Aerobic Anoxygenic Phototrophic Bacteria in the Marine Environments Revealed by Raman/Fluorescence-Guided Single-Cell Sorting and Targeted Metagenomics.

Aerobic anoxygenic phototrophic bacteria (AAPB) contribute profoundly to the global carbon cycle. However, most AAPB in marine environments are uncultured and at low abundance, hampering the recognition of their functions and molecular mechanisms. In this study, we developed a new culture-independent method to identify and sort AAPB using single-cell Raman/fluorescence spectroscopy. Characteristic Raman and fluorescent bands specific to bacteriochlorophyll a (Bchl a) in AAPB were determined by comparing multiple known AAPB with non-AAPB isolates. Using these spectroscopic biomarkers, AAPB in coastal seawater, pelagic seawater, and hydrothermal sediment samples were screened, sorted, and sequenced. 16S rRNA gene analysis and functional gene annotations of sorted cells revealed novel AAPB members and functional genes, including one species belonging to the genus Sphingomonas, two genera affiliated to classes Betaproteobacteria and Gammaproteobacteria, and function genes bchCDIX, pucC2, and pufL related to Bchl a biosynthesis and photosynthetic reaction center assembly. Metagenome-assembled genomes (MAGs) of sorted cells from pelagic seawater and deep-sea hydrothermal sediment belonged to Erythrobacter sanguineus that was considered as an AAPB and genus Sphingomonas, respectively. Moreover, multiple photosynthesis-related genes were annotated in both MAGs, and comparative genomic analysis revealed several exclusive genes involved in amino acid and inorganic ion metabolism and transport. This study employed a new single-cell spectroscopy method to detect AAPB, not only broadening the taxonomic and genetic contents of AAPB in marine environments but also revealing their genetic mechanisms at the single-genomic level.

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.

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.

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.

Temporal variation of antibiotic resistome and pathogens in food waste during short-term storage.

The massive food wastes pose a growing health concern for spreading of antibiotic resistance and pathogens due to food spoilage. However, little is known about these microbial hazards during collection, classification, and transportation before eventual treatment. Here, we profiled the temporal variations of antibiotic resistance genes (ARGs), pathogens, bacterial and fungal communities across four typical food wastes (vegetable, fish, meat, and rice) during storage at room temperature in summer (maximum 28-29 °C) of typical southeast city in China. A total of 171 ARGs and 32 mobile genetic elements were detected, and the absolute abundance of ARGs significantly increased by up to 126-fold with the storage time. Additionally, five bacterial pathogens containing virulence factor genes were detected, and Klebsiella pneumoniae was persistently detected throughout the storage time in all food types except rice. Moreover, fungal pathogens (e.g., Aspergillus, Penicillium, and Fusarium) were also frequently detected. Notably, animal food wastes were demonstrated to harbor higher abundance of ARGs and more types of pathogens, indicating a higher level of hazard. Mobile genetic elements and food types were demonstrated to mainly impact ARG profiles and pathogens, respectively. This work provides a comprehensive understanding of the microbial hazards associated with food waste recycling, and will contribute to optimize the food waste management to ensure biosecurity and benefit human health.

Effects of soil protists on the antibiotic resistome under long term fertilization.

Soil protists are key in regulating soil microbial communities. However, our understanding on the role of soil protists in shaping antibiotic resistome is limited. Here, we considered the diversity and composition of bacteria, fungi and protists in arable soils collected from a long-term field experiment with multiple fertilization treatments. We explored the effects of soil protists on antibiotic resistome using high-throughput qPCR. Our results showed that long term fertilization had stronger effect on the composition of protists than those of bacteria and fungi. The detected number and relative abundance of antibiotic resistance genes (ARGs) were elevated in soils amended with organic fertilizer. Co-occurrence network analysis revealed that changes in protists may contribute to the changes in ARGs composition, and the application of different fertilizers altered the communities of protistan consumers, suggesting that effects of protistan communities on ARGs might be altered by the top-down impact on bacterial composition. This study demonstrates soil protists as promising agents in monitoring and regulating ecological risk of antibiotic resistome associated with organic fertilizers.

Long-Term Fertilization History Alters Effects of Microplastics on Soil Properties, Microbial Communities, and Functions in Diverse Farmland Ecosystem.

Microplastics (MPs) pollution has caused a threat to soil ecosystem diversity and functioning globally. Recently, an increasing number of studies have reported effects of MPs on soil ecosystems. However, these studies mainly focused on soil bacterial communities and a few limited functional genes, which is why MPs effects on soil ecosystems are still not fully understood. Fertilization treatment often coinsides with MPs exposure in practice. Here, we studied effects of an environmentally relevant concentration of polyethylene on soil properties, microbial communities, and functions under different soil types and fertilization history. Our results showed that 0.2% PE MPs exposure could affect soil pH, but this effect varied according to soil type and fertilization history. Long-term fertilization history could alter effects of MPs on soil bacterial and fungal communities in diverse farmland ecosystems (P < 0.05). Soil fungal communities are more sensitive to MPs than bacterial communities under 0.2% PE MPs exposure. MPs exposure has a greater impact on the soil ecosystem with a lower microbial diversity and functional genes abundance and increases the abundance of pathogenic microorganisms. These findings provided an integrated picture to aid our understanding of the impact of MPs on diverse farmland ecosystems with different fertilization histories.

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.

Temporal Dynamics of Antibiotic Resistome in the Plastisphere during Microbial Colonization.

The increasing and simultaneous pollution of plastic debris and antibiotic resistance in aquatic environments makes plastisphere a great health concern. However, the development process of antibiotic resistome in the plastisphere is largely unknown, impeding risk assessment associated with plastics. Here, we profiled the temporal dynamics of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and microbial composition in the plastisphere from initial microbial colonization to biofilm formation in urban water. A total of 82 ARGs, 12 MGEs, and 63 bacterial pathogens were detected in the plastisphere and categorized as the pioneering, intermediate, and persistent ones. The high number of five MGEs and six ARGs persistently detected in the whole microbial colonization process was regarded as a major concern because of their potential role in disseminating antibiotic resistance. In addition to genomic analysis, D2O-labeled single-cell Raman spectroscopy was employed to interrogate the ecophysiology of plastisphere in a culture-independent way and demonstrated that the plastisphere was inherently more tolerant to antibiotics than bacterioplankton. Finally, by combining persistent MGEs, intensified colonization of pathogenic bacteria, increased tolerance to antibiotic, and potential trophic transfer into a holistic risk analysis, the plastisphere was indicated to constitute a hot spot to acquire and spread antibiotic resistance and impose a long-term risk to ecosystems and human health. These findings provide important insights into the antibiotic resistome and ecological risk of the plastisphere and highlight the necessity for comprehensive surveillance of plastisphere.

Dysbiosis of gut microbiome affecting small intestine morphology and immune balance: a rhesus macaque model.

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts. Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface; however, studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models. Here, we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal host-microbiome niche and immune balance with steady health status. Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment. Furthermore, the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment, and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed. In addition to inducing aberrant morphology of small intestinal villi, the depletion of gut commensal bacteria led to increased proportions of CD3 + T, CD4 + T, and CD16 + NK cells in peripheral blood mononuclear cells (PBMCs), but decreased numbers of Treg and CD20 + B cells. The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes, including IL-13, VCAM1, and LGR4.

Partial replacement of inorganic phosphorus (P) by organic manure reshapes phosphate mobilizing bacterial community and promotes P bioavailability in a paddy soil.

The optimization of more sustainable fertilization practice to relieve phosphorus (P) resource scarcity and increase P fertilizer utilization, a better understanding of the regulatory roles of microbes in P mobilization is urgently required to reduce P input. The genes phoD and pqqC are responsible for regulating organic and inorganic P mobilization, respectively. Using high-throughput sequencing, the corresponding bacterial communities harbored by these genes were determined. We conducted a 4-year rice-rice-crop rotation to investigate the responses of phoD- and pqqC-harboring bacterial communities to the partial replacement of inorganic P fertilizer by organic manure with reduced P input. The results showed that a combination of organic and inorganic fertilization maintained high rice yield, and also produced a more complex and stable phosphate mobilizing bacterial community, which contributed to phosphatase activities more than their gene abundances in the model analysis. Compared with the conventional mineral fertilization, organic-inorganic fertilization with the reduced P input slightly increased pqqC gene abundance while significantly enhanced the abundance of phoD-harboring bacteria, especially the genera Bradyrhizobium and Methylobacterium known as potential organic P mineralizers which can maintain high rice production. Moreover, the increased pH was the most impactful factor for the phoD- and pqqC-harboring bacterial communities, by promoting microbial P turnover and greatly increasing bioavailable P pools (H2O-Pi and NaHCO3-Pi, NaOH-Pi) in this P-deficient paddy soil. Hence, our study demonstrated that the partial replacement of mineral P with organic manure could reshape the inorganic phosphate solubilizing and alkaline-phosphomonoesterase encoding bacterial communities towards more resilient and effective to the high P utilization and productivity over intense cultivation, providing insights into the potential of soil microbes in the efficient management of agricultural P fertilization.

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.

D2O-Isotope-Labeling Approach to Probing Phosphate-Solubilizing Bacteria in Complex Soil Communities by Single-Cell Raman Spectroscopy.

Increasing the bioavailability of immobilized phosphorus (P) in soil by phosphate-solubilizing bacteria (PSB) is an effective strategy for sustainable agronomic use of P and for mitigating the P crisis. Here, D2O isotope labeling combined with single-cell Raman spectroscopy (Raman-D2O) was developed as an efficient activity-based approach to characterizing the presence and activity of PSB in a culture-independent way. On the basis of the finding that PSB were significantly more active than non-PSB in the presence of insoluble P, a C-D Raman band from active assimilation of D2O-derived D was established as a biomarker for both inorganic-phosphate-solubilizing bacteria and organic-phosphate-solubilizing bacteria. C-D ratios (intensities of C-D bands as percentages of the intensities of both the C-D and C-H bands) were further established as semiquantitative indicators of P-releasing activities because of the consistency between the C-D ratio and the concentration of solubilized phosphate or acid phosphatase activity as measured by conventional bulk assays. By applying Raman imaging, single-cell Raman-D2O clearly discerned PSB in a mixed-soil bacterial culture and even in complex soil communities. Remarkable heterogeneity of microbial activity, ranging from 2 to 30% (close to that in medium without P and that in medium with sufficient soluble P, respectively), was revealed at the single-cell level and clearly illustrated the subpopulation of soil bacteria active in solubilizing P. This work not only enables probing PSB and their P-releasing activities but also opens a window to explore more diverse microbial resources when obtaining related isotope-labeled substrates is prohibitive.

Identification and characterization of inorganic-phosphate-solubilizing bacteria from agricultural fields with a rapid isolation method.

The ability to solubilize fixed inorganic phosphorus (P) for plant growth is important for increasing crop yield. More P can be released by inoculating soil with inorganic-phosphate-solubilizing bacteria (iPSBs). We used 96-well microplates instead of traditional 200-mm petri dishes to rapidly screen iPSB strains for their solubilizing ability. We simultaneously obtained 76 iPSB isolates from 576 wells containing two agricultural soils. This method conveniently identified positive iPSB strains and effectively prevented fungal cross-contamination. Maximum-likelihood phylogenetic trees of the isolated strains showed that Bacillus megaterium was the most dominant iPSB, and strains Y99, Y95, Y924 and Y1412 were selected as representatives for the analysis of P solubilization. Succinic acid was the main organic acid of B. megaterium for releasing P. It was strongly correlated with the increase in soluble P concentration during 168 h of incubation of these four strains. pH was negatively exponentially correlated with the amount of soluble P in the medium, and the amount of succinic acid was strongly linearly correlated with the amount of P released (P < 0.001), suggesting that organic acid may mobilize microbial P. Our study provides an efficient and effective method for identifying and analyzing the growth of iPSB strains able to solubilize inorganic P and gives a better understanding of the mechanism of P solubilization.

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.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

[Effects of Shenqi compound recipe on cell cycle and apoptosis of ascites inducible hepatocarcinoma cell line H22].

AIM: To observe the effects of Shenqi compound recipe (SQ) on cell cycle and apoptosis of hepatocarcinoma cell line H22 in-vitro and in-vivo. METHODS: BALB/c mice bearing tumors were perfused with SQ for 10 days running. And then cytotoxic activity of mouse NK cells and macrophages and variation of cell cycle of H22 cells were observed. Inhibition of H22 cell proliferation and induction of the their apoptosis by SQ were analyzed by flow cytometry. RESULTS: The tumor-suppre ssive rate of SQ was 65.68%. SQ could make H22 cells arrested at the S phase and induce their apoptosis. CONCLUSION: SQ has significant anti-tumor effect both in-vitro and in-vivo. Its mechanism may be related to blocking of cell cycle and inducing apoptosis of H22 cells.