Struvite recovered from wastewater is a renewable source of phosphorus and nitrogen and can be used as fertilizer for plant growth. However, antibiotics and resistome can be enriched in the struvite derived from wastewater. Robust understanding of the potential risks after struvite application to soils has remained elusive. Here, we profiled antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in struvite, soil, rhizosphere and phyllosphere of Brassica using high-throughput quantitative PCR. A total of 165 ARGs and 10 MGEs were detected. Application of struvite was found to increase both the abundance and diversity of ARGs in soil, rhizosphere and phyllosphere. In addition, ARGs shared exclusively between Brassica phyllosphere and struvite were identified, indicating that struvite was an important source of ARGs found in phyllosphere. Furthermore, OTUs shared between rhizosphere and phyllosphere were found to significantly correlate with ARGs, suggesting that microbiota in leaf and root could interconnect and ARGs might transfer from struvite to the surface of plants via rhizosphere using bacteria as spreading medium. These findings demonstrated that struvite as an organic fertilizer can facilitate the spread of antibiotic resistance into human food chain and this environment-acquired antibiotic resistance should be put into human health risk assessment system.

•Manure-borne microorganism contribute largely to antibiotic resistance genes (ARGs) elevation in manured soil.•Indigenous soil microorganism prevent ARGs dissemination from manure to soil.•ARGs abundance in manured soil decreased over time.Manure application is a common practice that not only adds nutrients and organic matter to arable soils for crop growth, but also introduces antibiotic resistance genes (ARGs), posing a potential risk to human health. To investigate the mechanisms underlying the spread of ARGs in manured soil, especially the impact of manure-borne and indigenous soil microorganisms, a microcosm experiment with four specially designed treatments over a period of two months was conducted, including soil, soil with irradiated pig manure, irradiated soil with pig manure, and soil with pig manure. A total of 240 unique ARGs were detected via a high-throughput quantitative PCR (HT-qPCR) targeting almost all major classes of ARGs. Manure application significantly increased the diversity and abundance of ARGs in soil (P < 0.01), and also markedly shifted the bacterial composition that was significantly correlated with ARGs profiles. Manure-borne microorganisms contributed largely to the elevation of ARGs due to both the addition of manure-borne antibiotic resistant bacteria (ARB) in soil and potential horizontal gene transfer (HGT) via mobile genetic elements (MGEs) from manure-borne ARB to indigenous soil microorganisms. In contrast, indigenous soil microorganisms were demonstrated to prevent the dissemination of ARGs from manure to soil. The reason could be due to that indigenous soil microorganisms prevented the invasion and establishment of manure-borne ARB in soil. The abundance of ARG in manured soil decreased over time, but was still higher than that in control soil, indicating the persistence of ARGs in manured soil. These findings may shed light on the mechanisms underlying the spread and fate of ARGs in manured soil and also clues for ARGs mitigation.

The substantial cost of substrates is an enormous obstacle in the successful translation of biospectroscopy (IR or Raman) into routine clinical/laboratory practice (screening or diagnosis). As a cheap and versatile substrate, we compared the performance of readily available aluminium (Al) foil with low-E, Au-coated and glass slides for cytological and histological specimen analysis by attenuated total reflection Fourier-transform infrared (ATR-FTIR), transflection FTIR or Raman spectroscopy. The low and almost featureless background signal of Al foil enables the acquisition of IR or Raman spectra without substrate interference or sacrificing important fingerprint biochemical information of the specimen, even for very thin samples with thicknesses down to 2 μm. Al foil is shown to perform as well as, if not better than, low-E or Au-coated slide, irrespective of its relatively rough surface. Although transmission FTIR is not possible on Al foil, this work demonstrates Al foil is an inexpensive, readily available and versatile substrate suitable for ATR-FTIR, transflection FTIR or Raman spectrochemical measurements of diverse biological specimens. The features of Al foil demonstrated here could promote a transition towards accessible substrates that can be readily implemented in either research or clinical settings.

Massive production of nanomaterials poses a high risk to environmental ecology and human health. However, comprehensive understanding of nanotoxicity is still a major challenge due to the limitations of assessment methods, especially at the molecular level. We developed a new, sensitive, and robust fingerprinting surface-enhanced Raman spectroscopy (SERS) approach to interrogate both dose- and time-dependent phenotypic bacterial responses to zinc oxide nanoparticles (ZnO NPs). SERS enhancement was provided by biocompatible Au NPs. Additionally, a novel vacuum filtration-based strategy was adopted to fabricate bacterial samples with highly uniform SERS signals, ensuring the acquisition of robust and independent spectral changes from ZnO NPs-impacted bacteria without undesirable spectral variations. Combined with multivariate analysis, clear and informative spectral alteration profiles were obtained. Much greater alterations were found in low-dose ranges than high-dose ranges, indicating a reduction in the bioavailability of ZnO NPs with doses. Time-resolved bacterial responses provided important information on toxic dynamics, i.e., rapid action of ZnO NPs within 0.5 h was identified, and ZnO NPs at low doses and long exposure time exerted similar effects to high doses, indicating the concerns associated with low-dose exposure. Further analysis of biochemical changes revealed metabolic activity decrease over both incubation time and doses. Meanwhile, a short-term protection strategy of bacteria by producing lipid-containing outer membrane vesicles to mitigate the cell of toxic NPs was suggested. Finally, Zn2+ ions released from NPs were demonstrated to be irrelevant to bacterial responses on both dose and time scales. The new SERS methodology can potentially profile a large variety of toxic NPs and advance our understanding of nanotoxicity.

•SERS as a new tool to monitor dual-species biofouling development.•Dominant species within biofilms change with culture time.•Bacteria display differing behaviors in single and dual culture.•Cell concentration and competition for nutrients affect bacterial community.•SERS is also capable of early warning of cell attachment and biomass monitoring.Surface-enhanced Raman spectroscopy (SERS) was used to monitor the development of a dual-species biofilm formed by two model bacteria (Brevundimonas diminuta, BD and Staphylococcus aureus, SA) on a mixed cellulose ester membrane surface. The highly distinguishable SERS features of BD and SA as well as a semi-quantitative analysis of SERS were used to characterize dynamic changes in dominant species within the biofilm with culture time. SA dominated for the first 8 h but detached from the membrane after 24 h and were outcompeted by BD. SA also displayed differing behaviors in single and dual cultures, with no detachment in the former case but extensive detachment in the latter after 24 h. SERS results were in good agreement with that from scanning electron microscopy. Cell concentrations in solution and competition for limited nutrients accounted for changes in bacterial abundance in dual-species biofilms. Furthermore, bacterial attachment on the membrane as early as 1 h was detected by SERS, demonstrating its high sensitivity and capability for early diagnosis of biofouling. The extent of membrane biofouling was also monitored by plotting SERS peak intensity against culture time. This study suggests that SERS will provide insights into interspecies interactions in biofouling development and help the development of antifouling strategies.SEM of dual-species biofilm.

Silver nanoparticles (Ag NPs) are extensively used as an antibacterial additive in commercial products and their release has caused environmental risk. However, conventional methods for the toxicity detection of Ag NPs are very time consuming and the mechanisms of action are not clear. We developed a new, in situ, rapid, and sensitive fingerprinting approach, using surface-enhanced Raman spectroscopy (SERS), to study the antibacterial activity and mechanism of Ag NPs of 80 and 18 nm (Ag80 and Ag18, respectively), by using the strong electromagnetic enhancement generated by Ag NPs. Sensitive spectra changes representing various biomolecules in bacteria were observed with increasing concentrations of Ag NPs. They not only allowed SERS to monitor the antibacterial activity of Ag NPs of different sizes in different water media but also to study the antibacterial mechanism at the molecular level. Ag18 were found to be more toxic than Ag80 in water, but their toxicity declined to a similar level in the PBS medium. The antibacterial mechanism was proposed on the basis of a careful identification of the chemical origins by comparing the SERS spectra with model compounds. The dramatic change in protein, hypoxanthine, adenosine, and guanosine bands suggested that Ag NPs have a significant impact on the protein and metabolic processes of purine. Finally, by adding nontoxic and SERS active Au NPs, SERS was successfully utilized to study the action mode of the NPs unable to produce an observable SERS signal. This work opens a window for the future extensive SERS studies of the antibacterial mechanism of a great variety of non-SERS-active NPs.