AimThis study aimed to characterize the prevalence of fosfomycin resistance in clinical carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates.MethodsCommon drug resistance genes and virulence genes were amplified by polymerase chain reaction (PCR) and verified by whole-genome sequencing. The potential virulence of all the clinical CRKP strains was tested in a Galleria mellonella infection model.ResultsA total of 171 CR-hvKP isolates collected from a Chinese tertiary hospital were analyzed for fosfomycin resistance. 71 strains (41.5%) were sensitive to fosfomycin, and 100 strains (58.5%) were resistant to fosfomycin. All the clinical isolates were found to carry fosfomycin genes fosA and fosA6. Only one fosfomycin-resistant isolate was found to carry the gene fosA3. In addition to the modification of target enzyme murA, the mechanisms of fosfomycin resistance also involve the dysfunction of the transport system including glpt, uhpT, and their regulatory gene CyaA mutation deletion. The Galleria mellonella infection assays confirmed that all the CRKP strains exhibited hypervirulence.ConclusionCumulatively, fosfomycin resistance in CR-hvKP is mediated by diverse mechanisms and is highly prevalent in a tertiary hospital from Jiangxi Province, South China. It is therefore critical to continuously monitor the epidemiology of these fosfomycin-resistant CR-hvKP while simultaneously minimizing potential risks from fosfomycin-resistant CR-hvKP.

Accurately resolving evolutionary relationships among organisms is a cornerstone of evolutionary biology, with phylogenetic trees serving as critical tools for this purpose. While single-gene phylogenies are widely used, they often fail to capture the full complexity of evolutionary processes, particularly in organisms with complex or variable genomes. Concatenated gene alignments, built from multiple orthologous genes, offer a more robust framework but are challenging to generate, especially in taxa with incomplete or poorly annotated genomes such as fungi. To address this challenge, we present Buscogeny (Busco Phylogeny), an open-source command-line tool that automates the construction of concatenated gene alignments and phylogenetic trees using BUSCO-derived single-copy orthologs. Buscogeny integrates key features including genome quality assessment, ortholog extraction, multiple sequence alignment, gap filtering, and recombination filtering, followed by automated phylogenetic inference. By allowing users to select ortholog datasets of varying resolutions and tailor filtering thresholds, Buscogeny is adaptable to both closely related and more distantly related taxa. We demonstrate the utility of Buscogeny in phylogenetic analyses of both bacterial and fungal genomes, including complex groups such as Alternaria, where annotation and genome completeness vary significantly. The streamlined, reproducible workflow provided by Buscogeny makes BUSCO-based phylogenetic inference accessible to researchers across disciplines, enabling robust evolutionary insights from whole-genome data.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-025-00752-6.

Pasteurella multocida is a Gram-negative coccobacillus from the Pasteurellaceae family, commonly residing as a commensal organism in the respiratory tracts of healthy animals. However, it possesses multiple virulence factors and can cause severe respiratory diseases. This study aimed to characterize P. multocida and its virulence genes and evaluate the immunogenicity of an inactivated vaccine of serogroup B using different administration routes. A total of 250 samples were collected from animals showing respiratory symptoms. Using 5% blood agar, 27 P. multocida isolates were obtained, and 21 (8.4%) were confirmed via PCR targeting the kmt1 gene. Nineteen virulence-associated genes were screened, categorized into outer membrane, fimbrial, somatic antigen, and iron-binding genes. The plpB, tadD, gatG, and hgbA genes were detected in both serogroup B and E isolates, whereas ompA, toxA, pcgD, latB, nctB, ppgB, natG, hgbB, and exbB were absent in all isolates. The immunogenicity of an inactivated P. multocida vaccine was evaluated in mice using subcutaneous and intramuscular routes. Subcutaneous vaccination produced a significantly higher antibody titer at 3 and 5 weeks post-vaccination with a 0.5 mL dose; in contrast, intramuscular immunization resulted in a rapid increase after booster doses. Analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05) revealed statistically significant differences between treatments. The comparison between subcutaneous and intramuscular routes also showed a significant difference (p < 0.001). This study concludes that although P. multocida serogroup B harbors fewer virulence factors, it effectively induces an immune response in mice but fails to provide cross-protection against the local serogroup E strain.

Produced water (PW), a major by-product of the petrochemical industry, contains a complex mixture of contaminants that limit its reuse and pose environmental risks if discharged untreated. Numerous treatment technologies have been developed to remediate this water, with bioremediation standing out as one of the most promising novel approaches. One such bioremediation method is through the application of cyanobacteria, which are able to remove pollutants such as heavy metals from produced water, although the mechanism by which the pollutants are removed is still unknown. In this study, a well-characterized cyanobacterium, Synechococcus elongatus, was used as a model organism to establish a proof of concept for identifying genes responsive to PW exposure and heavy metal stress. RNA sequencing was performed to analyze transcriptomic changes in S. elongatus grown in BG-11 (control) and exposed to 3 mg/mL of iron (heavy metal (HM)) or 25% v/v PW in BG-11. Differential expression analysis revealed that 11 and 67 genes were ≥ fivefold upregulated, and 337 and 27 genes were ≥ fivefold downregulated under HM and PW exposure, respectively, compared to the control. Among the over-expressed genes, the plasma membrane transporter, nitrate ABC transporter permease, was identified, suggesting its important role in the bioremediation process of heavy metals from wastewater. These findings provide foundational insights into stress-responsive gene networks in cyanobacteria and inform future bioengineering strategies for enhancing bioremediation capabilities in S. elongatus and related strains.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-025-00715-x.

Aerobic composting of livestock manure concentrates heavy metals like cadmium (Cd), elevating environmental risks after land application. This study screened composite microbial strains for simultaneous promotion of compost maturation and Cd passivation, exploring their mechanisms on the composting process, microbial community succession, and Cd speciation transformation. Three Cd-resistant strains—Enterobacter hormaechei (LB3), Enterobacter cloacae (LB4), and Bacillus velezensis (J-1–2)—were isolated from chicken manure, formulated into two composite inoculants (T1: LB3 + LB4; T2: LB3 + LB4 + J-1–2), and compared with an uninoculated control (CK) during composting. Maturity parameters, Cd species distribution, and microbial community dynamics were monitored. Results showed composite inoculants significantly improved composting: T1 extended the thermophilic phase and enhanced organic matter degradation; T2 achieved optimal nitrogen retention, with the highest NO3−-N (1504 mg/kg, representing a 13.51% increase compared to CK) and lowest NH4+-N (153 mg/kg, a 23.12% reduction compared to CK). Microbial community analysis revealed Ace/Chao1 indices in T1/T2 were 1.5–1.8 times higher than CK in the heating phase, while the Shannon index at maturity was 10.13% and 22.40% higher, respectively. T2 had the highest Cd passivation efficiency (66.7%), with exchangeable Cd decreasing from 27 to 9%. Inoculants promoted composting and Cd immobilization via microbial community modulation and adsorption–complexation mediated by key genera (e.g., Thauera), providing an effective strategy for safe reuse of livestock manure and heavy metal pollution mitigation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-025-00730-y.

Fungal endophytes inhabiting the medicinal plants have been considered repertoire for bioactive metabolites. In the current study, the medicinal plant Polygala sinaica was used for the first time as a source for endophytic fungi, which were screened for novel bioactive compounds. The potent biologically active fungal isolate was morphologically identified and molecularly verified using 18S rDNA sequencing as F. oxysporum with accession # OR616565. Two compounds were isolated using flash chromatography, identified using GC/MS and NMR techniques, and quantified using HPLC. Identified compounds were bis-(2-ethylhexyl) phthalate (DEHP) (1) and dibutyl phthalate (DBP) (2) isolated for the first time from F. oxysporum. The ethyl acetate extract of F. oxysporum exhibited potent activity against different multi-drug resistant Gram-negative and Gram-positive bacteria and Candida tropicalis. The production of DEHP was studied in different modified Wickerham media, using oat flakes, rice, and tomato as carbon sources, while corn steep liquor (CSL) and soy protein were used as nitrogen sources. CSL-containing medium exhibited the highest DEHP production by F. oxysporum at an initial pH of 7.2, 0.1% inoculum size after 15 days of incubation under static conditions at 28 °C. The biosynthesis of DEHP by F. oxysporum would serve as an excellent safe and eco-friendly source for its production to be used medicinally and industrially on a large scale with less toxic effects. The current data brings insights into the potency of Fusarium oxysporum, an endophyte of Polygala sinaica, for the production of bis-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP).

Storing biochar in soil is an effective method for improving the soil environment and reducing atmospheric greenhouse gases. Some strains of Bacillus species have been utilized as plant growth-promoting bacteria or biological control agents against plant diseases and pests. By colonizing biochar with beneficial bacteria, highly functional biochar that contributes to increased crop yields can be developed. In this study, we investigated the survival of type strains of eight Bacillus species (Bacillus amyloliquefaciens, B. licheniformis, B. nakamurai, B. pumilus, B. siamensis, B. subtilis, B. thuringiensis, and B. velezensis) in rice husk biochar, based on the sporulation state of vegetative cells or endospores. The bacterial density in the biochar significantly reduced when inoculated with a high concentration of vegetative cells, whereas it remained high when inoculated with a high concentration of spores. When water-washed biochar was inoculated with a high concentration of vegetative cells, survival significantly improved compared to that when unwashed biochar was inoculated. When a high concentration of vegetative cells was inoculated into the biochar water extract (biochar: water = 1:10), the bacterial densities of most species, except B. nakamurai and B. licheniformis, were significantly lower than those in alkali-sterilized water at pH 10.2 (same pH as biochar) and normal sterilized water (pH 6.7). These results suggest that the survival of Bacillus vegetative cells in biochar is inhibited by water-soluble inhibitors contained in the biochar rather than by alkaline conditions. When endospores were inoculated onto unwashed biochar, all strains maintained high viability for at least 12 weeks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-025-00737-5.

We previously reported the mRNA levels of genes involved in the tricarboxylic acid cycle and the phosphoenolpyruvate-pyruvate-oxaloacetate node. Owing to the importance of the glycolytic pathway, massive mRNA sequencing of Streptomyces coelicolor grown in minimal medium with glucose as the sole carbon source was performed. The results revealed a large discrepancy between the mRNA levels of all the genes analysed; sco1947, encoding glyceraldehyde 3-phosphate dehydrogenase, had the highest relative levels with respect to those of the hrdB gene, with a decreasing trend over time in most cases. On the other hand, the genes encoding glucose kinase had the lowest expression levels. The existence of gene multiplicity in Streptomyces is well known, and S. coelicolor is no exception. Two, three, and even four paralogous genes were detected for some of the activities in this pathway, and not all of them were expressed under the growth conditions used. The results obtained showed the large differences in gene expression and in the activity of the enzymes involved in the glycolytic pathway, which may allow the design of strategies aimed at a better carbon flow in this microorganism.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10123-025-00744-6.