Background: We aimed to determine the evolutionary pathways of rifampicin resistance in Staphylococcus aureus, and the impact of resistance mutations in the rpoB gene on fitness. Methods: Three clinical strains and one reference strain were used to select for rifampicin-resistant S. aureus variants. The mutations responsible for rifampicin resistance in all of the selected isolates in vitro were investigated by polymerase chain reaction (PCR) and DNA sequencing. To compare the fitness cost of rpoB mutations against their corresponding original isolates, we performed bacterial growth curve assays, static biofilm assays, in vitro competition experiments and an infection model of Galleria mellonella larvae. Results: We obtained four rifampicin-resistant S. aureus isolates that showed high levels of resistance to rifampicin with a minimal inhibitory concentration (MIC) of 128 mg/L, and all isolates had a mutation at position 481 (H481F/Y) in RpoB. A broth microdilution assay indicated that mutation of H481F/Y did not affect susceptibility to common antibacterial drugs but slightly increased the vancomycin MIC. To identify the pathways involved in the development of rifampicin resistance, 32 variants (eight mutants for each strain) and four original isolates were selected for gene sequencing. Different generations of isolates were found to harbor various mutations sites. Compared with the corresponding original isolates, an in vitro fitness assay of the variant isolates showed that growth and virulence were reduced, with a statistically significantly decreased fitness, whereas the capacity for biofilm formation was elevated. Conclusions: Our findings suggested that the acquisition of rifampicin resistance in S. aureus was dynamic and was associated with a significant fitness cost.

Phytoremediation is an environment-friendly method for toxic elements remediation. The aim of this study was to improve the phytoremediation efficiency of Brassica juncea and the rhizosphere soil micro-ecology in cadmium (Cd) and arsenic (As) contaminated soil. A field experiment was conducted with six treatments, including a control treatment (CK), two treatments with two contents of Trichoderma harzianum (T1: 4.5 g m⁻²; T2: 9 g m⁻²), one biochar treatment (B: 750 g m⁻²), and two combined treatments of T1B and T2B. The results showed Trichoderma harzianum promoted the total chlorophyll and translocation factor of Brassica juncea, while biochar promoted plant biomass compared to CK. T2B treatment showed the best results, which significantly increased Cd accumulation by 187.49–308.92%, and As accumulation by 125.74–221.43%. As a result, the soil’s total Cd content was reduced by 19.04% to 49.64% and total As contents by 38.76% to 53.77%. The combined amendment increased the contents of soil available potassium, phosphorus, nitrogen, and organic matter. Meanwhile, both the activity of glutathione and peroxidase enzymes in plants, together with urease and sucrase enzymes in soil, were increased. Firmicutes (dominant bacterial phylum) and Ascomycota (dominant fungal phylum) showed positive and close correlation with soil nutrients and plant potentially toxic elements contents. This study demonstrated that phytoremediation assisted by biochar and Trichoderma harzianum is an effective method of soil remediation and provides a new strategy for enhancing plant remediation efficiency.

Cadmium (Cd) is a readily available metal in the soil matrix, which obnoxiously affects plants and microbiota; thus, its removal has become a global concern. For this purpose, a multifunctional nanoscale zerovalent—iron enriched biochar (nZVI/BC) was used to alleviate the Cd—toxicity in maize. Results revealed that the nZVI/BC application significantly enhanced the plant growth (57%), chlorophyll contents (65%), intracellular permeability (61%), and biomass production index (76%) by restraining Cd uptake relative to Cd control. A Cd stabilization mechanism was proposed, suggesting that high dispersion of organic functional groups (C–O, C–N, Fe–O) over the surface of nZVI/BC might induce complex formations with cadmium by the ion exchange process. Besides this, the regular distribution and deep insertion of Fe particles in nZVI/BC prevent self-oxidation and over-accumulation of free radicals, which regulate the redox transformation by alleviating Cd/Fe⁺ translations in the plant. Current findings have exposed the diverse functions of nanoscale zerovalent-iron-enriched biochar on plant health and suggest that nZVI/BC is a competent material, feasible to control Cd hazards and improve crop growth and productivity in Cd-contaminated soil.