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.

Chicken feedlot revisited: Co-dispersal of antibiotic and metal resistome under banning in-feed veterinary antibiotics.

Intensive livestock farming has been implicated as a notorious hotspot for antibiotic resistance genes (ARGs) due to the excessive or inappropriate use of in-feed antibiotics over the past few decades. Since China implemented a ban on the use of antibiotics in animal feed since 2020, the dissemination of ARGs in the vicinity of feedlots has remained unclear. This study presents a case study that aims to investigate the dispersal of antibiotics and ARGs from a chicken feedlot (established in 2020) to the adjacent aquatic and soil environments. Comparing the sample collected from upstream area, the water and sediment samples from midstream and downstream areas showed an increase in total antibiotic residues and metal content (Cu and Zn) by 4.2-5.3 fold and 1.3-22.6 fold, respectively. The downstream water samples exhibited a 2.49-2.93-fold increase in the abundance of ARGs and a 1.48-1.75-fold increase in the abundance of metal resistance genes (MRGs). The results of Pearson correlation and metagenome-assembled genome revealed a tendency for the co-occurrence of ARGs and MRGs. The dissemination of ARGs and MRGs is primarily driven by tetracycline, tylosin, Cu, and, Mn, with mobile genetic elements playing a more significant role than bacterial communities. These findings shed light on the overlooked co-dispersal pattern of ARGs and MRGs in the environment surrounding feedlots, particularly in the context of banning in-feed veterinary antibiotics.

Key taxa and mobilome-mediated responses co-reshape the soil antibiotic resistome under dazomet fumigation stress.

Agrochemicals are emergingly being implicated in the widespread dissemination of antibiotic resistance genes (ARGs) in agroecosystems. However, minimal research exists on the disturbance of fumigant on soil ARGs. Focusing on a typical fumigant dazomet in a simulated soil microcosm, we characterized the dazomet-triggered timely response and longstanding dynamic of ARGs at one-fold and two-fold field recommended doses using metagenome and quantitative PCR. Dazomet treatments reduced 13.17%-69.98% of absolute abundance of 16S rRNA gene and targeted ARGs, but, awfully, boosted diversity and relative abundance of ARGs up to 1.33-1.60 and 1.62-1.90 folds, respectively. Approximately 77.28% of changes in relative abundance of ARGs could be explained by bacterial community and mobile genetic elements (MGEs). Mechanistically, primary hosts of ARGs shifted from Proteobacteria (control) to Firmicutes and Actinobacteria (treatments) accompanied with corresponding changes in their abundance by combining community analysis, host tracking analysis and antibiotic resistant bacteria assay. Meanwhile, dazomet exposure significantly increased the incidence of MGEs and stimulated the conjugation of antibiotic-resistant plasmid. In addition, absolute abundance of targeted ARGs gradually recovered in the post-fumigation stage. Collectively, our results elucidate the dazomet-triggered emergence and spread of soil ARGs and highlight the importance of navigating toward rational use of fumigant in agricultural fields.

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.

Contrasting dynamics of manure-borne antibiotic resistance genes in different soils.

Antibiotic resistance genes (ARGs) are important biological contamination factors in soil systems, posing direct or indirect threats to soil health, food safety and human health. The ubiquitous pollution of ARGs is usually implicated with the application of organic manures in agricultural soil ecosystem. However, little is known about the transmission and fate of ARGs after manure input concerning different soils. Herein, the transmission potential and temporal dynamics of manure-associated ARGs was characterized with three different agricultural soils collected from Jiangxi (JX), Zhejiang (ZJ), and Jilin (JL), respectively. The results show that manure input did not affect the total abundance of ARGs in the receiving soils, but remarkedly alter the compositions of ARGs in soils. The manure-associated ARGs were significantly enriched in the manure-amended soils, including genes conferring resistance to sulfonamide, aminoglycoside, tetracycline, chloramphenicol, and trimethoprim with the fold of 1.97 - 27.86. Variance partitioning analysis showed that the major variances of ARG community was explained by mobile genetic elements and bacterial profile (> 76%) but not the concentrations of heavy metals and antibiotics. Furthermore, 31, 37, and 38 ARG subtypes were identified as the potential extrinsic ARGs derived from manures in the JX, ZJ, and JL soils, respectively, including 13 shared ARG subtypes. It was also found that the manure-associated ARGs (aadA, sul1, sul2, tetC, and tetG) declined with the incubation time in the JX and ZJ soils, whereas they firstly decreased and then increased in the JL soil. The abundance of these five ARGs in the JL soil was significantly higher than that in the JX and ZJ soils. Collectively, this finding revealed that soil type was responsible for the transmission and fate of manure-associated ARGs in agroecosystem.

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.

Resistance properties and adaptation mechanism of cadmium in an enriched strain, Cupriavidus nantongensis X1T.

Bacterial adaption to heavy metal stress is a complex and comprehensive process of multi-response regulation. However, the mechanism is largely unexplored. In this study, cadmium (Cd) resistance and adaptation mechanism in Cupriavidus nantongensis X1T were investigated. Strain X1T could resist the stress of 307 mg/L Cd2+ and remove 70% Cd2+ in 48 h. Spectroscopic analyses suggested interactions between Cd2+ with C-N, -COOH, and -NH ligands of extracellular polymeric substances. Whole-genome sequencing found that the resistance of Cd2+ in strain X1T was caused by the joint action of Czc and Cad systems. Cd2+ at 20 mg/L elicited differential expression of 1157 genes in strain X1T. In addition to the reported effects of uptake, adsorption, effluxion, and accumulation system, the oxidative stress system, Type-VI secretory protein system, Fe-S protein synthesis, and cysteine synthesis system in strain X1T were involved in the Cd2+ resistance and accumulation. The intracellular accumulation content of Cd2+ in strain X1T was higher than the extracellular adsorption content made strain X1T to be an important resource strain in the bioremediation of Cd-contaminated sewage. The results provide a theoretical network for understanding the complex regulatory system of bacterial resistance and adaptation of Cd against stressful environments.

The efficient persistence and migration of Cupriavidus gilardii T1 contribute to the removal of MCPA in laboratory and field soils.

The application of exogenous biodegradation strains in pesticide-polluted soils encounters the challenges of migration and persistence of inoculants. In this study, the degradation characteristics, vertical migration capacity, and microbial ecological risk assessment of an enhanced green fluorescent protein (EGFP)-tagged 2-Methyl-4-chlorophenoxyacetic acid (MCPA)-degrading strain Cupriavidus gilardii T1 (EGFP) were investigated in the laboratory and field soils. The optimum remediation conditions for T1 (EGFP) was characterized in soils. Meanwhile, leaching experiments showed that T1 (EGFP) migrated vertically downwards in soil and contribute to the degradation of MCPA at different depths. After inoculation with T1 (EGFP), a high expression levels of EGFP gene was observed at 28 d in the laboratory soil and at 45 d in the field soil. The degradation rates of MCPA were ≥ 60% in the laboratory soil and ≥ 48% in the field soil, indicating that T1 (EGFP) can efficiently and continuously remove MCPA in both laboratory and field conditions. In addition, the inoculation of T1 (EGFP) not only showed no significant impact on the soil microbial community structure but also can alleviate the negative effects induced by MCPA to some extent. Overall, our findings suggested that T1 (EGFP) strain is an ecologically safe resource for the in situ bioremediation of MCPA-contaminated soils.

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.

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.

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.

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.

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.