Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study.

BACKGROUND: The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-ß-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context. METHODS: For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain. Bacteria were extracted from a hospital wastewater treatment plant (Fujian Provincial Maternity and Children's Hospital, Fuzhou, China) as the bacterial recipient community. We mixed this recipient community with the E coli donor strain carrying the gfp-tagged plasmid, both with and without sodium hypochlorite (NaClO) as an environmental stressor, and conducted several culture-based and culture-independent conjugation assays. The conjugation events were observed microscopically and quantified by fluorescence-activated cell sorting. We analysed the taxonomic composition of the sorted transconjugal pool by 16S rRNA gene amplicon sequencing and assessed the stability of the plasmid in the isolated transconjugants and its ability to transfer back to E coli. FINDINGS: We show that the plasmid pX3_NDM-5 has a broad host range and can transfer across various bacterial phyla, including between Gram-negative and Gram-positive bacteria. Although environmental stress with NaClO did not affect the overall plasmid transfer frequency, it reduced the breadth of the transconjugant pool. The taxonomic composition of the transconjugal pool was distinct from that of the recipient communities, and environmental stress modulated the permissiveness of some operational taxonomic units towards the acquisition of pX3_NDM-5. Notably, pX3_NDM-5 transconjugants included the Gram-positive pathogen Enterococcus faecalis, and the plasmid could subsequently be reconjugated back to E coli. These findings suggest that E faecalis could act as a natural shuttle vector for the wide dissemination of pX3_NDM-5 plasmids. INTERPRETATION: Our culture-independent conjugation model simulates natural environmental conditions and challenges the established theory that Gram-negative and Gram-positive bacteria rarely exchange clinically important plasmids. The data show that plasmids disseminate more widely across genera and phyla than previously thought. These findings have substantial implications when considering the spread of antimicrobial resistance across One Health sectors. FUNDING: The Laboratory of Lingnan Modern Agriculture Project, the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province of China, and the Outstanding Young Research Talents Program of Fujian Agriculture and Forestry University.

Anaerobic production and biosynthesis mechanism of exopolysaccharides in Schizophyllum commune 20R-7-F01.

Exopolysaccharides (EPS) produced by microorganisms play a vital role in physiological and ecological processes. However, the mechanisms of EPS synthesis and release in anaerobic environments remain poorly understood. Here, we provide the first evidence of anaerobic EPS synthesis by the fungus Schizophyllum commune 20R-7-F01, isolated from coal-bearing sediments ~2.0 km below the seafloor. Under anaerobic conditions, the fungus exhibited significantly higher specific EPS production (1.57 times) than under aerobic conditions. Transcriptomic analysis revealed 2057 differentially expressed genes (DEGs) in the strain cultured anaerobically for 7 days compared to aerobically. Among these genes, 642 were significantly upregulated, while 1415 were significantly downregulated, mainly associated with carbon metabolism pathways. Genes involved in glycolysis and EPS synthesis, including hexokinase (HK), phosphoglucomutase (PGM), and (1 â†’ 3)-ß-glucan synthase (GLS), were significantly upregulated, while those related to the TCA cycle, respiratory chain, and pentose phosphate pathway were downregulated under anaerobic conditions. These findings highlight the oxygen-dependent regulation of EPS synthesis and suggest that EPS may serve as a key mechanism for fungal adaptation to anaerobic environments.

Transcriptome analysis reveals phenanthrene degradation strategy of Pseudomonas stutzeri LH-42.

Toxic polycyclic aromatic hydrocarbons (PAHs) are often released into the environment during the combustion and processing of fossil fuels and are capable of causing significant pollution to people and the environment. One of the representative substances of PAHs is phenanthrene, which is often studied as a model compound for PAHs treatment. In this study, we compared the results of transcriptome analysis of Pseudomonas stutzeri LH-42 in two different culture conditions under phenanthrene-induced culture (test group) and glucose-induced culture (control group), and analysed the key enzymatic mechanisms of Pseudomonas stutzeri LH-42 in the biodegradation of phenanthrene. In our experiments, the transcriptome results showed that a total of 380 genes were more than twofold differentially expressed in the test group, of which 187 genes were significantly up-regulated in expression under Phenanthrene induction. Among the 380 differentially expressed genes, 90 genes were involved in Phenanthrene biodegradation, mainly including genes involved in biometabolism, cellular chemotaxis, substrate transport, signal induction and other related processes. Based on the transcriptome sequence analysis of Pseudomonas stutzeri LH-42 at the time of phenanthrene induction, a total of 25 dioxygenase genes were identified, and the related genes were mainly concentrated in two relatively concentrated clusters of PAHs biodegradation genes. The transcriptome analysis resulted in a complete set of enzyme genes related to the phenanthrene biodegradation pathway. The analysis of key enzymes led to the inference of a possible phenanthrene biodegradation pathway: the salicylic acid degradation pathway. The results of this study provide a theoretical basis for in situ remediation of PAHs-contaminated environments using Pseudomonas stutzeri LH-42. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03473-7.

Enhancing the ability of two kinds of oil-degrading bacteria to treat oily sludge by optimising the growth conditions using a response surface methodology.

Biosurfactant are Surfactants produced by certain microorganisms. These biosurfactants increase the biodegradability of insoluble pollutants. In this study, the fermentation products of Pseudomonas stutzeri Lh-42 (PS) and Rhodococcus sp. PR-1 (RD) were studied by Oil spreading method, emulsifying activity and infrared spectrum analysis. It was proved that these fermentation products were biosurfactant. And then the fermentation conditions of PS, RD were optimised by Placket-Burman (PB) design, hill-climbing experiment and response surface methodology (RSM). N source and liquid loading were significant factors in the fermentation of PS, while C source and speed were significant factors in the fermentation of RD. The surface tension was found to be as low as 39.53 ± 0.25 mN/m for the fermentation conditions of PS with an N source of 4.62 ± 0.41 g and a liquid loading of 28.4 ± 0.3%. The surface tension was 40.70 ± 0.47 mN/m for the incubation conditions of RD with a C source of 26.94 ± 0.62 g and a rotational speed of 210 r/min. Finally, the experimental results for the degradation of oily sludge showed that the degradation rate of oily sludge was improved when the fermentation conditions were optimised. The results of the infrared spectroscopy analysis showed that the organic matter content of the oily sludge treated with PS bacteria was significantly reduced after the optimised fermentation. This study provides a theoretical reference for further use of these bacteria to produce biosurfactants to treat organic matter.

Change the original microbial community structure in the hydrolysis acidification tank to enhance the COD removal performance of oily wastewater.

The hydrolysis acidification tank mainly relies on microorganisms to treat oily sewage, but in many cases the chemical oxygen demand (COD) of the effluent from the hydrolysis acidification tank does not decrease or even increase. In this work, about 50 L of oily wastewater is treated in a facultative anaerobic hydrolysis acidification tank with a temperature of 29 °C, pH 6, high-throughput sequencing technology analyzes found that after long-term operation of the hydrolysis and acidification tank, the dominant bacterial Pseudomonas accounted for only 2.87%, at this time, the effluent COD of the hydrolysis and acidification tank was 450 mg/L. Pseudomonas stutzeri LH-42 a strain screened in the laboratory, was domesticated and colonized in the hydrolysis acidification tank. High-throughput sequencing and bioinformatics analysis showed that the proportion of Pseudomonas in the hydrolysis acidification tank reached 5.89%, the effluent COD of the hydrolysis and acidification tank was 200 mg/L. The above results indicate the importance of the proportion of Pseudomonas in the hydrolysis and acidification tank for the COD degradation of oily wastewater.

MicroRNA-134-3p inhibits ovarian cancer progression by targeting flap structure-specific endonuclease 1 in vitro.

Ovarian cancer (OC) is one of the most lethal gynecological malignancies in the world. The aim of the present study was to examine the role of microRNA (miR)-134-3p in OC. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-134-3p. Cell Counting Kit-8, TUNEL, flow cytometric and colony formation assays were performed to examine the effects of miR-134-3p on OC cell proliferation. Moreover, wound healing and Transwell assays were performed to examine the effects on migration and invasion. In addition, western blot analyses were used to assess protein expression. Finally, the target genes of miR-134-3p were analyzed by bioinformatics analysis and dual-luciferase reporter assay. The results revealed that miR-134-3p expression was low in OC cells compared with in normal ovarian cells. The overexpression of miR-134-3p decreased cell viability, facilitated cell apoptosis, inhibited cell proliferation and arrested the cell cycle in SKOV-3 and OVCAR-3 cells. Furthermore, transfection using a miR-134-3p mimic inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells, and decreased the protein expression levels of cyclooxygenase-2, matrix metalloproteinase (MMP)2 and MMP9. Bioinformatics analysis indicated that one of the potential target genes of miR-134-3p was flap structure-specific endonuclease 1 (FEN1), which was confirmed by dual-luciferase reporter assay. Moreover, overexpression of miR-134-3p decreased the expression levels of FEN1 in SKOV-3 and OVCAR-3 cells. Additionally, overexpression of FEN1 reversed the effects of the miR-134-3p mimic on the proliferation, migration and invasion of SKOV-3 and OVCAR-3 cells. Overall, the findings of the present study demonstrated that miR-134-3p may inhibit OC cell proliferation, migration and invasion by directly targeting FEN1.

NO and ABA Interaction Regulates Tuber Dormancy and Sprouting in Potato.

In plants, nitric oxide synthase (NOS)-like or nitrate reductase (NR) produces nitric oxide (NO), which is involved in releasing seed dormancy. However, its mechanism of effect in potato remains unclear. In this study, spraying 40 µM sodium nitroprusside (SNP), an exogenous NO donor, quickly broke tuber dormancy and efficiently promoted tuber sprouting, whereas 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), an NO scavenger, repressed the influence of NO on tuber sprouting. Compared with the control (distilled water), SNP treatment led to a rapid increase in NO content after 6 h and a decreased abscisic acid (ABA) content at 12 and 24 h. c-PTIO treatment significantly inhibited increase of NO levels and increased ABA production. In addition, N G -nitro-L-arginine methyl ester, an NOS inhibitor, clearly inhibited the NOS-like activity, whereas tungstate, an NR inhibitor, inhibited the NR activity. Furthermore, NO promoted the expression of a gene involved in ABA catabolism (StCYP707A1, encoding ABA 8'-hydroxylase) and inhibited the expression of a gene involved in ABA biosynthesis (StNCED1, encoding 9-cis-epoxycarotenoid dioxygenase), thereby decreasing the ABA content, disrupting the balance between ABA and gibberellin acid (GA), and ultimately inducing dormancy release and tuber sprouting. The results demonstrated that NOS-like or NR-generated NO controlled potato tuber dormancy release and sprouting via ABA metabolism and signaling in tuber buds.