Applied Microbiology and Biotechnology最新文献

The outbreak of certain bacteria can trigger severe diseases, even posing a threat to public safety, leading to significant social panic and economic losses. Thus, tracing the origin of bacteria is of great significance. Stable isotope analysis technology offers a new way to determine the geographical information of bacteria, yet related research still fails to meet the application requirements of this technology in practical cases of bacterial traceability. In this study, a systematic stable isotope analysis method for bacteria and their culture conditions, based on practical geographical environments, was established for the first time. Escherichia coli and Staphylococcus aureus were cultured with water from five regions and different culture media, and the stable isotope ratios of H/O/C/N in the two bacteria and the culture media were measured to explore the relationship between bacteria and their cultivation site. The results showed that there were linear relationships between the hydrogen and oxygen stable isotopes of the two bacteria and the culture water. The combined discriminant model constructed using multi-isotope (H/O/C/N) characteristics achieved a 100% accuracy rate in identifying the types of culture media. These results indicate that research on the isotope association between bacteria and their culture water can be used to infer the cultivation region, and the specific source of bacteria can be further inferred through the multi-isotope combination discriminant model. This study can provide a relatively complete research idea for bacterial geographical traceability research, and improve the efficiency and accuracy of bacterial traceability work in practical investigations.

Noxious odors from swine farms pose significant health and environmental concerns. Hydrogen sulfide (H₂S) is one of the major compounds causing noxious odors. Using biological inoculants offers a promising, efficient, and cost-effective method to reduce H₂S emissions from these farms. Several studies have found that Priestia megaterium (formerly Bacillus megaterium) can help mitigate the production of H₂S. However, detailed knowledge regarding its mechanisms of action is still limited. In this study, the potential of novel strain of P. megaterium, strain S188, for the reduction of H₂S in swine manure has been demonstrated. The artificial manure-based assay revealed that P. megaterium S188 significantly reduced the headspace concentration of H₂S. Moreover, transcriptomic analysis revealed that exposure to H₂S upregulated the expression of several cysteine and methionine biosynthesis-related genes in P. megaterium S188. Furthermore, treatment of swine manure with P. megaterium S188 was associated with significant modulation of the microbiota in the manure. Overall, this study provides insights into the putative mechanisms underlying the reduction in H₂S levels mediated by P. megaterium S188 and its potential as a biological inoculant for H₂S mitigation in swine farms.

• Priestia megaterium S188 has shown the ability to lower H₂S from swine manure.

• Transcriptomic analysis revealed upregulation of H₂S-related biosynthesis genes.

• P. megaterium S188 treatment is associated with changes in the manure microbiota.

Improving Escherichia coli’s thermotolerance through rational engineering is hindered by limited knowledge of the molecular mechanisms involved in supraoptimal thermal adaptation. To address this issue, we applied a reverse metabolic engineering strategy to develop a d-homolactic E. coli strain under non-aerated conditions. We first characterized the thermal reaction norms of the kinetic and stoichiometric parameters in strains evolved through continuous adaptive laboratory evolution, identifying those that maintained parental volumetric productivity and the lactate/glucose yield at high temperatures. Then, the genomic analysis of two thermally adapted strains was performed to determine the mutations acquired during thermal adaptation. Thermally adapted strains revealed convergent mutations in regulatory genes, notably metJ, lrp, and components of the RNA polymerase complex. Introducing these point mutations into the parental strain demonstrated that individual mutations in rpoB and metJ significantly improved growth at high temperatures, despite the presence of complex epistatic interactions in combinatorial analyses.

• ALE significantly enhanced the thermotolerance of homolactic Escherichia coli

• Mutations in rpoB and metJ notably enhance growth at high temperatures

• Lactate productivity and yield stayed similar to the parental strain

Lactiplantibacillus plantarum strains are increasingly recognized for their combined probiotic and antimicrobial activities, offering potential applications in gut health management and pathogen control. This study characterized the intracellular (Met-Int) and extracellular (Met-Ext) metabolomic profiles of L. plantarum UTNGt2 (Gt2), UTNGt3 (Gt3), and UTNGt28L (Gt28L) isolated from tropical fruits and evaluated their probiotic, antimicrobial, cytotoxic, and immunomodulatory properties in vitro. Metabolomic profiling was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with a SWATH (Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) acquisition method. Cytotoxicity and cell viability were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) release assays in colon epithelial cells, while cytokine responses (IL-10, IL-1β) were quantified to determine immunomodulatory effects. Antimicrobial mechanisms were examined by scanning and transmission electron microscopy (SEM/TEM) on Staphylococcus aureus ATCC1026. LC–MS/MS identified 117 Met-Int and 32 Met-Ext across the three strains, revealing shared metabolites (e.g., l-tryptophan, adenosine) and distinct strain-specific compounds (e.g., harmine, lincomycin, baicalein) associated with bioactivity. Pathway enrichment analysis indicated four enriched pathways in Gt2, eight in Gt3, and ten in Gt28L, reflecting differential specialization in amino-acid, carbohydrate, and cofactor metabolism. Gt3 exhibited the most diverse antimicrobial metabolite repertoire, whereas Gt28L showed the strongest anti-inflammatory effect, increasing IL-10 secretion by ~ 6.5-fold and reducing IL-1β by ~ 50% compared with control cells. All strains maintained > 85% cell viability with minimal LDH release. SEM/TEM analysis confirmed that Met-Ext fractions caused membrane disruption and intracellular damage in S. aureus. Overall, these results demonstrate strain-specific probiotic, antimicrobial, and immunomodulatory signatures, identifying Gt3 and Gt28L as promising candidates for therapeutic or food-grade applications.

Mounting evidence supports population-specific variation in the microbiome. This study applied PacBio HiFi 16S rRNA amplicon sequencing to profile the oral microbiome of 62 individuals (aged 18–55 years) from the four major South African population groups (Black, Coloured, Indian and White). Bioinformatics analysis was performed using QIIME2 with taxonomic classification based on the SILVA database. Across all groups, Firmicutes was the most dominant phylum (80.92%), followed by Proteobacteria (8.94%) and Bacteroidota (4.22%). A total of 236 genera and 376 species were identified. Veillonella was the most abundant genus (27.57%), followed by Streptococcus (25.08%) and Granulicatella (15.54%). Streptococcus (30.40%) and Veillonella (41.82%) were the most abundant in the Indian and Coloured populations, respectively. Significant microbiome variation was observed between groups (β-diversity, p < 0.001), highlighting distinct population-specific microbial profiles. The Coloured population exhibited the highest microbiome diversity, likely due to complex genetic makeup and diverse cultural influences. The White population displayed the lowest microbiome diversity, likely due to more uniform lifestyle patterns. Despite these differences, three bacterial species, namely, Streptococcus salivarius, Veillonella atypica, and Prevotella melaninogenica were present in the majority of individuals across the populations, suggesting a core component of the South African oral microbiome. No significant associations were observed between factors such as sex, age, diet antibiotic use, lifestyle and oral microbiome variation. Several undetermined factors, such as psychological factors, stress, level of exercise, host genetics and immunity, could have contributed to the observed diversity. Our results present the first report of oral microbiome analysis of healthy South African populations by PacBio HiFi sequencing and warrant further research to provide insight into how these differences influence health disparities and potential application in forensics.

• First report of the oral microbiome of healthy South Africans by PacBio HiFi sequencing

• S. salivarius, P. melaninogenica and V. atypica define South Africa’s core oral microbiome

• Population-specific microbiome signatures can guide forensics and health research

Bacterial blight (causal agent Pseudomonas syringae complex, Psc) is an endemic and economically important disease of northern highbush blueberry production in Canada and the Pacific Northwest of the USA. To date, there is no comprehensive survey of the disease in the region and detailed characterization of associated pathogens from Pacific western Canada. Therefore, we did comprehensive disease survey and characterization of associated pseudomonads population using pathogen morphology, biochemical tests, and molecular characterization. We isolated 380 strains of pseudomonads from symptomatic plants from 32 research and commercial fields in 10 diverse geographic locations in British Columbia. We used P. syringae specific (Psy) primers and identified 197 Psy-PCR positive isolates out of 380. We further sequenced Psy-PCR positive isolates of pseudomonads using four housekeeping genes and identified four phylogenomic species: P. syringae (40%), Pseudomonas avellanae (29%), Pseudomonas viridiflava (20%), and phylogenomic species A (7%). P. avellanae and P. viridiflava are new phylogenomic species of Psc causing bacterial blight in highbush blueberry. We found some patterns among geographical locations and highbush blueberry varieties in the frequency distribution of isolates of these phylogenomic species. Genetic fingerprinting with rep-PCR assays identified a very high genetic diversity of pseudomonads populations among geographical locations, varieties, and phylogenomic species. Biochemical characterization (LOPAT- levan, oxidase, pectolytic activity, arginine dihydrolase, and tobacco hypersensitivity) revealed that the vast majority of isolates were Pseudomonas Group Ia. Findings of this study provide insight into the population biology of pseudomonads infecting highbush blueberry, provide information for disease diagnosis, and exploit disease management options, including identifying sources of disease resistance.

• High prevalence of bacterial blight caused by P. syringae complex (Psc) in highbush blueberry in Pacific western Canada

• We report two new phylogenomic species of Psc, P. viridiflava and P. avellanae, that cause bacterial blight and canker disease in highbush blueberry

• The genetic diversity of the population of Psc was very high

Palm oil is the world’s most widely used vegetable oil, with a sizeable impact on the environment. As an alternative, microalgae are considered oil producers since they produce a variety of fatty acids (FA) depending on growth conditions. A collection of ten microalgae strains naturally producing oils similar in composition to palm oil was selected, and the effects of cultivation regime and varying light intensity on their growth and FA production and composition were analysed. To achieve high biomass density as well as total fatty acid (TFA) content, the optimum irradiance of 400 µmol photons m⁻² s⁻¹ in a photoautotrophic regime was determined for most of the strains. The growth rates of Scenedesmus and Desmodesmus strains in general were approximately twice as high as Chlamydomonas. The highest TFA content was found in S. obliquus CCALA 455 and D. subspicatus CCALA 467, grown photoautotrophically, reaching the values of about 66% and 58% of their dry weight, respectively. Moreover, the content of palmitic (PA), oleic (OA) and linoleic acid (LA) of about 39%, 30% and 14% of TFA, respectively, determined in D. subspicatus CCALA 467 was closest to that in palm oil (44% of PA, 39% of OA and 10% of LA). Eight of the ten microalgae strains were capable of heterotrophic growth, although their production under this regime has not been considered suitable in terms of TFA and individual FA content.

• The optimum irradiance of 400 µmol photons m⁻² s⁻¹ was determined

• CCALA 467 produces selected FAs in amounts close to those in palm oil

• TFA content (% of dry weight) in CCALA 467 is 1.6-fold higher than in the palm

This study presents Bacillus subtilis T7 as the first known strain of B. subtilis capable of simultaneous lignin depolymerization and direct hydrogen production—a dual metabolic capability not previously reported in this species. B. subtilis T7 demonstrated 63.38% lignin degradation and 56.53% Azure B decolorization over seven days, with HPLC detection of aromatic intermediates—ferulic acid (0.85 mg/L) and vanillin (0.666 mg/L)—confirming active lignin catabolism. Agar-based assays revealed robust hydrolytic enzyme activities, including proteases (23.3 mm), cellulases (24.5 mm), amylases (14.2 mm), and xylanases (11.6 mm), surpassing those of many reported Bacillus strains. Whole-genome analysis confirmed a cascade of carbohydrate-active enzymes (CAZymes) including AA10 (lytic polysaccharide monooxygenases), AA3 (oxidoreductases), AA6 (quinone reductases), and CE1 (acetyl xylan esterases). These enzymes are associated with enhanced cellulolytic and xylanolytic activities, as well as increased lignin degradation. Batch fermentation experiments demonstrated that B. subtilis T7 produced hydrogen yields ranging from 0.53 to 1.41 mol H₂/mol substrate across various feedstocks, including xylose, glucose, carboxymethyl cellulose (CMC), starch, and untreated food waste. Xylose exhibited the highest volumetric productivity, achieving 274 mL H₂/g volatile solids, along with the most rapid production kinetics, indicating efficient metabolic utilization of this pentose sugar. In contrast, untreated food waste yielded the maximum molar hydrogen output of 1.41 mol H₂/mol substrate, attributable to its heterogeneous carbohydrate composition and lower average molecular weight, which likely enhanced enzymatic accessibility and substrate solubilization. These findings indicate that B. subtilis T7 encodes a functional [FeFe]-hydrogenase operon, along with an expanded repertoire of oxidative CAZymes, enabling it to bioprocess waste biomass into hydrogen without the need for syntrophic partners.

• B. subtilis T7 show dual potential for lignin degradation and hydrogen production.

• The genome contains CAZymes (AA10, AA3, AA6, CE1) responsible for lignocellulose deconstruction.

• The strain T7 produced a hydrogen yield of 1.411 mol H₂/mol substrate from xylose.

Spread of antimicrobial resistance and lack of new antibiotics have brought attention to alternative strategies of combating pathogenic bacteria. One of these strategies takes advantage of the bacteriolytic activity of peptidoglycan hydrolases. The enzymes allow efficient elimination of pathogenic bacteria while preserving the natural microflora. Such enzymes must meet specific criteria of activity, stability, and safety to become efficient enzybiotics. In our previous work (10.1128/spectrum.03546-23), we have created three chimeric enzymes and demonstrated their high efficacy in the elimination of Enterococcus faecalis and Staphylococcus aureus. In this work, we investigated and addressed issues related to the stability and safety of these enzymes. To improve the stability, we engineered the linkers and optimized storage conditions. Moreover, we demonstrated that such enzymes do not have any cytotoxic effects on eukaryotic cells, Danio rerio or Galleria mellonella. We also investigated the prevalence of resistance development, a particularly important feature for new antimicrobials. In conclusion, we here propose efficient, safe, and stable chimeric enzybiotics to eliminate E. faecalis and S. aureus.

• Optimized linker design enhances enzyme stability.

• Generated chimeric lysins do not display cytotoxicity.

• Chimeras with minimal risk of resistance development were selected.

The current study investigated 17 bacterial strains for their ability to synthesize gold nanoparticles (AuNPs) from the aryldiazonium gold(III) salt (DS-AuCl₄). The study aims to investigate the ability of bacterial cell biomass in the stationary phase of growth to synthesize AuNPs at 28 °C and 37 °C. Eleven bacterial strains were isolated from soil and identified using the VITEK® 2 system and 16S rRNA sequencing. An additional six strains were obtained from the American Type Culture Collection (ATCC). The investigated Gram-positive and Gram-negative bacterial strains successfully produced anisotropic AuNPs at a cell density of 2.0 McFarland (6.0 × 10⁸ CFU/mL). Nanoparticle formation was faster when samples were incubated at 37 °C than at 28 °C across all bacterial strains. The results of UV-vis spectroscopy confirmed the presence of AuNPs, with peaks observed centered at 550 nm. High-resolution transmission electron microscopy (HR-TEM) revealed a variety of morphologies, including spheres, rods, triangles, pentagons, hexagons, irregular shapes, and flower-like structures. Gram-positive and Gram-negative bacteria synthesized AuNPs of sizes 38.7 ± 26.0 and 34.0 ± 18.6 nm, respectively. Lattice-spacing analysis confirmed the formation of metallic AuNPs. Energy-dispersed X-ray spectroscopy (EDS) validated the presence of gold in the samples, and X-ray photoelectron spectroscopy (XPS) confirmed the elemental composition of AuNPs at 84.0 eV. These nanoparticles have potential applications in cancer therapy and diagnosis, antibacterial treatments, and drug delivery.

• The AuNPs were synthesized using various bacterial strains

• The gold precursor is aryldiazonium gold(III) salt

• Various anisotropic morphologies were obtained

Created in BioRender. Ahmady, I. (2025)