Bin Liang

Isothiazolinones are widely used nonantibiotic antimicrobials with high electrophilic reactivity toward bacterial protein thiols. Although this reactivity leads to rapid degradation and low environmental persistence, their potential to drive cryptic microbial evolution remains poorly understood. Here, we focused on methylisothiazolinone (MIT), a widely used isothiazolinone, and conducted a 60-cycle experimental evolution of Pseudomonas aeruginosa PAO1 across a concentration gradient spanning environmentally relevant (10 μg/L) to preservative-use (8–16 mg/L) levels. We demonstrate an exposure-level-dependent bifurcation in evolutionary strategies. Trace-level MIT exposure enhanced horizontal gene transfer capacity (from 0.0520 ± 0.0006 to 0.0764 ± 0.0008) through membrane remodeling, including elevated membrane potential, reduced extracellular polymeric substances, and 2.79-fold induction of indole signaling. In contrast, preservative-level MIT exposure drove key mutations (e.g., mexR deletion) and metabolic-transcriptional rewiring, increasing minimal inhibitory concentrations of Meropenem by 8- to 16-fold with minimal fitness costs. Furthermore, the 16 mg/L-evolved lineages exhibited hypervirulence, causing 100% mortality within 24 h in a Galleria mellonella model compared to 90% ancestral survival. These findings demonstrate that even trace exposure to highly bioactive antimicrobials can reshape microbial evolution and accelerate resistance emergence, highlighting unrecognized evolutionary risks and providing a critical scientific basis for refining their risk assessment and management frameworks.

Reclaimed water (RW) is increasingly used for urban river replenishment, yet its ecological implications for multitrophic aquatic communities remain poorly understood. Here, we integrate multitrophic biodiversity, biotic integrity, food-web topology, environmental stress, and emerging contaminant (EC) risk to assess RW ecological effects in a typical urban receiving river. Downstream multitrophic Shannon and richness indices increased by 34.49% and 18.34%, respectively, whereas the multitrophic index of biotic integrity (M-IBI) declined by 13.58%. This reveals a clear decoupling between biodiversity elevation and in-situ ecological health. RW shifted riverine communities toward more tolerant and homogeneous assemblages. Downstream food webs gain stronger topological structure resilience driven by proliferation of phytoplankton and enhanced detritus utilization of bacteria, rather than an overall improvement in balance. RW replenishment intensifies comprehensive physicochemical stress and EC exposure risks. RW-derived multiple factors reshape riverine food webs via trophic-specific pathways. Specifically, consumer communities (fish, zoobenthos, and zooplankton) are constrained by reduced potential food transfer across the inferred food web, while producers (phytoplankton) and decomposers (bacteria) are predominantly impacted by ambient environmental shifts. These findings highlight that chemical compliance does not necessarily guarantee ecological sustainability of reclaimed water management, and biodiversity metrics alone are insufficient to comprehensively assess the ecological impacts of RW replenishment. This study offers new insights into multitrophic community response and underlying ecological effects under RW replenishment.

Landfill leachate is a chemically complex pollution source containing co-occurring antimicrobials, metals, organic matter, nutrients, and other stressors that may shape environmental resistomes. However, in such heterogeneous matrices, it remains difficult to identify which antimicrobial compounds are most consistently associated with antibiotic resistance gene (ARG) patterns. Here, we integrated targeted antimicrobial measurements with metagenomic ARG profiling in landfill leachate from 17 Chinese cities. We detected 52 antimicrobials with widespread risk quotient exceedances and 296 ARG subtypes representing a diverse resistome. Conventional pairwise and matrix-level analyses revealed fragmented antimicrobial–ARG relationships, indicating that concentration ranking, RQ-based screening, and one-variable-at-a-time comparisons were insufficient for this mixture-dominated system. Using ARG Shannon diversity as a low-dimensional descriptor of resistome organization, we applied a stability-oriented multivariable framework to identify antimicrobial features reproducibly associated with resistome variation. A linear support vector machine captured the strongest within-dataset covariation signal (cross-validated R² = 0.620 ± 0.342). Stability filtering identified clinafloxacin, clindamycin hydrochloride, and sulfathiazole as the compounds most consistently associated with ARG diversity. Covariate-adjusted analysis further distinguished a robust negative estimated effect for clinafloxacin from more context-dependent positive estimates for clindamycin hydrochloride and sulfathiazole. Together, these results move risk assessment beyond occurrence and RQ ranking by identifying specific antimicrobial signals linked to resistome variation, supporting integrated chemical–biological prioritization in complex pollution matrices.

Haloacetates, particularly trichloroacetate (TCAA), are pervasive contaminants, yet their biotransformation in anoxic, nutrient-limited environments remains poorly understood. Here, we elucidate a geographically widespread attenuation of haloacetates in nutrient-limited aquifers by field surveys across six Chinese provinces. Enrichment cultures metabolized TCAA as the sole carbon and energy source. Major metabolites (oxalate, acetate, CO₂) indicated complete hydrolytic dechlorination accompanied by fermentation and mineralization. Isotope labeling and chemical probe studies confirmed the simultaneous occurrence of TCAA dechlorination and nitrogen fixation under fixed nitrogen-limited conditions. Integrated metagenomic, metabolomic, and transcriptional analyses demonstrated nitrogen-regulated metabolic reconfiguration. Acetyl-CoA potentially associated with TCAA metabolism may enter the TCA cycle under nitrogen-limited conditions. Dehalogenase gene (had) homologues extensively co-occurred with homologues of nitrogenase genes (nifHDK) in oligotrophic environments. Potential cross-feeding interactions among unclassified Azospira sp., Ralstonia pickettii, and Azospira inquinata mediated carbon-nitrogen intermediate exchange. This study identifies a previously unrecognized process that enables simultaneous TCAA detoxification and nitrogen acquisition in oligotrophic aquifers, thereby proposing an energy-conserving and ecologically adaptive strategy for haloacetate bioremediation.