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.

Characterization of a novel carbendazim-degrading strain Rhodococcus sp. CX-1 revealed by genome and transcriptome analyses.

The persistence and ecotoxicity of carbendazim residues pose a potential risk to environmental ecology and human health. Here, a novel and highly efficient carbendazim-degrading bacterium Rhodococcus sp. CX-1, capable of utilizing carbendazim as its sole source of carbon and energy, was isolated from contaminated soil. The biodegradation characteristics and metabolic pathways were studied by mass spectrometry, genomic annotation, and transcriptome analysis. The degradation rate of carbendazim by strain CX-1 was 3.98-9.90 mg/L/h under different conditions, and the optimum degradation conditions were 40 °C and pH 7.0. The addition of carbon sources (glucose, fructose, and sucrose, 100 mg/L) could accelerate carbendazim degradation. HPLC-MS/MS identification suggested that carbendazim is first hydrolyzed into 2-aminobenzimidazole and then to 2-hydroxybenzimidazole, and is ultimately mineralized to carbon dioxide. The genome of strain CX-1 contained 6,511,628 bp nucleotides, 2 linear plasmids, 2 circular plasmids, and 6437 protein coding genes. Genome annotation and transcriptome analysis indicated that carbendazim degradation may be regulated by the degradation genes harbored in the chromosome and in plasmid 2, and two different degradation pathways of carbendazim by imidazole ring cleavage or benzene ring cleavage were predicted. This study provided new insight to reveal the biodegradation mechanism of carbendazim; furthermore, strain CX-1 is a promising bioresource for carbendazim bioremediation.

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.

Fungicides enhanced the abundance of antibiotic resistance genes in greenhouse soil.

Long-term substantial application of fungicides in greenhouse cultivation led to residual pollution in soils and then altered soil microbial community. However, it is unclear whether residual fungicides could affect the diversity and abundance of antibiotic resistance genes (ARGs) in greenhouse soils. Here, the dissipation of fungicides and its impact on the abundance of ARGs were determined using shotgun metagenomic sequencing in the greenhouse and mountain soils under laboratory conditions. Our results showed the greenhouse soils harbored more diverse and abundant ARGs than the mountain soils. The application of carbendazim, azoxystrobin, and chlorothalonil could increase the abundance of total ARGs in the greenhouse soils, especially for those dominant ARG subtypes including sul2, sul1, aadA, tet(L), tetA(G), and tetX2. The abundant ARGs were significantly correlated with mobile genetic elements (MGEs, e.g. intI1and R485) in the greenhouse soils but no significant relationship in the mountain soils. Meanwhile, the co-occurrence patterns of ARGs and MGEs, e.g., sul2 and R485, sul1 and transposase, were further verified via the genetic arrangement of genes on the metagenome-assembled contigs in the greenhouse soils. Additionally, host tracking analysis indicated that ARGs were mainly carried by enterobacteria in the greenhouse soils but actinomyces in the mountain soils. These findings confirmed that some fungicides might serve as the co-selectors of ARGs and elevated their abundance via MGEs-mediated horizontal gene transfer in the greenhouse soils.

Deposition, Distribution, Metabolism, and Reduced Application Dose of Thiamethoxam in a Pepper-Planted Ecosystem.

To decrease the application dose of thiamethoxam (TMX) to control the pepper whitefly (Bemisia tabaci Q), the deposition, dissipation, metabolism, and field efficacy of TMX were investigated in a pepper (Capsicum annuum var. grossum)-planted ecosystem using eight types of nozzles at six concentrations (56.25, 41.25, 26.25, 21.0, 15.75, and 10.5 g a.i./hm2). The initial deposition amount of TMX in the pepper plant first increased and then decreased with increasing application dose. The optimum spray conditions of TMX were found to be a droplet size of 200 µm volume median diameter and a spray volume of 350 L/hm2. Moreover, three metabolites, TMX-dm, clothianidin (CLO), and C5H8O2N3SCl, were detected in the pepper-planted system. The dissipation rate of TMX in the pepper-field ecosystem was leaves > stems > fruits > roots > soils. The results revealed the deposition and fate of TMX in the pepper-field ecosystem, and the application dose could be reduced by 20% based on the minimum recommended dose for controlling pepper whitefly.

Development of antibiotic resistance genes in soils with ten successive treatments of chlortetracycline and ciprofloxacin.

Antibiotic contamination caused by the long-term use of organic manure (OM) in greenhouse agricultural soils poses potential detrimental effects to the soil environment. By applying OM containing chlortetracycline (CTC) and/or ciprofloxacin (CIP) ten times in soil under laboratory conditions, we investigated the dissipation and accumulation characteristics of CTC and CIP in the soil, the changes in the microbial pollution-induced community tolerance (PICT), and the diversity and abundance of antibiotic resistance genes (ARGs) in the soil microbiome. The dissipation of CTC was rapid while CIP was accumulated in repeatedly treated soils; further, CIP could inhibit the dissipation of CTC. Meanwhile, the PICT to CTC and/or CIP significantly increased up to 15.0-fold after ten successive treatments compared to that in the first treatment. As the treatment frequency increased, significant upward trends in the abundances of tetracycline resistance genes tetA(G), tetX2, tetX, tetG, tetA(33), tetA, tetW, and tetA(P), fluoroquinolone resistance gene qnrA6, and multiple resistance gene mexF were revealed by both metagenomic and qPCR analyses. The findings demonstrated that repeated treatments with CTC and/or CIP can alter the dissipation rate, promote an increase in PICT to CTC and/or CIP, and increase the ARGs abundance in steps.

Exploring microbial community structure and biological function in manured soil during ten repeated treatments with chlortetracycline and ciprofloxacin.

The changes of enzyme activities, microbial community structure and function, and the diversity and resistance level of antibiotic-resistant bacteria (ARB) were studied in soil during ten repeated treatments with chlortetracycline (CTC) and/or ciprofloxacin (CIP) together with organic manure (OM) under laboratory conditions. The activities of neutral phosphatase (NPA) and catalase (CAT) displayed the suppression-recovery-stimulation trend in the OM&CTC treatment but the stimulation trend in the OM&CTC&CIP treatment. The NPA was stimulated but the CAT was little affected in the OM&CIP treatment. Soil microbial functional diversity displayed the suppression-recovery-stimulation trend in the OM&CTC and OM&CTC&CIP treatments and the stimulation-suppression trend in the OM&CIP treatment with the treatment frequency. Metagenomic analysis showed that the relative abundances of Actinobacteria and Firmicutes in the antibiotic treatment significantly increased by 0.5-235.6%, but that of Proteobacteria decreased by 0.2-27.3% compared to the control with the treatment frequency. Furthermore, the relative abundances of dominant bacterial genera including Streptomyces, Actinomadura, Mycobacterium, and Streptococcus in the antibiotic treatment significantly increased by 1.1-10433.3% compared to the control. Meanwhile, repeated antibiotic treatments induced a significant increase in the diversity and resistance level of ARB isolates, especially in the OM&CTC treatment. It is concluded that repeated treatments with CTC and/or CIP can alter enzyme activities, microbial community structure and function, and increase the diversity and resistance level of ARB isolates.

Exploring bacterial communities and biodegradation genes in activated sludge from pesticide wastewater treatment plants via metagenomic analysis.

Activated sludge (AS) has been regarded as the main driver in the removal of organic pollutants such as pesticides due to a high diversity and abundance of microorganisms. However, little is known about the biodegradation genes (BDGs) and pesticide degradation genes (PDGs) harbored in the AS from wastewater treatment plants (WWTPs). In this study, we explored the bacterial communities and BDGs/PDGs in the AS from five WWTPs affiliated with pesticide factories across four consecutive seasons based on high-throughput sequencing. The AS in pesticide WWTPs exhibited unique bacterial taxa at the genus level. Furthermore, a total of 17 BDGs and 68 PDGs were explored with a corresponding average relative abundance of 0.002-0.046% and 2.078-7.143% in each AS sample, respectively, and some BDGs/PDGs clusters were also identified in the AS. The bacterial communities and BDGs/PDGs were season-dependent, and the total variations of 50.4% and 76.8% were jointly explained by environmental variables (pesticide types, wastewater characteristics, and temperature). In addition, network analysis and distribution patterns suggested that the potential hosts of BDGs/PDGs were Thauera, Stenotrophomonas, Mycobacterium, Hyphomicrobium, Allochromatium, Ralstonia, and Dechloromonas. Our findings demonstrated the linkages of bacterial communities and BDGs/PDGs in the AS, and depended on the seasons and the pesticide wastewater characteristics.

Dissemination of antibiotic resistance genes and human pathogenic bacteria from a pig feedlot to the surrounding stream and agricultural soils.

The dissemination of antibiotic resistance genes (ARGs), human pathogenic bacteria (HPB), and antibiotic-resistant HPB (ARHPB) from animal feedlot to nearby environment poses a potentially high risk to environmental ecology and public health. Here, a metagenomic analysis was employed to explore the dissemination of ARGs, HPB, and ARHPB from a pig feedlot to surrounding stream and agricultural soils. In total, not detectable (ND)-1,628.4 µg/kg of antibiotic residues, 18 types of ARGs, 48 HPB species, and 216 ARB isolates were detected in all samples. Antibiotic residues from pig feedlot mainly migrated into stream sediments and greenhouse soil. The dominant ARGs and HPB species from pig feedlot spread into stream sediments (tetracycline resistance genes, Clostridium difficile, and Mycobacterium tuberculosis), stream water (multidrug resistance (MDR) genes, Shigella flexneri, and Bordetella pertussis), and greenhouse soil (MDR genes, Bacillus anthracis, and Brucella melitensis). It is concerning that 54.4% of 216 ARB isolates from all samples were potential ARHPB species, and genome sequencing and functional annotation of 4 MDR HPB isolates showed 9 ARG types. Our findings revealed the potential migration and dissemination of antibiotic residues, ARGs, HPB, and ARHPB from pig feedlot to surrounding stream and agricultural soils via pig sewage discharge and manure fertilization.

Repeated treatments of ciprofloxacin and kresoxim-methyl alter their dissipation rates, biological function and increase antibiotic resistance in manured soil.

The dissipation of ciprofloxacin (CIP, 1.0 and 10.0mg/kg) and kresoxim-methyl (KM, 1.0 and 2.0mg/kg) in manure-amended soil, the variations in soil enzyme activities and microbial functional diversities, and CIP-induced bacterial community tolerances were studied using a chromatographic analysis, enzyme colorimetric and titration analyses, and the BIOLOG EcoPlate method. Three successive treatments of individual and combined samples of CIP and KM at low and high concentrations were performed at 60d intervals. The dissipation half-life of CIP increased, but that of KM decreased in manured soil with treatment frequency; furthermore, the combined treatment altered the dissipation rates of CIP and KM. A stronger inhibitory effect on the activities of soil neutral phosphatase and urease was observed in the individual KM treatment than in the individual CIP treatment. A similar inhibitory trend was also found in soil neutral phosphatase activity in the combined treatment at high concentration compared to that at low concentration, but the activity of soil catalase was enhanced in the early stages of the KM or CIP treatments. Meanwhile, the inhibitory trend on the overall activity and functional diversity of soil microorganisms was observed in the individual KM or CIP treatment, and the combined treatment exerted a greater suppression effect than that in the individual treatment. Bacterial community resistance to CIP increased significantly with increasing treatment frequency and concentration, and furthermore antibiotic resistance developed faster in the combined treatment than in the individual treatment. It was concluded that the repeated treatments of CIP and KM could alter their dissipation rates and soil enzyme activities, suppress microbial functional diversity, and increase bacterial community resistance to CIP in manured soil.