts that harbor and propagate these genes. When the host organisms decline, the genes they carry decline with them, offering a mechanistic explanation for the observed reductions in effluent ARG loads.

An equally important finding concerned where in the system these effects occurred. The researchers found no significant differences in antibiotic resistance gene abundance in the sludge fraction between the cold and control systems, indicating that temperature mainly influenced ARG persistence in the liquid phase. This distinction matters because the liquid effluent is what gets discharged into receiving rivers and coastal waters, while sludge is typically handled separately, often through anaerobic digestion and land application. The selective reduction of ARGs in the liquid phase suggests that low-temperature operation specifically curtails the release of resistance genes into the aquatic environment, which is one of the principal pathways by which resistance spreads from human settlements into natural ecosystems.

The temperature effect extended beyond resistance genes to the removal of fecal indicator organisms. Reductions of Escherichia coli, the standard marker of fecal contamination, were significantly greater at 5 degrees Celsius than at the control temperature, indicating that cold operation also enhanced the removal of this pathogen indicator from the effluent. This finding adds to the appeal of cold operation from a water quality perspective, although the researchers caution that the picture is not uniformly positive. Low temperature impaired nutrient removal, a critical function of activated sludge systems that protects receiving waters from eutrophication. Nitrifying bacteria and other nutrient-cycling microorganisms are notoriously sensitive to cold, and their slowed activity under low-temperature conditions translated into poorer nutrient removal performance.

Crucially, however, the overall effluent quality remained within discharge limits even during the cold-temperature runs, meaning that the trade-off between enhanced ARG suppression and reduced nutrient removal did not push the system out of regulatory compliance in this study. This balance is central to any practical consideration of the findings. Treatment plant operators cannot simply chill their reactors without consequence, but the study demonstrates that the penalty may be more manageable than feared, while the benefit, a significant reduction in clinically relevant resistance genes entering the environment, could be substantial. The authors frame temperature as an important operational driver of antibiotic resistance dissemination, a variable that deserves deliberate attention rather than passive acceptance.

The broader significance of this work lies in its contribution to the growing field of evidence-based wastewater management for antibiotic resistance control. As concerns mount over the role of treatment plants as conduits for resistance genes into rivers, irrigation systems, and drinking water sources, engineers and regulators are searching for interventions that are feasible within existing infrastructure. This study suggests that temperature, a parameter already monitored and in some contexts already manipulated, could be part of that toolkit, particularly in climates or seasons where colder operation is achievable. It also underscores a fundamental ecological principle: the fate of antibiotic resistance genes in engineered environments is governed not only by the genes themselves but by the fitness of the microbial communities that carry them. By shifting the thermal environment, operators can shift the competitive balance of those communities, and with it, the resistance burden flowing out of the plant and into the world downstream.

Subject of Research: Effect of temperature on antibiotic resistance gene dynamics in conventional activated sludge wastewater treatment

Article Title: Influent-driven antibiotic resistance dynamics are constrained by low temperatures in conventional activated sludge systems

Article References: Influent-driven antibiotic resistance dynamics are constrained by low temperatures in conventional activated sludge systems. (n.d.). https://doi.org/10.1007/s00253-026-14036-0

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14036-0

Keywords: antibiotic resistance genes, wastewater treatment, activated sludge, temperature, intI1, blaIMP, qnrS, Escherichia coli, Acinetobacter, bacterial community, nutrient removal, environmental microbiology