Indoor environments are primary settings of human activity and play a central role in shaping population health through microbial exposure. However, how short-duration climatic regimes influence indoor airborne microbiomes and resistomes remains largely unknown. Here, we investigated how the plum rain season, characterized by persistent rainfall and humid conditions, affects airborne bacterial communities, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and potential human pathogens in a dormitory building. The plum rain season significantly reduced airborne bacterial richness and altered community composition, with more pronounced effects indoors than outdoors. Airborne bacterial communities during this period also exhibited less complex co-occurrence networks and broader niche breadth, suggesting season-associated changes in bacterial ecological features. Although the number of detected ARGs in indoor air remained largely unchanged, the plum rain season significantly increased the relative abundances of ARGs (3.02% vs. 1.52%) and MGEs (0.42% vs. 0.14%) indoors compared with the post-plum rain period, whereas such seasonal enrichment was not observed outdoors. Indoor air during the plum rain season also showed higher relative abundances of high-risk ARGs. Concurrently, the relative abundance of potential human pathogens increased from 7.34% to 13.38% indoors, with Achromobacter xylosoxidans being the dominant potential pathogen. Furthermore, indoor ARG enrichment was associated with higher MGE abundance, increased A. xylosoxidans abundance, and shifts in airborne bacterial community composition. Together, these findings suggest that the plum rain season promotes indoor airborne resistome enrichment and potential exposure to airborne microbial hazards, underscoring the need to incorporate regional climatic regimes into indoor environmental health assessment.

Protozoa, as primary predators of soil bacteria, represent an overlooked natural driver in the dissemination of antibiotic resistance genes (ARGs). However, the effects of protozoan predation on ARGs dissemination at the community level, along with the underlying mechanisms, remain unclear. Here we used fluorescence-activated cell sorting, qPCR, combined with metagenomics and reverse transcription quantitative PCR, to unveil how protozoa (Colpoda steinii and Acanthamoeba castellanii) influence the plasmid-mediated transfer of ARGs to soil microbial communities. Protozoan predation reduced the absolute abundance of plasmids but promoted the expression of conjugation-associated genes, leading to a 5-fold and 4.5-fold increase in conjugation frequency in the presence of C. steinii and A. castellanii, respectively. Excessive oxidative stress, increased membrane permeability, and the provoked SOS response closely associated with the increased conjugative transfer. Protozoan predation also altered the plasmid host range and selected for specific transconjugant taxa along with ARGs and virulence factors carried by transconjugant communities. This study underscores the role of protozoa in the plasmid-mediated conjugative transfer of ARGs, providing new insights into microbial mechanisms that drive the dissemination of environmental antibiotic resistance.

Soil microbiomes play a crucial role in maintaining ecological functions and are of great importance for soil health. Some of them could bring benefits to plants for growth promotion. Despite numerous studies have focused on specific beneficial bacteria and their interactions with soils and plants, we still lack a comprehensive understanding of beneficial communities in plant-soil continuums and their responses to agricultural activities. To address this gap, we carried out a microcosm experiment using 16S rRNA amplicon sequencing to explore the effects of organic fertilization on beneficial communities in plant–soil continuums and assess their potential multifunctionality. Our findings reveal that organic fertilization had a positive impact on the beneficial functionality of bacterial communities in plant-soil continuums. This improvement was primarily attributed to the optimized soil physicochemical conditions resulting from organic fertilization. Additionally, organic fertilization increased the complexity of bacterial co-occurrence networks in both soils and the endosphere. Keystone taxa in the endosphere undergone a shift of functions toward pathogen suppression as the result of organic fertilization. Furthermore, this study revealed that plants exhibited a preference for internalizing beneficial bacteria over other type of bacteria. We also provided new insights for evaluating the multifunctionality of microbiomes, and found that the functionality of beneficial communities in plant-soil continuums is enhanced by organic fertilization. All these findings suggested that organic fertilization can be an effective strategy for maintaining plant and soil health.

Since Alexander Fleming's discovery of penicillin in 1928, numerous antibiotics have been discovered and produced industrially. Approximately, 30% to 90% of antibiotics cannot be absorbed by humans and animals and, are released into the environment in different forms. As a result, antibiotics are much more concentrated in human-affected environments than in natural environments, forming a pervasive selection pressure and accelerating the evolution and emergence of antibiotic resistance genes (ARGs). With ongoing research, we are gradually gaining more insights into how human activities impact ARGs. However, in natural environments with little anthropogenic disturbance, microbial interactions are the main drivers of antibiotic resistance, which have been largely overlooked. Protists are central to a wide array of food web processes and biogeochemical cycles, and deserve more attention. It is well documented that protists prey on bacteria, the main carriers of ARGs. Thus, protists can indirectly affect resistome by changing bacterial community structure through predation. Likewise, antibiotics are secreted by bacteria as a means of avoiding predation by protozoa, increasing the selective pressure on the environment. Therefore, understanding the relationship between protists and bacterial antibiotic resistance will help us improve the ability to predict and manage microbes to better guide agricultural production, reduce antibiotic overuse and prevent or mitigate the emergence of new antibiotic resistance. This paper first introduces the origin, evolution and spread of antibiotic resistance, and then summarizes that the main factors driving its evolution vary from the pre-antibiotic era to the antibiotic era. In the second section, this paper reviews that autotrophic protists may contribute to the reduction of antibiotic resistance: (1) By bioadsorption, bioaccumulation, and biodegradation, autotrophic protists can remove antibiotics from the environment, reducing selection pressure and slowing the speed of evolution; (2) autotrophic protists can secrete some secondary metabolites, including alternatives to antibiotics, quorum sensing inhibitors, and efflux pump inhibitors, all of which contribute to reducing antibiotic resistance. Besides, phagocytic protists also play an important role. In addition to serving as the hotspot for horizontal gene transfer (HGT), they also stimulate bacteria to secrete antibiotics through predation and increase the frequency of HGT between different bacteria. Since individual level studies do not represent the actual situation of communities in natural environments, research on the community level is required. Therefore, the third section is focused on the community level. Protists display a myriad of sizes, morphologies and nutritional modes, making it difficult to study them with the traditional method. The ultra-deep high-throughput sequencing combined with statistical approaches provides new insights into the conundrum of how protists regulate bacterial antibiotic resistance at the community level. It is noteworthy that studies based on sequencing data also have their limitations. First of all, data requirements are high, that is, a large number of samples and key environmental variables need to be covered. Secondly, the results are data-driven and need to be verified by other experiments. The last section discusses perspectives regarding the role of protists in preventing and controlling the risk of bacterial antibiotic resistance: (1) Screening secondary metabolites from autotrophic protists, searching for substances that can reduce antibiotic resistance, and realizing industrial production; (2) protists should be considered as one of the indicators of environmental monitoring, especially in hospitals, preventing intracellular bacteria from becoming super-resistant bacteria via HGT; (3) it is necessary to develop novel disinfection techniques to kill antibiotic resistant bacteria protected by protist cysts; and (4) the regulating mechanisms between protists and bacterial antibiotic resistance should be explored at the community level.

Microplastics (MPs) ubiquitous in environments promote the dissemination of antibiotic resistance genes (ARGs), threatening ecosystem safety and human health. However, quantitative assessments of the health risks of ARGs (HRA) in plastisphere and an in-depth exploration of their driving mechanisms are still lacking. Here, the microbiomes, ARGs, and community assembly processes of five types of MPs in an urbanizing watershed were systematically investigated. By fully considering the abundance, clinical availability, human pathogenicity, human accessibility, and mobility of 660 ARGs in plastisphere, the HRA on MPs were quantified and compared. Polyethylene had the highest HRA among the five MP types, and urbanization further increased its risk index. In addition to abiotic factors, more complex biotic factors have been shown to drive HRA in plastisphere. Specifically, dispersal limitation accounted for the increasing diversity and interaction of bacteria that determined HRA in plastisphere. Further analysis of metabolic functions indicated that a higher HRA was accompanied by decreased normal metabolic functions of plastisphere microbiota due to the higher fitness costs of ARGs. This study advances the quantitative surveillance of HRA in plastisphere and understanding of its driving mechanisms. This will be helpful for the management of both MPs and ARGs treatments for human health.

Protists, functionally divided into consumers, phototrophs, and parasites act as integral components and vital regulators of microbiomes in soil-plant continuums. However, the drivers of community structure, assembly mechanisms, co-occurrence patterns, and the associations with human pathogens and different protistan trophic groups remain unknown. Here, we characterized the phyllosphere and soil protistan communities associated with three vegetables under different fertilization treatments (none and organic fertilization) at five growth stages. In this study, consumers were the most diverse soil protist group, had the role of inter-kingdom connector, and were the primary biomarker for rhizosphere soils which were subjected to decreasing deterministic processes during plant growth. In contrast, phototrophs had the greatest niche breadth and formed soil protistan hubs, and were the primary biomarkers for both bulk soils and the phyllosphere. Parasites had minimal input to microbial co-occurrence networks. Organic fertilization increased the relative abundance (RA) of pathogenic protists and the number of pathogen-consumer connections in rhizosphere soils but decreased protistan richness and the number of internal protistan links. This study advances our understanding of the ecological roles and potential links between human pathogens and protistan trophic groups associated with soil-plant continuums, which is fundamental to the regulation of soil-plant microbiomes and maintenance of environmental and human health.

Protists are a trophically diverse and biogeochemically significant component of water environments and are widely reported as hosts of bacteria. However, the potential role of protists in wastewater treatment plants (WWTPs) as reservoirs for human pathogens does not appear to have received adequate attention. Here, a combination of fluorescence-activated cell sorting and Illumina sequencing was applied to characterize the dynamics of the internalized bacterial community of the enriched protists from the influents and effluents of five WWTPs. The results showed that Proteobacteria (mainly Betaproteobacteria) dominate the intracellular bacterial communities of protists in both influents and effluents of WWTPs, accounting for 72.6% of the total intracellular bacterial communities. The most frequently detected genus was Sulfuricurvum in the influent samples, Chryseobacterium and Pseudomonas were most prevalent in the effluent samples. Compared with the influents, a more diverse and abundant intracellular bacterial community was observed in the effluents. Moreover, the potential intracellular bacterial pathogens were 26 times higher in effluents than in influents, with Pseudomonas fluorescens and Pseudomonas putida significantly enriched in effluents. This work provides insights into the dynamics of bacterial communities and potential pathogens harbored by protists in the influents and effluents from WWTPs, contributing to the improved evaluation of biosafety in WWTPs.

Food safety of leafy greens is an emerging public health issue as they can harbor opportunistic human pathogens (OHPs) and expose OHPs to consumers. Protists are an integral part of phyllosphere microbial ecosystems. However, our understanding of protist-pathogen associations in the phyllosphere and their consequences on public health remains poor. Here, we examined phyllosphere protists, human pathogen marker genes (HPMGs), and protist endosymbionts from four species of leafy greens from major supermarkets in Xiamen, China. Our results showed that Staphylococcus aureus and Klebsiella pneumoniae were the dominant human pathogens in the vegetable phyllosphere. The distribution of HPMGs and protistan communities differed between vegetable species, of which Chinese chive possessed the most diverse protists and highest abundance of HPMGs. HPMGs abundance positively correlated with the diversity and relative abundance of phagotrophic protists. Whole genome sequencing further uncovered that most isolated phyllosphere protists harbored multiple OHPs which carried antibiotic resistance genes, virulence factors, and metal resistance genes and had the potential to HGT. Colpoda were identified as key phagotrophic protists which positively linked to OHPs and carried diverse resistance and virulence potential endosymbiont OHPs including Pseudomonas nitroreducens, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. We highlight that phyllosphere protists contribute to the transmission of resistant OHPs through internalization and thus pose risks to the food safety of leafy greens and human health. Our study provides insights into the protist-OHP interactions in the phyllosphere, which will help in food safety surveillance and human health.

Warming affects microbial functioning of soil and the phyllosphere across global ecosystems. However, little is known about the impact of increasing temperature on antibiotic resistome profiles in natural forests. To address this issue, we investigated antibiotic resistance genes (ARGs) in both soil and the plant phyllosphere using an experimental platform established in a forest ecosystem that delivers a temperature difference of 2.1 °C along an altitudinal gradient. Principal Coordinate Analysis (PCoA) showed that there were significant differences in the composition of soil and plant phyllosphere ARGs at different altitudes (P = 0.001). The relative abundance of phyllosphere ARGs and mobile genetic elements (MGEs) and soil MGEs increased with temperature. More resistance gene classes increased in abundance in the phyllosphere (10 classes) than soil (2 classes), and a Random Forest model analysis suggested that phyllosphere ARGs were more sensitive to temperature change than soil. Increasing temperature as a direct consequence of an altitudinal gradient, and the relative abundance of MGEs were the main drivers that shaped the profiles of ARGs in the phyllosphere and soil. Biotic and abiotic factors affected phyllosphere ARGs indirectly via MGEs. This study enhances our understanding of the influence of altitude gradients on resistance genes in natural environments.

Spread of antibiotic resistance or the presence of antibiotic resistance genes (ARGs) in pathogens is a globally recognized threat to human health. Numerous studies have shown that application of organic fertilizers may increase the risk of ARGs, however, the risk of resistance genes associated with biofertilizers is largely unknown. To investigate whether biofertilizer application introduces ARGs to the soil, we used high-throughput quantitative polymerization chain reaction (HT-qPCR) to explore the effect of biofertilizer application over three years on soil ARGs in three orchards with different locations in China. Redundancy analysis showed specific and significant differences in the beta diversity of soil bacteria and fungi between treatments (fertilizer vs. no fertilizer). One-way ANOVA analysis revealed findings of the main driver of the significant difference in microbial community structure between fertilizer and control treatment was the change in soil properties following the application of biofertilizer. A total of 139 ARGs and 27 MGEs (mobile genetic elements), and 46 ARGs and 6 MGEs from 11 major taxa were detected in biofertilizer and soil samples, respectively. Only the samples from Guangxi had significant differences in the detected number of ARGs and MGEs between fertilization and control. Through structural equation modeling (SEM), we found that soil properties indirectly affected ARGs by shaping bacterial diversity, while bacterial abundance directly affected ARGs. Biofertilizer application did not significantly alter the relative abundance of ARGs in soil due to the complexity of the soil environment and competition between exogenous and native microorganisms. This study provided new insights into the spread of the antibiotic resistome of the soil through biofertilizer applications.

With growing concerns about antibiotic resistance, the tracking of antibiotic resistance genes (ARGs) in urban waterways will facilitate our increased understanding of the impact of urbanization on ARGs dissemination. In the current study, we assessed the ARGs profiles and antibiotic resistome in water samples along the Jiulong River basin, a distance of 250 km, to better understand the impact of anthropogenic activities. A total of 244 ARGs and 12 MGEs were detected from 21 sampling sites. Both relative and absolute abundance of the observed resistome decreased with increasing distance from urban areas. Ordinary least-squares (OLS) regression revealed that both the relative and absolute resistome abundance were positively correlated with city size. The resistome had several inputs and outputs and Fast Expectation Maximization Microbial Source Tracking (FEAST), suggested that the majority of the antibiotic resistome originated from anthropogenic activities. A total of 8 ARGs and 20 microbial OTUs were considered as biomarkers that differentiated the location of sampling sites. Bacterial communities were significantly correlated with ARGs according to Procrustes analysis and Mantel test, which was also supported by a co-occurrence network. Variation partitioning analysis revealed that ARG profiles were driven by multiple factors. Although antibiotic resistome abundance significantly increased near urban conurbations, overall resistome abundance decreased as the river flowed downstream. Our study highlights the effect of conurbation size on antibiotic resistance profiles within the river basin and the potential resilience of rivers to recover from ARGs contamination.

The demand for facial masks remains high. However, little is known about discarded masks as a potential refuge for contaminants and to facilitate enrichment and spread of antibiotic resistance genes (ARG) in the environment. We address this issue by conducting an in-situ time-series experiment to investigate the dynamic changes of ARGs, bacteria and protozoa associated with discarded masks. Masks were incubated in an estuary for 30 days. The relative abundance of ARGs in masks increased after day 7 but levelled off after 14 days. The absolute abundance of ARGs at 30 days was 1.29 × 1012 and 1.07 × 1012 copies for carbon and surgical masks, respectively. According to normalized stochasticity ratio analysis, the assembly of bacterial and protistan communities was determined by stochastic (NST = 62%) and deterministic (NST = 40%) processes respectively. A network analysis highlighted potential interactions between bacteria and protozoa, which was further confirmed by culture-dependent assays, that showed masks shelter and enrich microbial communities. An antibiotic susceptibility test suggested that antibiotic resistant pathogens co-exist within protozoa. This study provides an insight into the spread of ARGs through discarded masks and highlights the importance of managing discarded masks with the potential ecological risk of mask contamination.

Investigating the contribution and associations of environmental microbes to ecological health and human well-being is in great demand with the goal of One Health proposed. To achieve the goal, there is an urgent need for accurate approaches to obtaining a large amount of high-resolution molecular information from various microbes. In this study, we developed a high-throughput library construction chip (HiLi-Chip) for profiling environmental microbial communities and evaluated its performance. The HiLi-Chip showed high conformity with the conventional Pacbio method in terms of α-diversity, community composition of abundant bacteria (>83%), as well as rare taxa (>84%) and human pathogens detection (>67%), indicating its advantages of accuracy, high-throughput, cost-efficiency, and broad practicability. It is suggested that the optimal strategy of the HiLi-Chip was a 2.4 μL PCR mixture per sample (∼2.4 ng DNA) with a 216-sample × 24-replicate format. We have successfully applied the HiLi-Chip to the Jiulongjiang River and identified 51 potential human bacterial pathogens with a total relative abundance of 0.22%. Additionally, under limited nutrients and similar upstream environments, bacteria tended to impose competitive pressures, resulting in a more connected network at the downstream river confluence (RC). Whereas narrow niche breadth of bacteria and upstream environmental heterogeneity probably promoted niche complementary and environment selection leading to fewer links at RC in the midsection of the river. Core bacteria might represent the entire bacterial community and enhance network stability through synergistic interactions with other core bacteria. Collectively, our results demonstrate that the HiLi-Chip is a robust tool for rapid comprehensive profiling of microbial communities in environmental samples and has significant implications for a profound understanding of environmental microbial interactions.

Pathogens can colonize plant endosphere and, be transferred into human beings through the food chain. However, our understanding of the influences of agricultural activities, such as fertilization, on endophytic microbial communities and human pathogens is still limited. Here, we conducted a microcosm experiment using the combination of 16 S rRNA gene amplicon sequencing and high-throughput qPCR array to reveal the effects of manure fertilization on microbiomes of soils and plants and how such impact is translated into endophytic pathogens. Our results showed that manure fertilization significantly altered soil microbiomes, whereas with less influence on endophytic microbial communities. Soil is a vital source of both bacterial communities and human pathogens for the plant endosphere. The abundance of pathogens was increased both in soils and endosphere under manure fertilization. These findings provide an integrated understanding of the impact of manure fertilization on endophytic pathogens.

研究了溶解性有机物(DOM)在水厂全流程处理工艺中的演变,并利用FT-ICR MS技术深入分子层面探究小分子DOM的分子组成、质荷比、芳香性等变化.结果 表明,DOM总浓度呈下降趋势,小分子DOM(分子质量＜1 ku)在水厂中占比最大且去除效果最差.小分子DOM中CHO-DOM(仪含C、H、O,占比为48.75％ ～53.13％)的多样性和相对丰度占主导地位,木质素/CRAM类化合物(68.12％ ～72.05％)占显著优势.预氯化对小分子DOM的去除量低、生成量高,混凝沉淀对CHO-DOM和CHON-DOM(仅含C、H、O、N)有较好的去除效果,普通砂滤和生物活性炭过滤对DOM的影响较小,加氯消毒工艺影响最大,去除了大量芳香性/不饱和度高的小分子DOM,CHO-DOM的相对丰度趋于平缓,CHON-DOM中C13H20NO9跃升至最大丰度,CHOS-DOM(仅含C、H、O、S)的相对丰度明显增加,出现多个突出的高H/C化合物.总体上小分子DOM向高H/C、低O/C转移,出水中碳水化合物类、单宁酸类占比下降,稠环化合物类基本被去除,脂质类、类蛋白类和木质素/CRAM类占比增加,小分子DOM的芳香性或不饱和度大大降低.

The reaction between organic matter and disinfectants leads to the formation of disinfection byproducts (DBPs) in drinking water. With the improvement of detection technology and in-depth research, more than 1000 kinds of DBPs have been detected in drinking water. Nitrogenous DBPs (N-DBPs) are more genotoxic and cytotoxic than the regulated DBPs. The main methods are enhanced coagulation, pretreatment, and depth technologies which based are on conventional technology. Amino acids (AAs) are widely found in surface waters and play an important role by providing precursors from which toxic nitrogenous disinfection by-products (N-DBPs) are generated in chlorinated drinking water. The formation of N-DBPs, including dichloroacetonitrile, trichloroacetonitrile, and trichloronitromethane (TCNM), was investigated by analyzing chlorinated water using ozone (OZ), permanganate (PM), and ferrate (Fe(VI)) pre-oxidation processes. This paper has considered the control of pre-oxidation over N-DBPs formation of AAs, OZ, PM, and Fe(VI) pre-oxidation reduced the haloacetonitrile formation in the downstream chlorination. PM pre-oxidation decreased the TCNM formation during the subsequent chlorination, while Fe(VI) pre-oxidation had no significant influence on the TCNM formation, and OZ pre-oxidation increased the formation. OZ pre-oxidation formed the lowest degree of bromine substitution during subsequent chlorination of aspartic acid in the presence of bromide. Among the three oxidants, PM pre-oxidation was expected to be the best choice for reducing the estimated genotoxicity and cytotoxicity of the sum of the measured haloacetonitriles (HANs) and TCNM without bromide. Fe(VI) pre-oxidation had the best performance in the presence of bromide.

Dissolved organic nitrogen (DON) represents a unique challenge in prechlorinated raw water distribution systems (PRWDSs) because of its contribution to the formation of harmful nitrogen-disinfection byproducts, influence upon biogeochemical processes, and unclear molecular characteristics. Here, Fourier transform ion cyclotron resonance mass spectrometry in combination with high-throughput sequencing was applied to elucidate the molecular changes of DON and biofilm microbial communities in a PRWDS in Yixing, China. Our study revealed that dynamic characteristics of DON are significantly correlated with the biofilm. The accumulation of refractory lignin-like compounds and CnHmOpN1 contributes to the higher recalcitrance molecular characteristics of DON in the effluent associated with Alphaproteobacteria, Planctomycetes, and Bacteroidetes. Additionally, with the help of prechlorination, the biofilm may change the DON characteristics and lead to higher oxygenation, higher m/z, and lower saturation during transportation. Despite the promotion of CnHmOpN1 and CnHmOpN3 at the early stage, we suggest that appropriate concentration of chlorine can add to the front end of raw water distribution pipes. Prechlorination may control the nitrification process and stabilize the rapid growth of diversity and concentration of low molecular weight DON, especially the refractory CnHmOpN1 in the effluent, which may help to improve treatment efficiency of drinking water treatment plants.

Understanding the transformation pattern of nitrogen (N) pollutants and its pathways in the prechlorinated raw water distribution system (PRWDS) is vital for controlling the stablitiy and safety of raw water qulity. This study investigated the N transformation, N functional genes and their correlations to find the N transformation pathways along the PRWDS. Results suggested that simultaneous nitrification, anaerobic ammonium oxidation and denitrification (SNAD) contribute to the N transformationin the PRWDS. Along the pipeline, anammox 16S rRNA (9.18 x 10(7)-8.41 x 10(8) copies/g), limited by prechlorination, was the most abundant N functional genes and anammox process was the main pathway of ammonia nitrogen (NH4+-N). The decreasing NH4+-N was connected with Planctomycetes, Nitrospira and abundance of nxrA attributing to the joint effort of anammox and declined nitrification. The concentration of nitrate (NO3--N) increasing at first and then decreasing, was correlated positively with Sphingomonas. because of the declined nitritication and increased denitrification. Besides, the NO3--N -> NO2--N process was considered to be primary NO3--N transformation pathways. Increases in the concentration of dissolved organic nitrogen (DON) and nitrite (NO2--N) observed in the PRWDS had positive correlation with relative abundance of Pseudomonas. We believe that prechlorination shaped the particular bacterialcharacteristics in biofllms and influenced the N transformation pathways indirectly, resulting in the varying N transformation rules in PRWDSs. Moreover, systematic and extended research is particularly vital for determining the effects of changes in source water quality and environmental conditions on bacterial community structure and N conversion along PRWDSs. (C) 2020 Published by Elsevier Ltd.

After the discovery of complete ammonia-oxidizing (comammox) Nitrospira, detection and assessments of the contribution of comammox Nitrospira communities to nitrogen cycling are in great demand. PCR-based approach, a common method for the detection of comammox, depends strongly on accurate amplification of the amoA genes from the original DNA samples using appropriate primers. In this study, we reported an evaluation of the performance of two commonly used primer sets, Ntsp-amoA 162F/359R and comaA/B-244f/659r, for amplifying the comammox amoA genes from three representative wetland soils in China [Sangsang (SS), Sanjiang (SJ), and Xianghai (XH)]. Our results demonstrated the two primer sets could both successfully amplify the clades with high relative abundances (RA), and further revealed a broadly similar diversity and community composition of dominant comammox operational taxonomic units (OTUs) (RA ≥ 1%) in each of the three wetland soils. However, the clades with low RA, such as the clade A (1.26%) in SJ and the clade B (11.54%) in XH that were recovered by metagenomics analysis, failed to be amplified using comaA/B-244f/659r, but were successfully amplified and sequenced using Ntsp-amoA 162F/359R. It indicated that, compared to comaA/B-244f/659r, Ntsp-amoA 162F/359R was more sensitive to the clades with low RA. However, it is worth noting that Ntsp-amoA 162F/359R would overestimate the RA of some rare clades. For example, the RAs of clade B in XH were overestimated by 32-fold. Furthermore, high levels of non-target amplification were detected via gel electrophoresis using both primer sets, especially for comammox Clade B amoA genes, implying that we should treat qPCR results based on these primers with caution. Taken together, our study comprehensively compared the performance of the two primer sets on the sensitivity and specificity of amplifying comammox amoA genes in three wetland soils, pointing out the necessity of further development of new primers for the efficient and accurate detection of comammox in various environments.

The transformation of dissolved organic nitrogen (DON) in the drinking water treatment plants could be closely associated with nitrogenous disinfection by-product (N-DBP) formation. In this study, we have assessed the molecular transformation of DON and its impact on N-DBP formation in a full scale drinking water treatment plant. Based on the result of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analysis, DON compounds with low molecular weight (<1 kDa) were classified as CHON, CHON2 and CHON3 according to the number of nitrogen atoms. Via the analytical window of van Krevelen diagrams, we found that the molecular structural features of CHON, CHON2 and CHON3 were not altered before the chlorination process. In detail, the CHON2 and CHON3 compositions were concentrated on the regions assigned to a lignin-structure while CHON compositions were also distributed in other compounds including proteins, carbohydrates and tannin. Furthermore, CHON formation was more difficult to be removed before the V-filter process. For N-DBP, chlorine-containing DON (Cl-DON) composition was likely to be removed through flocculation and sedimentation processes, whereas N-nitrosamine compounds were removed in V-filter and biological activated carbon filter processes. The health risks of aromatic structure N-nitrosamines due to the pre-chlorination of the raw water should be further studied.