Chulabhorn Graduate Institute

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.

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.

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.

Aeromonas veronii is an important bacterial pathogen responsible for motile Aeromonas septicemia (MAS) in aquaculture and is commonly controlled through antibiotic use. The increasing prevalence of antimicrobial resistance (AMR) has driven the need for alternative control strategies, among which bacteriophages have emerged as promising biocontrol agents. Here, we characterized two lytic A. veronii-specific bacteriophages, DJ6712 and FW6709, previously isolated from wastewater and aquaculture environments. Whole-genome sequencing and comparative genomic analyses were performed to investigate their genomic features, evolutionary relationships, and suitability for therapeutic applications. DJ6712 and FW6709 possess double-stranded DNA genomes of approximately 60 – 62kb, each with a GC content of 62%. Genome annotation revealed genes associated with phage structure, DNA metabolism, and host lysis, including endolysin and Rz-like spanin proteins. Importantly, no antibiotic resistance genes, virulence factors, or lysogeny-related elements were detected. Phylogenetic analysis based on the conserved terminase large subunit (TerL) indicated that DJ6712 is most closely related to Aeromonas phage vB_C4, whereas FW6709 shows the closest evolutionary relationship to Aeromonas phage pAEv1. Whole-genome similarity analyses using VIRIDIC and Mash further demonstrated that DJ6712 and FW6709 are closely related to Aeromonas phage vB_C4 and Aeromonas phage BUCT551, respectively, within the family Casjensviridae. However, intergenomic similarity values below the commonly accepted species demarcation threshold (95%) support that DJ6712 and FW6709 represent genetically distinct A. veronii phage isolates within established phage lineages. Overall, these results expand the limited genomic knowledge of A. veronii-specific bacteriophages and support further investigation of DJ6712 and FW6709 for potential phage-based control of MAS in aquaculture.