Active antibiotic resistome in soils unraveled by single-cell isotope probing and targeted metagenomics.

Antimicrobial resistance (AMR) in soils represents a serious risk to human health through the food chain and human-nature contact. However, the active antibiotic-resistant bacteria (ARB) residing in soils that primarily drive AMR dissemination are poorly explored. Here, single-cell Raman-D2O coupled with targeted metagenomics is developed as a culture-independent approach to phenotypically and genotypically profiling active ARB against clinical antibiotics in a wide range of soils. This method quantifies the prevalence (contamination degree) and activity (spread potential) of soil ARB and reveals a clear elevation with increasing anthropogenic activities such as farming and the creation of pollution, thereby constituting a factor that is critical for the assessment of AMR risks. Further targeted sorting and metagenomic sequencing of the most active soil ARB uncover several uncultured genera and a pathogenic strain. Furthermore, the underlying resistance genes, virulence factor genes, and associated mobile genetic elements (including plasmids, insertion sequences, and prophages) are fully deciphered at the single-cell level. This study advances our understanding of the soil active AMR repertoire by linking the resistant phenome to the genome. It will aid in the risk assessment of environmental AMR and guide the combat under the One Health framework.

Spatiotemporal Changes of Antibiotic Resistance, Potential Pathogens, and Health Risk in Kindergarten Dust.

The microorganisms present in kindergartens are extremely important for children's health during their three-year preschool education. To assess the risk of outdoor dust in kindergartens, the antibiotic resistome and potential pathogens were investigated in dust samples collected from 59 kindergartens in Xiamen, southeast China in both the winter and summer. Both high-throughput quantitative PCR and metagenome analysis revealed a higher richness and abundance of antibiotic resistance genes (ARGs) in winter (P < 0.05). Besides, the bloom of ARGs and potential pathogens was evident in the urban kindergartens. The co-occurrence patterns among ARGs, mobile genetic elements (MGEs), and potential pathogens suggested some bacterial pathogens were potential hosts of ARGs and MGEs. We found a large number of high-risk ARGs in the dust; the richness and abundance of high-risk ARGs were higher in winter and urban kindergartens compared to in summer and peri-urban kindergartens, respectively. The results of the co-occurrence patterns and high-risk ARGs jointly reveal that urbanization will significantly increase the threat of urban dust to human beings and their risks will be higher in winter. This study unveils the close association between ARGs/mobile ARGs and potential pathogens and emphasizes that we should pay more attention to the health risks induced by their combination.

Long-term seawall barriers lead to the formation of an urban coastal lagoon with increased antibiotic resistome.

Urbanization has increased the spread of antibiotic resistance genes (ARGs) impacting urban aquatic ecosystems and threatening human health. However, an overview of the antibiotic resistome in artificial coastal lagoons formed by coastal seawall construction is unclear. This study investigated the resistome of sediment in a coastal lagoon, established for over 60 years and found that the composition of the resistome in the lagoon sediments associated with the seawall significantly differed from that of marine sediment external to the seawall. Moreover, the diversity, number, relative abundance, and absolute abundance of the antibiotic resistome in the lagoon sediments were significantly higher compared to marine sediment. Network analyses revealed that more co-occurrences were found in lagoon sediment between bacterial communities, ARGs and mobile genetic elements (MGEs) than in marine sediments, suggesting that bacteria in lagoon sediments may be associated with multiple antibiotic resistances. Random forest and structural equation models showed that an increase in the absolute abundance of MGEs had a concomitant effect on the absolute abundance and diversity of ARGs, whereas increasing salinity decreased the absolute abundance of ARGs. This study provides a basis to assess the risk of resistome diffusion and persistence in an artificial coastal lagoon.

Increasing Antimicrobial Resistance and Potential Human Bacterial Pathogens in an Invasive Land Snail Driven by Urbanization.

Our understanding of the role urbanization has in augmenting invasive species that carry human bacterial pathogens and antimicrobial resistance (AMR) remains poorly understood. Here, we investigated the gut bacterial communities, antibiotic resistance genes (ARGs) and potential antibiotic-resistant pathogens in giant African snails (Achatina fulica) collected across an urbanization gradient in Xiamen, China (n = 108). There was a lack of correlation between the microbial profiles of giant African snails and the soils of their habitats, and the resistome and human-associated bacteria were significantly higher than those of native snails as well as soils. We observed high diversity (601 ARG subtypes) and abundance (1.5 copies per 16S rRNA gene) of giant African snail gut resistome. Moreover, giant African snails in more urban areas had greater diversity and abundance of high-risk ARGs and potential human bacterial pathogens (e.g., ESKAPE pathogens). We highlight that urbanization significantly impacted the gut microbiomes and resistomes of these invasive snails, indicating that they harbor greater biological contaminants such as ARGs and potential human bacterial pathogens than native snails and soils. This study advances our understanding of the effect of urbanization on human bacterial pathogens and AMR in a problematic invasive snail and should help combat risks associated with invasive species under the One Health framework.

Antibiotic resistome in groundwater and its association with mountain springs and river.

The distribution of antibiotic resistance genes (ARGs) in water sources potentially threatens drinking water safety. However, the sources of antibiotic resistome in groundwater are still under-investigated. Here, we evaluated the profiles of antibiotic resistome in peri-urban groundwater and its associated water sources (river and mountain spring) to characterize the antibiotic resistome from natural water sources on groundwater resistome. A total of 261 antibiotic resistome were detected in groundwater, mountain spring, and river samples. The relative abundances of ARGs and mobile genetic elements (MGEs) were significantly higher in the river samples than in spring water and groundwater samples. The resistome profiles were similar between groundwater and spring water but differed from the river samples. According to source tracking results, the groundwater resistome was likely to be derived from springs (28.0%-50.0%) and rivers (28.6%-48.6%), which share the same trend for the source tracking of bacterial communities. Bacterial α-diversity, bacterial ß-diversity, and MGEs directly or indirectly affected the ARGs in groundwater samples. Although the abundance of groundwater resistome was not elevated by river and spring water, groundwater resistomes were diverse and may be derived from both river and spring water. We highlight the importance of groundwater resistome and its association with potential water sources, providing a better understanding and basis for the effective control of the ARG proliferation and dissemination in groundwater from exogenous water bodies in the future.

Short-Term Benzalkonium Chloride (C12) Exposure Induced the Occurrence of Wide-Spectrum Antibiotic Resistance in Agricultural Soils.

Antibiotic resistance genes (ARGs) are global pollutants that pose a potential risk to human health. Benzalkonium chloride (C12) (BC) disinfectants are thought to exert selection pressure on antibiotic resistance. However, evidence of BC-induced changes in antibiotic resistance in the soil environment is lacking. Here, we established short-term soil microcosms to investigate ARG profile dynamics in agricultural soils amended with sulfamethazine (SMZ, 10 mg kg-1) and gradient concentrations of BC (0-100 mg kg-1), using high-throughput quantitative PCR and Illumina sequencing. With the increase in BC concentration, the number of ARGs detected in the soil increased, but the normalized ARG abundance decreased. The added SMZ had a limited impact on ARG profiles. Compared to broad-spectrum fungicidal BC, the specificity of SMZ significantly affected the microbial community. Network analysis found that low-medium BC exposure concentrations resulted in the formation of small but strong ARG co-occurrence clusters in the soil, while high BC exposure concentration led to a higher incidence of ARGs. Variation partitioning analysis suggested that BC stress was the major driver shaping the ARG profile. Overall, this study highlighted the emergence and spread of BC-induced ARGs, potentially leading to the antimicrobial resistance problem in agricultural soils.

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.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables.

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1-2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Arsenic contribution of poultry manure towards soils and food plants contamination and associated cancer risk in Khyber Pakhtunkhwa, Pakistan.

Exposure to high level of arsenic (As) through the ingestion of contaminated soil, dust and food plants can pose health risk to humans. This study investigates the total arsenic (As), arsenobetaine (AsB), monomethylarsenate (MMA), dimethylarsenate (DMA), arsenite (As3+) and arsenate (As5+) concentrations in poultry feed, manure, agricultural soils and food plants collected from Khyber Pakhtunkhwa Province, Pakistan. The total mean As concentrations in the edible parts of food plants ranged from 0.096 mg kg-1 to 1.25 mg kg-1 with percentile (P) values (P25-0.039, P50-0.0765, P75-0.165 1 mg kg-1 to P25-0.95, P50-1.23, P75-1.6 1 mg kg-1) and exceeded the food safety limit (0.1 mg kg-1) of Food & Agriculture Organization (FAO) and World Health Organization (WHO) in all plant species except Pisum sativum (pea) and Mentha arvensis (mint). The risk to human health was assessed through the average daily intake (ADI), hazards quotient (HQ), health risk index (HRI) and lifetime cancer risk (LTCR). The highest average daily intake of As via the ingestion of Malva neglecta (mallow, a leafy plant) was observed for adults and children. The ADI for adults and children (2.36 × 10-4 mg kg-1 day-1 and 6.33 × 10-4 mg kg-1 day-1) was about 13% and 5%, respectively, of the Bench Mark Dose Limit (BMDL0.5) of 3.00 × 10-3 mg kg-1 day-1 set by WHO. The HRI was 3 times more in the children (2.1) than the adults (0.79), posing non-cancer health risks (health risk index > 1) for children. The LTCR values were slightly higher (1.53 × 10-4) relative to USEPA and WHO limits (1 × 10-6 to 1 × 10-4) for children whereas a minimal cancer risk was observed for adults via consumption of selected food plants. The results showed that poultry manure can contaminate food plants that may lead to cancer and non-cancer risks in agricultural areas, Pakistan. Thus, it is important to minimize As concentration in poultry feed to safeguard human health and environment from adverse effects.

Viral Community and Virus-Associated Antibiotic Resistance Genes in Soils Amended with Organic Fertilizers.

Antibiotic resistance is a global health concern. Long-term organic fertilization can influence the antibiotic resistome of agricultural soils, posing potential risks to human health. However, little is known about the contribution of viruses to the dissemination of antibiotic resistance genes (ARGs) in this context. Here, we profiled the viral communities and virus-associated ARGs in a long-term (over 10 years) organic fertilized field by viral metagenomic analysis. A total of 61,520 viral populations (viral operational taxonomic units, vOTUs) were retrieved, of which 21,308 were assigned at the family level. The viral community structures were significantly correlated with the bacterial community structures (P < 0.001) and the dosage of applied sewage sludge (r2 = 0.782). A total of 16 unique ARGs were detected in soil viromes, and the number of virus-associated ARG subtypes was higher in sewage sludge treatments (except for 1 SS) than others. The network analysis showed that the application of the organic fertilizer increased the bacteria-virus interactions, suggesting that the chances of ARG exchange between viruses and their hosts may increase. Overall, our results provide a novel understanding about virus-associated ARGs and factors affecting the profile of viral community in fertilized soil.

Influence of Legacy Mercury on Antibiotic Resistomes: Evidence from Agricultural Soils with Different Cropping Systems.

Agricultural soils are important reservoirs for antibiotic resistance genes (ARGs), which have close linkage to human health via crop production. Metal stress in environments may function as a selection pressure for antibiotic resistomes. However, there is still a lack of field studies focusing on the effect of historical mercury (Hg) contamination on antibiotic resistomes in agricultural soils. Here, we explored the ARG profile in soils with different cropping systems (paddy and upland) and linked them to legacy Hg exposure. We found that ARG profiles were significantly different between paddy and upland soils. However, both paddy and upland soils with long-term field Hg contamination harbored higher diversity and abundance of ARGs than non-polluted soils. The co-occurrence network reveals significant associations among Hg, Hg resistance genes, mobile genetic elements (MGEs), and ARGs. Together with path analysis showing legacy Hg possibly affecting soil resistomes through the shifts of soil microbiota, Hg resistance genes, and MGEs, we suggest that legacy Hg-induced potential co-selection might elevate the ARG level. Redundancy analysis further supports that legacy Hg pollution had a significant association with ARG variations in the paddy and upland soils (P < 0.01). Collectively, our results highlight the underappreciated role of legacy Hg as a potential persistent selecting agent in contributing to soil ARGs in agroecosystems.

Deciphering Potential Roles of Earthworms in Mitigation of Antibiotic Resistance in the Soils from Diverse Ecosystems.

Earthworms are capable of redistributing bacteria and antibiotic resistance genes (ARGs) through soil profiles. However, our understanding of the earthworm gut microbiome and its interaction with the antibiotic resistome is still lacking. Here, we characterized the earthworm gut and soil microbiome and antibiotic resistome in natural and agricultural ecosystems at a national scale, and microcosm studies and field experiments were also employed to test the potential role of earthworms in dynamics of soil ARGs. The diversity and structure of bacterial communities were different between the earthworm gut and soil. A significant correlation between bacterial community dissimilarity and spatial distance between sites was identified in the earthworm gut. The earthworm gut consistently had lower ARGs than the surrounding soil. A significant reduction in the relative abundance of mobile genetic elements and dominant bacterial phylotypes that are the likely hosts of ARGs was observed in the earthworm gut compared to the surrounding soil, which might contribute to the decrease of ARGs in the earthworm gut. The microcosm studies and field experiments further confirmed that the presence of earthworms significantly reduced the number and abundance of ARGs in soils. Our study implies that earthworm-based bioremediation may be a method to reduce risks associated with the presence of ARGs in soils.

Developing Surrogate Markers for Predicting Antibiotic Resistance "Hot Spots" in Rivers Where Limited Data Are Available.

Pinpointing environmental antibiotic resistance (AR) hot spots in low-and middle-income countries (LMICs) is hindered by a lack of available and comparable AR monitoring data relevant to such settings. Addressing this problem, we performed a comprehensive spatial and seasonal assessment of water quality and AR conditions in a Malaysian river catchment to identify potential "simple" surrogates that mirror elevated AR. We screened for resistant coliforms, 22 antibiotics, 287 AR genes and integrons, and routine water quality parameters, covering absolute concentrations and mass loadings. To understand relationships, we introduced standardized "effect sizes" (Cohen's D) for AR monitoring to improve comparability of field studies. Overall, water quality generally declined and environmental AR levels increased as one moved down the catchment without major seasonal variations, except total antibiotic concentrations that were higher in the dry season (Cohen's D > 0.8, P < 0.05). Among simple surrogates, dissolved oxygen (DO) most strongly correlated (inversely) with total AR gene concentrations (Spearman's ρ 0.81, P < 0.05). We suspect this results from minimally treated sewage inputs, which also contain AR bacteria and genes, depleting DO in the most impacted reaches. Thus, although DO is not a measure of AR, lower DO levels reflect wastewater inputs, flagging possible AR hot spots. DO measurement is inexpensive, already monitored in many catchments, and exists in many numerical water quality models (e.g., oxygen sag curves). Therefore, we propose combining DO data and prospective modeling to guide local interventions, especially in LMIC rivers with limited data.

Phenotypic Tracking of Antibiotic Resistance Spread via Transformation from Environment to Clinic by Reverse D2O Single-Cell Raman Probing.

The rapid spread of antibiotic resistance threatens our fight against bacterial infections. Environments are an abundant reservoir of potentially transferable resistance to pathogens. However, the trajectory of antibiotic resistance genes (ARGs) spreading from environment to clinic and the associated risk remain poorly understood. Here, single-cell Raman spectroscopy combined with reverse D2O labeling (Raman-rD2O) was developed as a sensitive and rapid phenotypic tool to track the spread of plasmid-borne ARGs from soil to clinical bacteria via transformation. Based on the activity of bacteria in assimilating H to substitute prelabeled D under antibiotic treatment, Raman-rD2O sensitively discerned a small minority of phenotypically resistant transformants from a large pool of recipient cells. Its single-cell level detection greatly facilitated the direct calculation of spread efficiency. Raman-rD2O was further employed to study the transfer of complex soil resistant plasmids to pathogenic bacteria. Soil plasmid ARG-dependent transformability against five clinically relevant antibiotics was revealed and used to assess the spreading risk of different soil ARGs, i.e., ampicillin > cefradine and ciprofloxacin > meropenem and vancomycin. The developed single-cell phenotypic method can track the fate and risk of environmental ARGs to pathogenic bacteria and may guide developing new strategies to prevent the spread of high-risk ARGs.

High-throughput diagnosis of human pathogens and fecal contamination in marine recreational water.

Waterborne pathogens and their associated diseases are major threats to public health, and surveillance of pathogens and identification of the sources of pollution are imperative for preventing infections. However, simultaneously quantitative detection of multiple pathogens and pollution sources in water environments is the major challenge. In this study, we developed and validated a highly sensitive (mostly >80%) and highly specific (>99%) high-throughput quantitative PCR (HT-qPCR) approach, which could simultaneously quantify 68 marker genes of 33 human pathogens and 23 fecal markers of 10 hosts. The HT-qPCR approach was then successfully used to investigate pathogens and fecal pollution in marine recreational water samples of Xiamen, China. Totally, seven pathogenic marker genes were found in 13 beach bathing waters, which targeted Acanthamoeba spp., Clostridium perfringens, enteropathogenic Escherichia coli, Klebsiella pneumoniae, Vibrio cholera/V. parahaemolyticus and Legionella spp.. Fecal markers from human and dog were the most frequently detected, indicating human and dog feces were the main contamination in the recreational waters. Nanopore sequencing of full-length 16S rRNA gene revealed that 28 potential human pathogens were detected and electrical conductivity, salinity, oxidation-reduction potential and dissolved oxygen were significantly correlated with the variation in bacterial community. Our results demonstrated that HT-qPCR approach had the potential rapid quantification of microbial contamination, providing useful data for assessment of microbial pathogen associated health risk and development of management practices to protect human health.

Rapid Antibiotic Susceptibility Testing of Pathogenic Bacteria Using Heavy-Water-Labeled Single-Cell Raman Spectroscopy in Clinical Samples.

Speeding up antibiotic susceptibility testing (AST) is urgently needed in clincial settings to guide fast and tailored antibiotic prescription before treatment. It remains a big challenge to achieve a sample-to-AST answer within a half working day directly from a clinical sample. Here we develop single-cell Raman spectroscopy coupled with heavy water labeling (Raman-D2O) as a rapid activity-based AST approach directly applicable for clinical urine samples. By rapidly transferring (15 min) bacteria in clinical urine for AST, the total assay time from receiving urine to binary susceptibility/resistance (S/R) readout was shortened to only 2.5 h. Moreover, by overcoming the nonsynchronous responses between microbial activity and microbial growth, together with setting a new S/R cutoff value based on relative C-D ratios, S/R of both pathogenic isolates and three clinical urines against antibiotics of different action mechanisms determined by Raman-D2O were all consistent with the slow standard AST assay used in clincial settings. This work promotes clinical practicability and faciliates antibiotic stewardship.

RNA Stable Isotope Probing of Potential Feammox Population in Paddy Soil.

Anaerobic ammonium oxidation coupled to iron reduction (Feammox) is a recently discovered pathway contributing to nitrogen loss in various ecosystems such as paddy soils and sediments. However, little is known about the microbes driving Feammox in an agricultural ecosystem. Here, we demonstrated the occurrence of Feammox in paddy soils of Southern China using a 15N isotopic tracing technique, and examined the microbial communities associated with Feammox using RNA based stable isotope probing (RNA-SIP) combined with Illumina sequencing. Feammox was detected in all collected soils with direct N2 production as the dominant Feammox pathway. It was estimated that approximately 6.91% of the applied nitrogen fertilizers were lost through Feammox in the paddy soils. RNA-SIP results showed that the composition of enriched active microbial communities were dependent on soil properties, especially the soil pH and grain size. Geobacter were enriched in most soils across various properties. The abundance of enriched GOUTA19 were significantly higher in soils with low pH than those in soils with medium pH and high pH, and the relative abundance of active Nitrososphaeraceae and Pseudomonas only increased in soils with medium and high pH during 4-day of incubation. These results suggested Feammox is a ubiquitous and important process for N loss. Geobacter, GOUTA19, Nitrososphaeraceae and Pseudomonas were active during the incubation that favored Feammox and the growth of Feammox microbes, suggesting these microbes were potentially associated with Feammox in natural agricultural soils.

AsChip: A High-Throughput qPCR Chip for Comprehensive Profiling of Genes Linked to Microbial Cycling of Arsenic.

Arsenic (As) is a ubiquitous toxic element adversely affecting human health. Microbe-mediated cycling of As is largely mediated by detoxification and energy metabolism in microorganisms. We here report the development of a novel high-throughput qPCR (HT-qPCR) chip (AsChip) for comprehensive profiling of genes involved in microbial As cycling (here collectively termed "As genes"). AsChip contained 81 primer sets targeting 19 As genes and the 16S rRNA gene as a reference gene. Gene amplicon sequencing showed high identity (>96%) of newly designed primers corresponding to their targets. AsChip displayed high sensitivity (plasmid template serial dilution test; r = -0.99), with more than 96% of all PCR assays yielding true positive signals. R2 coefficients for standard curves and PCR amplification efficiencies averaged 0.98 and 0.99, respectively. A high correlation between CT values obtained by AsChip and conventional qPCR was obtained ( r = 0.962, P < 0.001). Finally, we successfully applied AsChip on soil samples from a chromium-copper-arsenic-contaminated field site and identified diverse As genes with total abundance average of 0.4 As gene copies per 16S rRNA. Our results indicate that AsChip constitutes a robust tool for comprehensive quantitative profiling of As genes in environmental samples.

Functional Single-Cell Approach to Probing Nitrogen-Fixing Bacteria in Soil Communities by Resonance Raman Spectroscopy with 15N2 Labeling.

Nitrogen (N) fixation is the conversion of inert nitrogen gas (N2) to bioavailable N essential for all forms of life. N2-fixing microorganisms (diazotrophs), which play a key role in global N cycling, remain largely obscure because a large majority are uncultured. Direct probing of active diazotrophs in the environment is still a major challenge. Herein, a novel culture-independent single-cell approach combining resonance Raman (RR) spectroscopy with 15N2 stable isotope probing (SIP) was developed to discern N2-fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N2-fixing bacteria), along with a marked 15N2-induced Cyt c band shift, generated a highly distinguishable biomarker for N2 fixation. 15N2-induced shift was consistent well with 15N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N2-fixing bacteria in both artificial and complex soil communities were discerned and imaged at the single-cell level. The linear band shift of Cyt c versus 15N2 percentage allowed quantification of N2 fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.

Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes.

The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons.IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments.