Chulabhorn Graduate Institute

Agrobacterium tumefaciens, a soil bacterium, was used as a model organism to study the mechanisms of triclosan (TCS) resistance in environmental bacteria. Adaptive laboratory evolution tests were performed to select TCS-resistant strains by challenging a wild-type (WT) strain with increasing concentrations of TCS (8, 12, 16, and 20 µg/mL). Two high-dose-resistant strains, HDR-12a and HDR-20a, were isolated and used for detailed examination. In comparison to the minimum inhibitory concentration of the WT strain (10 µg/mL), HDR-12a (20 µg/mL), and HDR-20a (32 µg/mL) showed increased resistance to TCS. Whole-genome sequencing and transcriptomic analysis performed to identify mechanisms underlying the different degrees of TCS resistance among the two evolved A. tumefaciens strains revealed a nucleotide base change (missense mutation, Asn157Thr) in the transcriptional repressor triR gene as the key mechanism of TCS resistance in HDR-20a. This change reduced the DNA-binding ability of TriR, causing overexpression of the triABC operon that encodes the TCS-specific efflux pump. In contrast, HDR-12a had no mutation in the triR gene. HDR-12a exhibited transcriptomic changes in several genes involved in ATP-binding cassette (ABC) transporters and in the metabolism of sulfur, fatty acids, and carbohydrates. However, it remains unclear whether these transcriptomic changes are directly responsible for TCS resistance in HDR-12a. Both the TCS-adapted strains also showed increased resistance to chloramphenicol and erythromycin. Overall, these results demonstrate that TCS pollution in environmental hotspots can select for adaptive and cross-resistant bacteria.IMPORTANCETCS is widely used as a preservative and disinfectant in many personal healthcare products. TCS is subsequently released into aquatic and terrestrial environments. The emergence and spread of multidrug-resistant pathogens from the use of antimicrobials like TCS and the misuse of antibiotic drugs now pose a serious global public health threat. Understanding how resistance develops has implications for preventing the emergence of antimicrobial resistance. The adapted TCS-resistant strains showed cross-resistance to chloramphenicol and erythromycin. This study provides insight into how environmental exposure to triclosan can drive adaptive and cross-resistance mechanisms in a soil bacterium, highlighting its relevance to environmental antimicrobial resistance and public health risk.

A mild photoinduced isomerization of isatogens to benzoxazin-4-ones is described. Upon irradiation at 370 nm, a broad range of 2-aryl- and 2-alkyl-substituted isatogens undergo efficient scaffold reorganization under catalyst-free conditions to afford benzoxazin-4-ones in good to excellent yields. The transformation tolerates diverse substitution patterns, including heteroaryl and strained alkyl groups, and is readily scalable. Mechanistic experiments support participation of an excited-state pathway, and inclusion of catalytic 2-picolinic acid provided reproducible outcomes. For a nitro-substituted substrate, a distinct reaction pathway is observed, leading to the formation of a toluene-derived adduct. These results establish isatogens as versatile photochemical precursors to benzoxazinone scaffolds under operationally simple conditions.

Antimicrobial-resistant bacteria are emerging biological contaminants that threaten both ecosystems and human health. Antimicrobial resistance (AMR) in livestock waste is increasingly recognized as an environmental hazard of global concern, with implications for food safety and public health. This study examined the occurrence and environmental behavior of extended-spectrum β-lactamase (ESBL)-producting and colistin-resistant Escherichia coli under environmentally relevant conditions in livestock waste from pig, cow, goat, chicken, and duck farms in Thailand. Cefotaxime-resistant E. coli were detected in all animal types, with pig, cow, and duck farms serving as major reservoirs. Untreated pig wastewater contained the highest levels of resistant E. coli (70% prevalence; 5.13-7.55 log10 CFU/100 mL), while treatment reduced but did not eliminate contamination. Among 78 confirmed isolates, all were resistant to ampicillin and cefotaxime, 5.1% to ceftazidime, and none to carbapenems. Phenotypic colistin resistance occurred in 6.4% of isolates. All isolates carried blaCTX-M genes, mainly blaCTX-M group 1 (85.9%), while blaCTX-M group 9 (15.4%) was also detected. Plasmid-mediated mcr genes (mcr-1 to mcr-3) were identified in 5.1% of isolates, exclusively from untreated pig wastewater, confirming these effluents as localized hotspots for resistance dissemination. Multiple antibiotic resistance indices ranged 0.29-0.43, with the highest values in pig farm isolates, indicating moderate to high levels of multidrug resistance. Overall, livestock wastewater represents a significant source of microbial contaminants. This study provides field-based evidence to support risk assessment and mitigation of antimicrobial-resistant bacteria within the One Health framework, emphasizing the importance of improving waste management to protect environmental and public health.

Stenotrophomonas maltophilia is a multidrug-resistant Gram-negative opportunistic pathogen. Its antimicrobial resistance arises from intrinsic traits and acquired mechanisms. Overexpression of drug efflux transporters is one of the key mechanisms mediating high-level antibiotic resistance. These transporters are regulated by transcriptional factors, including SoxR, a superoxide-responsive regulator containing an iron-sulfur [2Fe-2S] cluster. SoxR controls the multidrug efflux pumps MfsA and SmeVWX, as well as superoxide dismutase SodA1. To examine how soxR mutations affect antimicrobial resistance, random mutagenesis was performed, and mutated genes were ectopically expressed in a ΔsoxR mutant. Transformants were selected for ciprofloxacin resistance, a substrate for both MfsA and SmeVWX. Sequencing of 21 resistant colonies revealed five distinct mutation patterns (A–E), each containing multiple mutations. The ΔsoxR mutant expressing these soxR variants showed increased resistance to multiple antibiotics targeted by the efflux pumps, correlating with elevated MfsA and SmeVWX expression. This indicates constitutive SoxR activation. To assess the role of the [2Fe-2S] cluster, cysteine-to-serine substitutions at three of the four cluster-binding residues abolished SoxR’s ability. Computational structure prediction and molecular dynamics simulations supported these findings, revealing that mutations enhanced solvent exposure of the [2Fe-2S] cluster. This suggests that the mutations shift SoxR into a conformation more prone to activation. Furthermore, a single amino acid substitution at residue R49 was sufficient to activate SoxR and confer resistance. Together, our findings demonstrate that S. maltophilia can enhance antimicrobial resistance through soxR mutations, particularly under antibiotic pressure. This highlights the role of SoxR in the antimicrobial resistance of S. maltophilia.

The edible mushroom Pleurotus citrinopileatus Singer contains bioactive compounds that may be used for the management of obesity. Despite extensive research, secondary metabolites with lipase inhibition and suppression of lipid accumulation remain unexplored. Herein, we report the isolation, structure elucidation, and biological evaluation of secondary metabolites from the fruiting bodies of P. citrinopileatus. Based on spectroscopic analysis and a comparison with spectroscopic data reported in the literatures, six compounds were identified: ergosterol (1), cerevisterol (2), uracil (3), a diastereomeric mixture of 5'-deoxy-5'-methylaminoadenosine (4), diethylene glycol monobutyl ether (5), and tryptophan (6). Compounds 1-6 showed mild lipase inhibitory activity ranging from 1.7 to 11.3%. In contrast to a previous report, ergosterol 1 did not inhibit lipid accumulation in 3T3-L1 adipocytes at 25, 50, and 75 µM. This finding provides valuable insight about secondary metabolites from the fruiting bodies of this mushroom and reports their lipase inhibitory and anti-lipid accumulation activities.