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-D 2 O) 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-D 2 O were all consistent with the slow standard AST assay used in clincial settings. This work promotes clinical practicability and faciliates antibiotic stewardship.

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.

Nitrogen (N) fixation is the conversion of inert nitrogen gas (N) to bioavailable N essential for all forms of life. N-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 N stable isotope probing (SIP) was developed to discern N-fixing bacteria in a complex soil community. Strong RR signals of cytochrome c (Cyt c, frequently present in diverse N-fixing bacteria), along with a marked N-induced Cyt c band shift, generated a highly distinguishable biomarker for N fixation. N-induced shift was consistent well with N abundance in cell determined by isotope ratio mass spectroscopy. By applying this biomarker and Raman imaging, N-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 N percentage allowed quantification of N fixation extent of diverse soil bacteria. This single-cell approach will advance the exploration of hitherto uncultured diazotrophs in diverse ecosystems.

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.

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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.

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.

In this paper, we derive the Lax pair associated with the Whitham–Broer–Kaup equations. Based on the Lax pair obtained, we construct the Darboux transformation with multi-parameters and obtain the one- and multi-soliton solutions. In addition, we qualitatively analyze various types of interaction behavior between two solitary waves along with graphical demonstration, in order to provide valuable information on the shallow water motion.