Impact of aeration rate on the transfer range of antibiotic-resistant plasmids during manure composting.

Conjugative plasmids are important vectors of mobile antibiotic resvistance genes (ARGs), facilitating their horizontal transfer within the environment. While composting is recognized as an effective method to reduce antibiotics and ARGs in animal manure, its impact on the bacterial host communities containing antibiotic-resistant plasmids remains unclear. In this study, we investigated the permissiveness of bacterial community during composting when challenged with multidrug-resistant conjugative RP4 plasmids, employing Pseudomonas putida as the donor strain. Ultimately, this represents the first exploration of the effects of aeration rates on the range of RP4 plasmid transfer hosts. Transconjugants were analyzed through fluorescent reporter gene-based fluorescence-activated cell sorting and Illumina sequencing. Overall, aeration rates were found to influence various physicochemical parameters of compost, including temperature, pH, total organic matter, total nitrogen, and potassium. Regarding RP4 plasmid host bacteria, the dominant phylum was determined to shift from Bacteroidetes in the raw material to Proteobacteria in the compost. Notably, a moderate-intensity aeration rate (0.05 L/min/L) was found to be more effective in reducing the diversity and richness of the RP4 plasmid host bacterial community. Following composting, the total percentage of dominant transconjugant-related genera decreased by 66.15-76.62%. Ultimately, this study determined that the aeration rate negatively impacts RP4 plasmid host abundance primarily through alterations to the environmental factors during composting. In summary, these findings enhance our understanding of plasmid host bacterial communities under varying composting aeration rates and offer novel insights into preventing the dissemination of ARGs from animal manure to farmland.

Does increasing the organic fertilizer application rate always boost the antibiotic resistance level in agricultural soils?

The amendment of organic fertilizer derived from livestock manure or biosolids is a significant driver of increasing antibiotic resistance in agricultural soils; however, it remains unclear whether increasing organic fertilizer application rates consistently enhances soil antibiotic resistance levels. Herein, we collected soils with long-term amendment with three types of organic fertilizers at four application rates (15, 30, 45, and 60 t/ha/y) and found that the higher the fertilization rate, the higher the antibiotic resistance gene (ARG) abundance. However, when the fertilization rate exceeded 45 t/ha/y, the ARG abundance ceased to significantly increase. Moreover, the soil ARG abundance was positively correlated with total nitrogen (TN) content and bacterial abundance, especially Firmicutes, and negatively affected by pH and bacterial diversity. Soil TN/bacterial abundance and pH/bacterial diversity reached maximum and minimum values at the 45 t/ha/y fertilization rate, respectively. Meanwhile, at this fertilization rate, Firmicutes enrichment peaked. Therefore, an organic fertilization rate of 45 t/ha/y appeared to represent the threshold for soil antibiotic resistance in this study. The underlying mechanism for this threshold was closely related to soil TN, pH, bacterial abundance, and diversity. Taken together, the findings of this study advance the current understanding regarding the soil resistome under different fertilization rates, while also providing novel insights into organic fertilizer management in agricultural practices.

Aerosolization behavior of antimicrobial resistance in animal farms: a field study from feces to fine particulate matter.

Antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in animal feces can be released into the atmosphere via aerosolization, posing a high health risk to farm workers. So far, little attention has been paid to the characterization of the aerosolization process. In this study, fecal and fine particulate matter (PM2.5) samples were collected from 20 animal farms involving swine, cattle, layers, and broilers, and the ARGs, ARB, and human pathogenic bacteria (HPB) were loaded in these two media. The results showed that approximately 70% of ARGs, 60% of ARBs, and 43% of HPBs were found to be preferential aerosolization. The bioaerosolization index (BI) of target 30 ARGs varied from 0.04 to 460.07, and the highest value was detected from tetW. The highest BI values of erythromycin- and tetracycline-resistant bacteria were for Kocuria (13119) and Staphylococcus (24746), respectively, and the distribution of BI in the two types of dominant ARB was similar. Regarding the bioaerosolization behavior of HPB, Clostridium saccharolyticum WM1 was the most easily aerosolized pathogen in swine and broiler farms, and Brucella abortus strain CNM 20040339 had the highest value in cattle and layer farms. Notably, the highest BI values for ARGs, ARB, and HPB were universally detected on chicken farms. Most ARGs, ARB, and HPB positively correlated with animal age, stocking density, and breeding area. Temperature and relative humidity have significant effects on the aerosolization behavior of targets, and the effects of these two parameters on the same target are usually opposite. The results of this study provide a basis for a better understanding of the contribution of animal feces to airborne ARGs and HPBs in farms, as well as for controlling the transport of the fecal microbiome to the environment through the aerosolization pathway.

A web-based novel model for predicting prognostic value in patients with invasive micropapillary carcinoma in breast cancer: a real-world data retrospective cohort study.

Invasive micropapillary carcinoma (IMPC) accounts for less than 2% of all invasive breast cancers and usually associates with poor survival, so we investigated the prognostic factors for IMPC using a large population-based database and designed a web-based novel model. Clinicopathological prognostic factors were evaluated using the Surveillance, Epidemiology, and End Results (SEER) database. Multivariate Cox regression analysis was performed to evaluate the prognostic value of variables on the overall survival. A web-based nomogram was finally constructed to predict the survival probability. The model was validated in an external dataset. A web-based model, combined with age, radiation, clinical stage, and hormone receptor (HR) immunochemistry status four prognostic factors, was constructed. The C-index (0.714, 95% CI 0.683-0.741), calibration curves, and decision curves showed that this model was superior in prediction. By determining the cut-off values, high-risk group and low-risk group were divided. The Kaplan-Meier survival curves showed that these two groups had significantly different survival rates (P < 0.0001). The result of C-index, calibration curves, and Kaplan-Meier survival curves were consistent in the validation cohort. The novel nomogram with four risk factors resulted in accurate prognostic prediction for IMPC.

Aerosolization Behaviour of Fungi and Its Potential Health Effects during the Composting of Animal Manure.

Compost is an important source of airborne fungi that can adversely affect occupational health. However, the aerosol behavior of fungi and their underlying factors in composting facilities are poorly understood. We collected samples from compost piles and the surrounding air during the composting of animal manure and analyzed the aerosolization behavior of fungi and its potential health effects based on the fungal composition and abundance in two media using high-throughput sequencing and ddPCR. There were differences in fungal diversity and richness between the air and composting piles. Ascomycota and Basidiomycota were the two primary fungal phyla in both media. The dominant fungal genera in composting piles were Aspergillus, Thermomyces, and Alternaria, while the dominant airborne fungal genes were Alternaria, Cladosporium, and Sporobolomyces. Although the communities of total fungal genera and pathogenic/allergenic genera were different in the two media, fungal abundance in composting piles was significantly correlated with abundance in air. According to the analysis on fungal composition, a total of 69.10% of the fungal genera and 91.30% of pathogenic/allergenic genera might escape from composting pile into the air. A total of 77 (26.64%) of the fungal genera and six (20%) of pathogenic/allergenic genera were likely to aerosolize. The influence of physicochemical parameters and heavy metals on the aerosol behavior of fungal genera, including pathogenic/allergenic genera, varied among the fungal genera. These results increase our understanding of fungal escape during composting and highlight the importance of aerosolization behavior for predicting the airborne fungal composition and corresponding human health risks in compost facilities.

Enhancing control of multidrug-resistant plasmid and its host community with a prolonged thermophilic phase during composting.

The plasmid-mediated horizontal transfer of antibiotic resistance genes (ARGs) among bacteria facilitates the evolution and dissemination of antibiotic resistance. Broad-host-range plasmids can be transferred to different bacterial hosts in soil, plant rhizospheres, and wastewater treatment plants. Although composting is an effective way to convert organic waste into fertilizer and reduce some ARGs, few studies have focused on its effects on the spread of ARG-carrying plasmids and their bacterial host communities during composting. In this study, a fluorescently labeled Pseudomonas putida (P. putida) harboring a broad-host-range plasmid RP4 carrying three ARGs was inoculated into a raw material microcosm and composted with different durations of the thermophilic phase. The fate of the donor and RP4 in composting was investigated. The prolonged thermophilic composting removed 95.1% of dsRed and 98.0% of gfp, and it inhibited the rebound of P. putida and RP4 during the maturation phase. The spread potential of RP4 decreased from 10-4 to 10-6 transconjugants per recipient after composting. In addition, we sorted and analyzed the composition of RP4 recipient bacteria using fluorescence-activated cell sorting combined with 16S rRNA gene amplicon sequencing. The recipient bacteria of RP4 belonged to eight phyla, and Firmicutes, accounting for 75.3%-90.1%, was the dominant phylum in the transconjugants. The diversity and richness of the RP4 recipient community were significantly reduced by prolonged thermophilic periods. Overall, these findings provide new insights for assessing the contribution of composting in mitigating the dissemination of plasmid-mediated ARGs, and the prolonged thermophilic phase of composting can limit the transfer of multidrug-resistant plasmids.

Diversity and abundance of antibiotic resistance genes in rhizosphere soil and endophytes of leafy vegetables: Focusing on the effect of the vegetable species.

Antibiotic resistance genes (ARGs) in the endophytes of vegetables represent a potential route of human exposure to the soil resistome. However, the effect of vegetable species on the endophytic ARG profiles is unclear, hampering our understanding of how ARGs migrate into the soil-vegetable system and their potential health risks. Here, we planted four leafy vegetables (cilantro, endive, lettuce, and pak choi), which are commonly eaten raw, and analyzed the resistomes and microbiomes in three sample types (rhizosphere soil, root, and leaf endophytes). A total of 150 ARG subtypes were detected using high-throughput quantitative PCR. Vegetable species had a significant effect on ARG diversity and abundance, and pak choi accumulated more ARGs in its associated microbiome than the other three vegetables. The bacterial community was the primary factor shaping ARG profiles and was significantly correlated with ARG subtypes. We identified aadE, tet(34), and vanSB as shared ARGs among leaves of the four vegetables; the bacterial families correlated with tet(34) and vanSB were also shared across the vegetables and belonged to Proteobacteria. This study deepens our understanding of how endophytic ARG profiles vary among different vegetables and highlights the potential health risk associated with consuming these vegetables raw.

Sulfamethoxazole affects the microbial composition and antibiotic resistance gene abundance in soil and accumulates in lettuce.

Pot experiments were set up to simulate the soil contamination by three initial concentrations of sulfamethoxazole (SMX) (S1, 100 mg/kg; S2, 200 mg/kg; S3, 300 mg/kg). The content of SMX in soil and its accumulation in lettuce were analysed. Additionally, the effects of SMX on soil microorganisms and antibiotic resistance genes were studied by Illumina high-throughput sequencing and droplet digital polymerase chain reaction (ddPCR). The results demonstrated that the SMX content in soil reduced by 97%, 86% and 75% in the S1, S2 and S3 treatment groups after 120 days, respectively. The accumulated SMX in lettuce was positively correlated with the initial concentration of SMX in soil. SMX contamination significantly reduced the bacterial diversity and altered the composition of bacterial and fungal communities in soil. The dominant bacterial and fungal genera in the SMX-contaminated soil were obviously different from those in the control soil. The relative abundance of sul1 (sulfonamide resistance gene) remarkably increased in the SMX-contaminated soil, while that of other ARGs, such as sul2 and tetracycline and quinolone resistance genes, showed no significant change.

[Distribution Characteristics of Antibiotic Resistance Genes and Mobile Genetic Elements in Beijing Vegetable Base Soils].

The distribution characteristics of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in five vegetable base soils from Beijing, China, were assessed. The composition of ARGs and MGEs in soil samples were analyzed by HT-qPCR. We detected 92-121 ARGs and 4-6 MGEs. The ARGs and MGEs in vegetable base soils from different districts were separated from each other. The dominant ARGs shared by vegetable bases were oprD, acrA-04, and acrA-05 of a multidrug, mphA-01 of MLSB, and ß-Lactamase fox5, vanC-03 of vancomycin. The shared MGE among the five vegetable base soils was intI1. A total of seven antibiotics were detected in the soil of several vegetable bases. The dominant antibiotics included enoxacin (ENR), norfloxacin (NOR), oxytetracycline (OTC), and sulfamethoxazole (SMX). The numbers and abundance of antibiotics in the soil of vegetable bases from the Shunyi district were the highest, followed by those from Tongzhou and Changping. Correlation analysis showed that there was a significant positive correlation between the abundance of ARGs and the abundance of antibiotics in the soil of vegetable bases (P<0.05). These results provide basic theoretical data for controlling the transmission of ARGs.

[Bacterial Diversity and Community Structure Antibiotic-resistant Bacteria in Bioaerosol of Animal Farms].

Confined animal feeding operations generate high concentrations of airborne antibiotic-resistant bacteria, including pathogenic strains that may pose a health risk to both animals and farm workers and pollute the local air environment. In this study, tetracycline and erythromycin-resistant bacteria were used as examples to study the biodiversity and community structure of airborne antibiotic-resistant bacteria in animal farms. The Anderson sampler was used to collect bioaerosols samples from the inside environment and outside atmospheric environments. A comparative analysis of biological differences of antibiotic-resistant bacteria was conducted on fine and coarse particles, bioaerosol samples inside the house, fecal samples, and inside and outside bioaerosol samples based on the result of the Illumina MiSeq sequencing. The key genus that drives the above differences was also studied. Results showed that the biodiversity of airborne erythromycin-resistant bacteria was higher than that of airborne tetracycline-resistant bacteria, and the biodiversity of bioaerosol samples in the house was higher than that in fecal samples. There were no significant differences in the biodiversity and community structure of airborne antibiotic-resistant bacteria between fine and coarse particles. Actinobacteria is one of the key bacteria responsible for the differences between erythromycin-resistant bacteria and other bacterial populations. Staphylococcus is one of the key genera of tetracycline-resistant flora that is distinguished from erythromycin resistance and all bacterial flora. The results of the community structure showed that there was no significant difference in the dominant flora and the community structure of tetracycline and erythromycin-resistant bacteria. The community structure of feces and bioaerosol samples is different at the genus level, and the dominant bacteria are likewise different. The results of this study provide basic data for the accurate assessment of the current status of antibiotic-resistant bacteria in animal farms and their ecological risks.

[Microbial Community Structure and the Distribution of Antibiotic Resistance Genes in Soil Contaminated by Sulfamethoxazole].

A pot experiment was carried out to simulate soil contaminated by sulfamethoxazole at different concentrations. The community structure of soil microorganisms was investigated using Illumina high-throughput sequencing, and 64 subtypes of antibiotic resistance genes (ARGs) resistant to six classes of antibiotic were also analyzed by PCR and droplet digital PCR. The results showed that soil contamination with sulfamethoxazole had no significant effect on fungal diversity after 120 days (P>0.05) whereas bacterial diversity was significantly reduced (P<0.05). The microbial community structure of the contaminated soil changed significantly, with the dominant bacterial and fungal genera being significantly different from the control soil. Sulfamethoxazole contamination resulted in an increase in ARG diversity, and the abundance of the sulfonamide resistance gene sul1 increased significantly (P<0.05). However, the abundance of the sulfonamide resistance gene sul2, the quinolone resistance genes floR and cmlA1, and the tetracycline resistance genes tet(34), tetG2, tetG1,tetM, and tetA/P did not show significant changes in the contaminated soil (P>0.05).