Pneumatic nano-sieve for CRISPR-based detection of drug-resistant bacteria.

The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieved an on-chip concentration factor of ∼15-fold for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU mL-1. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.

Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria.

The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.

Ceramsite made from remediated soil: A risk assessment of its potential role serving as urban street cushion.

To promote the reuse of remediated soil (RS) and facilitate the cleanup of rainwater in sponge city, we investigated the effects of ceramsite made from RS serving as urban street cushion. Ceramsite was prepared by RS or pollution-free soil (PS) and showed no difference in physical properties. Compared with gravel, ceramsite had purification effects on effluents, reducing the content of chemical oxygen demand, total nitrogen, and ammoniacal nitrogen. However, the content of total phosphorus and the concentration of Cr(VI) and arsenic slightly increased in ceramsite groups, inferring potential risk. Microbial community analysis proved that ceramsite promoted microbial growth and increased microbial diversity. A long-term risk assessment indicated that ceramsite was good at fixing heavy metals during leaching process. Taken together, ceramsite prepared from RS could serve as excellent urban street cushion with little potential risk to surroundings.

Extended lag phase indicates the dormancy of biphenyl degrading Rhodococcus biphenylivorans TG9 under heat stress.

Microbial remediation is a green and sustainable technology, but harsh environmental conditions could lead to microbial dormancy, such as entering a viable but non-culturable (VBNC) state. However, the evidence of VBNC is controversial and limited. In this study, heat stress (60 °C), one of the leading challenges for mesophilic degrading bacteria, was mimicked to investigate the physiological response of Rhodococcus biphenylivorans TG9. After 2 h of heat stress, the culturable TG9 cell count decreased from 108 cells/mL to undetectable while the viable cell count was still 105 cells/mL. The biphenyl degradation efficiency of stressed TG9 dropped by 50% compared to that of cells at logarithmic phase. During heat stress, the respiratory activity of TG9 declined dramatically while the intracellular ATP level initially increased and then decreased. Notably, the corresponding indicators recovered when restored to 30 °C. These characteristics were in consistent with bacteria entering into VBNC state. Furthermore, fluorescence activated cell sorting together with single cell as seed culture detection verified the unculturability and viability of VBNC state of TG9 cells. Also, we found that single cells in VBNC state could resuscitate and regrowth with significantly extended lag phase (LP). Our results highlight the potential of TG9 for microbial remediation and hint LP duration as an indicator for survival state of bacteria.

Electron-bifurcation and fluoride efflux systems in Acetobacterium spp. drive defluorination of perfluorinated unsaturated carboxylic acids.

Enzymatic cleavage of C-F bonds in per- and polyfluoroalkyl substances (PFAS) is largely unknown but avidly sought to promote systems biology for PFAS bioremediation. Here, we report the reductive defluorination of α, ß-unsaturated per- and polyfluorocarboxylic acids by Acetobacterium spp. Two critical molecular features in Acetobacterium species enabling reductive defluorination are (i) a functional fluoride efflux transporter (CrcB) and (ii) an electron-bifurcating caffeate reduction pathway (CarABCDE). The fluoride transporter was required for detoxification of released fluoride. Car enzymes were implicated in defluorination by the following evidence: (i) only Acetobacterium spp. with car genes catalyzed defluorination; (ii) caffeate and PFAS competed in vivo ; (iii) models from the X-ray structure of the electron-bifurcating reductase (CarC) positioned the PFAS substrate optimally for reductive defluorination; (iv) products identified by 19 F-NMR and high-resolution mass spectrometry were consistent with the model. Defluorination biomarkers identified here were found in wastewater treatment plant metagenomes on six continents.

Single-cell-level microfluidics assisted with resuscitation-promoting factor technology (SMART) to isolate novel biphenyl-degrading bacteria from typical soils in eastern China.

Soil microorganisms represent one of the largest biodiversity reservoirs. However, most low-abundance, slow-growing or dormant microorganisms in soils are difficult to capture with traditional enrichment culture methods. These types of microorganisms represent a valuable "microbial seed bank". To better exploit and utilize this "microbial dark matter", we developed a novel strategy that integrates single-cell-level isolation with microfluidics technology and culture with resuscitation-promoting factor (Rpf) to isolate biphenyl-degrading bacteria from four typical soils (paddy soil, red soil, alluvial soil and black soil) in eastern China. Multitudinous bacteria were successfully isolated and cultured; some of the identified clades have not been previously linked to biphenyl biodegradation, such as Actinotalea, Curtobacterium and Rothia. Soil microcosmic experiments validated that some bacteria are responsible for biphenyl degradation in soil. In addition, genomic sequencing and Illumina MiSeq sequencing of 16S rRNA genes indicated that exogenous Rpf mainly promotes the recovery and growth of bacteria containing endogenous Rpf-encoding genes. In summary, this study provides a novel strategy for capturing target functional microorganisms in soils, indicates potential bioresources for the bioremediation of contaminated soils, and enhances our current understanding of the mechanisms involved in the response to exogenous Rpf.

The amendment of Organic matters enhances the anaerobic dechlorination of Polychlorinated Biphenyls in Paddy Soil.

The toxicity of high-chlorinated polychlorinated biphenyls (PCBs) can be efficiently reduced through anaerobic dechlorination. However, this approach suffers a lot in face of in situ microbial remediations, like a shortage of biomass. In this study, we showed that the amendment of organic matters could help microbiota in paddy soil with anaerobic dechlorination and greatly shortened the lag period. The presence of organic matters offered a better environment for dechlorinating bacteria. They provided not only a more strictly anaerobic milieu but also copious carbon sources. By using high-throughput 16S rRNA gene sequencing, genera Dehalobacter, Dehalobacterium, and Desulfitobacterium capable of dechlorination were identified in enriched cultures. Taken together, this study proved that extra organic matters can promote anaerobic dechlorination in paddy soil slurry microcosm systems, which provides new insights into the bioremediation of PCB-contaminated soils.

Metagenomic study of humic acid promoting the dechlorination of polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that degrade slowly in the environment. Humic acid (HA), the main component of soil organic matter, or more specifically, the quinone moieties in HA, is generally regarded as an "electron shuttle" between pollutants and microorganisms, which could promote microbial remediation of contamination. In this study, we examined the dechlorination of PCB153 by adding HA and anthraquinone-2,6-disulfonate (AQDS, a model compound of quinones) to systems containing PCB dechlorinators, analyzed the composition and functional gene network of the microbial community by metagenomics, and explored the role of HA by modifying or substituting carbon sources or electron donors. However, this study found that HA accelerated microbial dechlorination of PCBS, while AQDS did not. Moreover, HA without quinone activity still promoted dechlorination, but not without carbon source or electron donor. Metagenomic analysis showed that HA did not promote the growth of PCB dechlorinator (Dehalococcoides), but the transmembrane electron carriers in the HA group were higher than those in the AQDS group and the control group, so HA may have promoted the electron transport process. This study is helpful for microbial remediation of PCB contamination, and provides new insights into the role that HA plays in the biogeochemical cycle.

Antibiotic tolerance and degradation capacity of the organic pollutant-degrading bacterium Rhodococcus biphenylivorans TG9T.

Antibiotics are ubiquitous in soil due to natural ecological competition, as well as emerging contaminants due to anthropogenic inputs. Under environmental factors like antibiotic stress, some bacteria, including those that degrade environmental pollutants, can enter a dormant state as a survival strategy, thereby limiting their metabolic activity and function. Dormancy has a critical influence on the degradative activity of bacteria, dramatically decreasing the rate at which they transform organic pollutants. To better understand this phenomenon in environmental pollutant-degrading bacteria, we investigated dormancy transitions induced with norfloxacin in Rhodococcus biphenylivorans TG9T using next-generation proteomics, proteogenomics, and additional experiments. Our results suggest that exposure to norfloxacin inhibited DNA replication, which led to damage to the cell. Dormant cells then likely triggered DNA repair, particularly homologous recombination, for continued survival. The results also indicated that substrate transport (ATP-binding cassette transporter), ATP production, and the tricarboxylic acid (TCA) cycle were repressed during dormancy, and degradation of organic pollutants was down-regulated. Given the widespread phenomenon of dormancy among bacteria involved in pollutant removal systems, this study improves our understanding of possible implications of antibiotic survival strategies on biotransformation of mixtures containing antibiotics as well as other organics.