Phagotrophic protists preserve antibiotic-resistant opportunistic human pathogens in the vegetable phyllosphere.

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.

Quantifying health risks of plastisphere antibiotic resistome and deciphering driving mechanisms in an urbanizing watershed.

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.

Movement of protistan trophic groups in soil-plant continuums.

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.

Distinct patterns of the soil and phyllosphere antibiotic resistome in natural forest ecosystems under an altitudinal gradient.

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.

Characterizing potential pathogens from intracellular bacterial community of protists in wastewater treatment plants.

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.

Effects of manure fertilization on human pathogens in endosphere of three vegetable plants.

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.

HiLi-chip: A high-throughput library construction chip for comprehensive profiling of environmental microbial communities.

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.

Conurbation size drives antibiotic resistance along the river.

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.

Effects of biofertilizer on soil microbial diversity and antibiotic resistance genes.

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.

Discarded masks as hotspots of antibiotic resistance genes during COVID-19 pandemic.

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.

Evaluation of Two Primer Sets for Amplification of Comammox Nitrospira amoA Genes in Wetland Soils.

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.

Investigation of nitrogen pollutants transformation and its pathways along the long-distance prechlorinated raw water distribution system.

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 × 107-8.41 × 108 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 biofilms 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.

Effects of Pre-Oxidation on Haloacetonitrile and Trichloronitromethane Formation during Subsequent Chlorination of Nitrogenous Organic Compounds.

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.

Molecular Characteristics of Dissolved Organic Nitrogen and Its Interaction with Microbial Communities in a Prechlorinated Raw Water Distribution System.

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.

Transformation and fate of dissolved organic nitrogen in drinking water supply system: A full scale case study from Yixing, China.

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.

Effects of pipe material on nitrogen transformation, microbial communities and functional genes in raw water transportation.

Raw water transportation pipelines are vital in an urban water supply system for transporting raw water to drinking water treatment plants. This study investigated the effects of pipe material on nitrogen transformation, microbial communities and characteristics of related function genes in paint-lined steel pipe (PLSP) and cement-lined steel pipe (CLSP) raw water model systems. We established quantitative relationships between specific functional genes and change rates of nitrogen pollutants, which were verified by field investigation on nitrogen pollutant transformations in real raw water transportation systems. The results showed that the CLSP produced higher ammonia nitrogen (NH4+-N) transformation rates and higher effluent concentrations of nitrate nitrogen (NO3--N) and dissolved organic nitrogen (DON) than the PLSP. Both pipes achieved high and stable nitrite nitrogen (NO2--N) and low total nitrogen (TN) removal efficiency. Nitrification was found to be the dominant process in both model systems, especially in the CLSP. Characteristics of microbial communities and nitrogen functional genes, which were analysed by high-throughput pyrosequencing and quantitative polymerase chain reaction (qPCR), respectively, varied between the two pipe systems. Nitrogen transformation pathways, identified by path analysis, were also different between the PLSP and CLSP due to different microbial community characteristics and synergistic effects of nitrogen functional genes. In the CLSP, (NH4+-N→NO2--N) with part denitrification, was the primary transformation pathway of ammonia nitrogen (NH4+-N), while only ammonia oxidization contributed to NH4+-N transformation in the PLSP. (NO2--N→NO3--N) was the main pathway involved in NO2--N transformation and NO3--N accumulation. The TN removal showed complex relationships with nitrification, denitrification and nitrogen fixation processes. These findings provided molecular-level insights into nitrogen pollutant transformations during the transportation of raw water through different types of pipes and technical support for the selection of raw water pipe materials. In our study area, the Taihu basin, China, PLSP was better than CLSP for distributing raw water in a short transportation distance, due to the lower effluent concentrations of DON and NO3--N and less abundance of microorganisms.

Variation of microbial communities and functional genes during the biofilm formation in raw water distribution systems and associated effects on the transformation of nitrogen pollutants.

This study aimed to investigate the variation of microbial communities and functional genes during the biofilm formation in raw water distribution systems without prechlorination and associated effects on the transformation of nitrogen pollutants by using a designed model pipe system. The results showed the transformation of nitrogen pollutants was obvious during the biofilm formation. The richness and diversity of the microbial communities changed significantly. The higher abundance of Nitrospirae in biofilm samples significantly contributed to biological nitrification. In particular, the stable content of Bacteroidetes in the biofilm and soluble microbial products released by the biomass might have enhanced the increase in dissolved organic nitrogen. In addition, the variation tendency of nitrogen functional gene abundances and their strong effects on NH4+-N, NO2--N, and NO3--N transformation were clearly observed. These findings provide new insights into the evolution of microbial communities and functional genes during the initial operation period of real-world raw water distribution pipes and highlight management and possible safety issues in the subsequent water treatment process.