Mechanisms Underlying the Overlooked Chiral Fungicide-Driven Enantioselective Proliferation of Antibiotic Resistance in Earthworm Intestinal Microbiome.

From a "One Health" perspective, the global threat of antibiotic resistance genes (ARGs) is associated with modern agriculture practices including agrochemicals application. Chiral fungicides account for a considerable proportion of wildly used agrochemicals; however, whether and how their enantiomers lead to differential proliferation of antibiotic resistance in agricultural environments remain overlooked. Focused on the soil-earthworm ecosystem, we for the first time deciphered the mechanisms underlying the enantioselective proliferation of antibiotic resistance driven by the enantiomers of a typical chiral fungicide mandipropamid (i.e., R-MDP and S-MDP) utilizing a multiomic approach. Time-series metagenomic analysis revealed that R-MDP led to a significant enhancement of ARGs with potential mobility (particularly the plasmid-borne ARGs) in the earthworm intestinal microbiome. We further demonstrated that R-MDP induced a concentration-dependent facilitation of plasmid-mediated ARG transfer among microbes. In addition, transcriptomic analysis with verification identified the key aspects involved, where R-MDP enhanced cell membrane permeability, transfer ability, biofilm formation and quorum sensing, rebalanced energy production, and decreased cell mobility versus S-MDP. Overall, the findings provide novel insights into the enantioselective disruption of microbiome and resistome in earthworm gut by chiral fungicides and offer significant contributions to the comprehensive risk assessment of chiral agrochemicals in agroecosystems.

A novel bidirectional regulation mechanism of mancozeb on the dissemination of antibiotic resistance.

The high abundance of antibiotic resistance genes (ARGs) in the fungicide residual environment, posing a threat to the environment and human health, raises the question of whether and how fungicide promotes the prevalence and dissemination of antibiotic resistance. Here, we reported a novel mechanism underlying bidirectional regulation of a typical heavy-metal-containing fungicide mancozeb on the horizontal transfer of ARGs. Our findings revealed that mancozeb exposure significantly exerted oxidative and osmotic stress on the microbes and facilitated plasmid-mediated ARGs transfer, but its metallic portions (Mn and Zn) were potentially utilized as essential ions by microbes for metalating enzymes to deal with cellular stress and thus reduce the transfer. The results of transcriptome analysis with RT-qPCR confirmed that the expression levels of cellular stress responses and conjugation related genes were drastically altered. It can be concluded mancozeb bidirectionally regulated the ARGs dissemination which may be attributed to the diverse effects on the microbes by its different portions. This novel mechanism provides an updated understanding of neglected fungicide-triggered ARGs dissemination and crucial insight for comprehensive risk assessment of fungicides.

Fungicide exposure accelerated horizontal transfer of antibiotic resistance genes via plasmid-mediated conjugation.

Co-pollution of soil with pesticide residues and antibiotic resistance genes (ARGs) is increasing due to the substantial usage of pesticides and organic fertilizers in greenhouse-based agricultural production. Non-antibiotic stresses, including those from agricultural fungicides, are potential co-selectors for the horizontal transfer of ARGs, but the underlying mechanism remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic resistant plasmid RP4 were established to examine conjugative transfer frequency under stress from four widely used fungicides: triadimefon, chlorothalonil, azoxystrobin, and carbendazim. The mechanisms were elucidated at the cellular and molecular levels using transmission electron microscopy, flow cytometry, RT-qPCR, and RNA-seq techniques. The conjugative transfer frequency of plasmid RP4 between Escherichia coli strains increased with the rising exposure concentrations of chlorothalonil, azoxystrobin, and carbendazim, but was suppressed between E. coli and Pseudomonas putida by a high fungicide concentration (10 µg/mL). Triadimefon did not significantly affect conjugative transfer frequency. Exploration of the underlying mechanisms revealed that: (i) chlorothalonil exposure mainly promoted generation of intracellular reactive oxygen species, stimulated the SOS response, and increased cell membrane permeability, while (ii) azoxystrobin and carbendazim primarily enhanced expression of conjugation-related genes on the plasmid. These findings reveal the fungicide-triggered mechanisms associated with plasmid conjugation and highlight the potential role of non-bactericidal pesticides on the dissemination of ARGs.

Deposition and dissipation of difenoconazole in pepper and soil and its reduced application to control pepper anthracnose.

The initial deposition amount, dissipation dynamics, retention rate, and field control efficacy of difenoconazole in pepper-soil system were studied with different application dosages, planting regions and patterns. The initial deposition amount of difenoconazole under the same application dosage showed the following order: fruits < cultivated soils < lower stems < upper stems < lower leaves < upper leaves, open field < greenhouse, and Changjiang < Cixi < Hefei < Langfang, respectively, which increased with increasing application dosage. The dissipation rates in leaves, stems, fruits and cultivated soils exhibited an initially fast and then slow trend, while the retention rates displayed a tendency of first increasing and then stabilizing with increasing application dosages. After 7 d of difenoconazole application, the retention rates at five concentrations were 10.3%- 39.1%, and the field efficacy mostly reached the minimum effective dose. These results suggested that difenoconazole could be reduced by 25% based on the minimum recommended dose meeting the requirements of field control efficacy for controlling pepper anthracnose.

Carbendazim shapes microbiome and enhances resistome in the earthworm gut.

BACKGROUND: It is worrisome that several pollutants can enhance the abundance of antibiotic resistance genes (ARGs) in the environment, including agricultural fungicides. As an important bioindicator for environmental risk assessment, earthworm is still a neglected focus that the effects of the fungicide carbendazim (CBD) residues on the gut microbiome and resistome are largely unknown. In this study, Eisenia fetida was selected to investigate the effects of CBD in the soil-earthworm systems using shotgun metagenomics and qPCR methods. RESULTS: CBD could significantly perturb bacterial community and enrich specific bacteria mainly belonging to the phylum Actinobacteria. More importantly, CBD could serve as a co-selective agent to elevate the abundance and diversity of ARGs, particularly for some specific types (e.g., multidrug, glycopeptide, tetracycline, and rifamycin resistance genes) in the earthworm gut. Additionally, host tracking analysis suggested that ARGs were mainly carried in some genera of the phyla Actinobacteria and Proteobacteria. Meanwhile, the level of ARGs was positively relevant to the abundance of mobile genetic elements (MGEs) and some representative co-occurrence patterns of ARGs and MGEs (e.g., cmx-transposase and sul1-integrase) were further found on the metagenome-assembled contigs in the CBD treatments. CONCLUSIONS: It can be concluded that the enhancement effect of CBD on the resistome in the earthworm gut may be attributed to its stress on the gut microbiome and facilitation on the ARGs dissemination mediated by MGEs, which may provide a novel insight into the neglected ecotoxicological risk of the widely used agrochemicals on the gut resistome of earthworm dwelling in soil. Video abstract.

Analysis method development and health risk assessment of pesticide and heavy metal residues in Dendrobium Candidum.

Herbal medicines that are widely used worldwide are easily contaminated by pesticides and heavy metals, threatening human health. In this study, a modified QuEChERS pre-treatment method combined with HPLC/GC-MS/MS was established for the determination of 24 pesticide residues in Dendrobium candidum. The average recoveries of 24 pesticides in D. candidum were 76.9-110.0% with the relative standard deviation (RSD) of 0.28-11.40%, and their limits of detection (LOD) and limits of quantitation (LOQ) were 0.005-10 and 0.011-22 µg kg-1, respectively. The results showed that 83.33% of all samples had detected pesticide residues with the concentrations of 0.06-312.83 µg kg-1. Meanwhile, microwave digestion combined with ICP-MS was used to detect the residues of 8 heavy metals in D. candidum. The average recoveries of 8 heavy metals were 82.7-108.1% with an RSD of 1.4-8.0%, and their LOD and LOQ were 0.0001-0.05 mg kg-1 and 0.0003-0.2 mg kg-1, respectively. The results indicated that 8 heavy metals were all detected in all samples, and the highest concentration of Zn was 11.97 mg kg-1. Furthermore, the health risk assessment showed that the risk of the detected pesticides and heavy metals in samples to humans, specifically to the general population including adults and children, was acceptable.

The jasmonic acid-amino acid conjugates JA-Val and JA-Leu are involved in rice resistance to herbivores.

The phytohormone jasmonic acid (JA) plays a core role in plant defence against herbivores. When attacked by herbivores, JA and its bioactive derivatives are accumulated at the damage site, and subsequently perceived by the jasmonate co-receptors COI1 and JAZ proteins. The (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is known to be the main active JA derivative controlling vascular plant responses to herbivores as well as other JA-regulated processes. However, whether other endogenous JA-amino acid conjugates (JA-AAs) are involved in herbivore-induced defence responses remain unknown. Here, we investigated the role of herbivore-elicited JA-AAs in the crop plant rice. The levels of five JA-AAs were significantly increased under the armyworm, leaf folder and brown planthopper attack. Of the elicited JA derivatives, JA-Ile, JA-Val and JA-Leu could serve as ligands to promote the interaction between rice COI1 and JAZs, inducing OsJAZ4 degradation in vivo. JA-Val or JA-Leu treatment increased the expression of JA- and defence-related pathway genes but not JA-Ile levels, suggesting that these JA-AAs may directly function in JA signalling. Furthermore, the application of JA-Val or JA-Leu resulted in JA-mediated plant growth inhibition, while enhancing plant resistance to herbivore attack. This study uncovers that JA-Val and JA-Leu also play a role in rice defence against herbivores.

Reduced bacterial network complexity in agricultural soils after application of the neonicotinoid insecticide thiamethoxam.

Pesticides may alter soil microbial community structure or diversity, but their impact on microbial co-occurrence patterns remains unclear. Here, the effect of the widely used neonicotinoid insecticide thiamethoxam on the bacterial community in five arable soils was deciphered using the 16S rRNA gene amplicon sequencing technique. The degradation half-life of thiamethoxam in nonsterilized soils was significantly lower than that in sterilized soils, suggesting a considerable contribution from biodegradation. Soil bacterial community diversity diminished in high concentration thiamethoxam treatment and its impact varied with treatment concentration and soil type. Bacterial co-occurrence network complexity significantly decreased after exposure to thiamethoxam. Under thiamethoxam stress, the relative changes in bacterial co-occurrence networks were closely related (the majority of p-values < 0.05) to the soil physicochemical properties, yet the diversity and dominant phyla were slightly related (the majority of p-values > 0.05). Additionally, three bacterial genera, Sphingomonas, Streptomyces, and Catenulispora, were identified to be relevant to the degradation of thiamethoxam in soils. This finding deciphers the succession of the bacterial community under thiamethoxam stress across multiple soils, and emphasizes the potential role of physicochemical properties in regulating the ecotoxicological effect of pesticides on the soil microbiome.

Foam shares antibiotic resistomes and bacterial pathogens with activated sludge in wastewater treatment plants.

Foaming is a common operational problem that occurs in activated sludge (AS) from many wastewater treatment plants (WWTPs), but the characteristic of antibiotic resistance genes (ARGs) and human pathogenic bacteria (HPB) in foams is generally lacking. Here, we used a metagenomic approach to characterize the profile of ARGs and HPB in foams and AS from full-scale WWTPs receiving pesticide wastewater. No significant difference in the microbial communities was noted between the AS and foam samples. The diversity and abundance of ARGs in the foams were similar to those in the pertinent AS samples. Procrustes analysis suggested that the bacterial community is the major driver of ARGs. Metagenomic assembly also indicated that most ARGs (e.g., multidrug, rifamycin, peptides, macrolide-lincosamide-streptogramin, tetracycline, fluoroquinolone, and beta-lactam resistance genes) were carried by chromosomes rather than mobile genetic elements. Moreover, the relative abundances of HPB, Pseudomonas putida and Mycobacterium smegmatis, were enriched in the foam samples. Nine HPB were identified as carriers of 21 ARG subtypes, of which Pseudomonas aeruginosa could carry 12 ARG subtypes. Overall, this study indicates the prevalence of ARGs, HPB, and ARG-carrying HPB in foams, which highlights the potential risk of foams in spreading ARGs and HPB into the surrounding environments.

Determination and Dietary Intake Risk Assessment of Pesticide Residues in Fritillariae Thunbergii Bulbs and Cultivated Soils.

BACKGROUND: Pesticide residues in traditional Chinese medicines pose a potential risk to human health. However, little is known about the characteristics of pesticide residues in the fritillariae thunbergii bulbs (FTB). OBJECTIVE: This study aims to establish a method for the determination of pesticide multi-residues in FTB and then measured their residual levels in the FTB collected from nine cultivation regions. METHODS: A modified QuEChERS method coupled with GC/UPLC was used to determine the residues of 24 pesticides in the FTB and soil samples. RESULTS: The recoveries of these pesticides at three concentrations were 72.17-112.48% in the FTB and 70.92-113.74% in the soil with RSD < 11.83%, and the LOD and LOQ ranged from 0.005-10 µg/kg and 0.011-22 µg/kg, respectively. A total of 13 pesticide residues were detected in the FTB samples with the residual levels of 0.0011-509.63 µg/kg, which were all below the referred MRLs in other Chinese herbs or food. Meanwhile, dietary intake risk assessment showed that the risk of pesticide residues in the FTB was acceptable to consumers. CONCLUSIONS: This study developed a method for the determination of pesticide multi-residues in the FTB and cultivated soil samples, and furthermore the dietary intake risk of pesticide residues in the FTB is safe. HIGHLIGHTS: A modified QuEChERS method was established for the determination of 24 pesticides in the FTB with high sensitivity, accuracy, and precision.

Exposure to fungicide difenoconazole reduces the soil bacterial community diversity and the co-occurrence network complexity.

Difenoconazole is a triazole fungicide that is widely used worldwide and has been frequently detected in agricultural soils, but its ecotoxicological effect on soil bacterial community remains unknown. Here, the degradation of difenoconazole and its effect on soil bacterial communities were investigated at three concentrations in five different agricultural soils. Difenoconazole degraded faster in non-sterilized soils than in sterilized soils, suggesting that biodegradation is a major contributor to the dissipation of difenoconazole in soils. Exposure to high concentrations of difenoconazole decreased the soil bacterial community diversity in most soils, and this influence was aggravated with the increasing concentration. The effect of difenoconazole on soil bacterial community diversity was also enhanced with the increasing content of organic matter and total nitrogen in soils. Moreover, difenoconazole exposure also reduced the soil bacterial community network complexity and exhibited a concentration-dependent characteristic. In addition, a core bacterial community (57 operational taxonomic units, OTUs) was identified, and some core OTUs were strongly linked to the degradation of difenoconazole in soils. It is concluded that high concentrations of difenoconazole may have a significant effect on the soil bacterial communities, and co-occurrence networks may improve the ecotoxicological risk assessment of fungicides on soil microbiome.

Tracking resistomes, virulence genes, and bacterial pathogens in long-term manure-amended greenhouse soils.

Organic manure has been implicated as an important source of antibiotic resistance genes (ARGs) in agricultural soils. However, the profiles of biocide resistance genes (BRGs), metal resistance genes (MRGs) and virulence genes (VGs) and their bacterial hosts in manure-amended soils remain largely unknown. Herein, a systematic metagenome-based survey was conducted to comprehensively explore the changes in resistomes, VGs and their bacterial hosts, mobile genetic elements (MGEs), and pathogenic bacteria in manure-amended greenhouse soils. Many manure-borne ARGs, BRGs, MRGs, VGs, and bacterial pathogens could be transferred into soils by applying manures, and their abundance and diversity were markedly positively correlated with greenhouse planting years (manure amendment years). The main ARGs transferred from manures to soils conferred resistance to tetracycline, aminoglycoside, and macrolide-lincosamide-streptogramin. Both statistical analysis and gene arrangements showed a good positive co-occurrence pattern of ARGs/BRGs/MRGs/VGs and MGEs. Furthermore, bacterial hosts of resistomes and VGs were significantly changed in the greenhouse soils in comparison with the field soils. Our findings confirmed the migration and dissemination of resistomes, VGs, and bacterial pathogens, and their accumulation and persistence were correlated with the continuous application of manures.

Enterobacteriaceae predominate in the endophytic microbiome and contribute to the resistome of strawberry.

Antibiotic resistance genes (ARGs) harbored by plant microbiomes have been implicated as a potential risk to public health via food chain, especially directly edible fruits and vegetables. Here, we investigated the microbiome and antibiotic resistome in soil-strawberry ecosystem using shotgun metagenomic sequencing. The results showed that the enterobacterial population dominated the endophytes of strawberry fruits. Moreover, 85 subtypes of ARGs, including several clinically important ARGs, were detected in the strawberry fruit metagenomes. Additionally, host tracking analysis in combination with antibiotic-resistant bacterial isolate screening suggested that fruit-borne ARGs were mainly carried by members of the Enterobacteriaceae family. Unexpectedly, most of fruit-borne isolates were found to be resistant to several clinically important antimicrobials, e.g., erythromycin and cephalexin. Our findings provide broad insights into endophytic antibiotic resistomes of direct edible strawberry fruits and their potential hosts, and highlight the potential exposure risks of plant microbiomes to the human food chain.