Search Results (236)

This study proposed a biosafe strategy to prevent staphylococcal enterotoxin production in cooked bacon, particularly in microenvironments at the product–package interface that may favor toxinogenesis. Challenge tests were conducted on bacon samples with varying water activity, inoculated with Staphylococcus aureus, and treated with a biopreservative produced in an axenic cultivation of Lacticaseibacillus paracasei DTA-83. Staphylococcal enterotoxin production was assessed using an ELISA test. Data about microbial growth and staphylococcal enterotoxin production were evaluated under simulated storage using the MicroLab_ShelfLife protocol. Results showed that staphylococcal enterotoxin production occurred independently of water activity, indicating that it alone does not ensure microbial safety. Even at a water activity level traditionally considered inhibitory, enterotoxin formation was observed, emphasizing the critical role of the product–package interface. However, a 1.0% biopreservative applied to the product surface effectively inhibited S. aureus growth and completely suppressed staphylococcal enterotoxin production under all conditions, including temperature abuse. In conclusion, the formation of enterotoxin by S. aureus at low water activity reveals a critical safety risk at the product–package interface. Targeted application of a 1.0% biopreservative at this vulnerable site proved highly effective, reinforcing its role as a practical and non-intrusive hurdle strategy to enhance microbial safety in shelf-stable meat products. Full article

Coxiella burnetii, the causative agent of Q fever, is a highly infectious pathogen capable of invading diverse cell types, from alveolar macrophages to trophoblasts. Within host cells, it establishes a replicative niche named Coxiella-containing vacuole (CCV). This is driven by effector proteins secreted by the bacterium into the host cell cytoplasm via a Type 4b Secretion System (T4SS). Advances in axenic culture and mutagenesis allowed the characterization of Coxiella effector proteins, revealing their host targets and strategies of cellular subversion. This review highlights recent insights into Coxiella effector proteins and their manipulation of host processes, from vesicular trafficking to innate immunity. Full article

Previous studies have suggested correlations between the microbiota of the black soldier fly and larval growth and bioconversion ability, primarily through functional inference. However, the concrete impact of the microbiota remains to be demonstrated. To address this, we assembled two synthetic microbial communities (SynComs) derived from endogenous bacteria and evaluated their effects on larval growth. SynComs were administered to axenic larvae reared on sterilised diet (gnotobiotic) or as a probiotic in non-sterile treatments. Larvae were reared on vegetable-based (pre-consumer vegetable residues) or on animal-based (chicken hatchery residues) substrates. The SynComs were administered at two concentrations (5 × 10⁷ and 10⁸ CFU per isolate) in the substrate prior to neonate introduction. SynComs improved the growth of axenic larvae compared to untreated controls, although not to the levels observed in conventionally reared larvae. In non-sterile conditions, the combined SynComs increased growth on vegetable-based substrate, but no effect was observed on the animal-based substrate, suggesting a substrate-dependent effect. These results highlight microbiota’s critical role in larval development and the potential of microbiome engineering in insect rearing systems. This preliminary study opens the way for optimisation of SynCom assemblies, which could be enhanced through pre-testing of individual isolates and selecting microbial combinations tailored to specific substrates. Full article

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small amounts of feces containing cysts or trophozoites to the culture medium. Implantation success is lower when starting from cysts, and the mechanisms and early events of excystation remain poorly understood. In this study, we describe the sequence of events involved in excystation and identify factors potentially important for culture establishment. Cysts were obtained from orangutan feces and genetically confirmed as B. coli. Only viable cysts, determined by trypan blue or methylene blue exclusion, were used. After artificial digestion with pepsin and trypsin, cysts were incubated at 28 °C for up to 72 h in DMEM supplemented with L-glutamine, yeast extract, fetal bovine serum, and starch granules. Excystation began with a fissure in the cyst wall, allowing for bacterial entry. This appeared to stimulate the trophozoites, the increased motility of which progressively weakened and ruptured the wall, allowing for their emergence. Wall rupture and bacterial entry were critical for activation., whereas starch type had no apparent influence. Excystation occurred within the first hours; otherwise, cysts degenerated. Full article

Giardia lamblia is a flagellated protozoan and the etiological agent of giardiasis, a leading cause of epidemic and sporadic diarrhoea globally. The clinical and public health relevance of giardiasis underscores the need for robust methodologies to investigate and manage this pathogen. This study reviews the main methodologies described in the literature for studying the life cycle of G. lamblia, focusing on isolation, purification, axenization, excystation, and encystation. A systematic literature review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) statement. Searches were performed in MEDLINE, ScienceDirect, and Web of Science Core Collection databases. A total of 43 studies were included, revealing 58 methods for isolation and purification, 7 for excystation, 2 for axenization, and 5 for encystation. Isolation and purification methods exhibited significant variability, often involving two phases: an initial separation (e.g., filtration and centrifugation) followed by purification using a density gradient for faecal samples or immunomagnetic separation for water samples. Method effectiveness differed depending on the sample source and type, limiting comparability across studies. In contrast, methods used for other life cycle stages were more consistent. These findings underscore the need for standardised methodologies to enhance the reproducibility and reliability of research outcomes in this field. Full article

Seed microbes play crucial roles in plant health, but studying their diversity is challenging due to host DNA contamination. This study aimed to optimise methodologies for investigating seed microbiomes across diverse plant species, focusing on the efficacy of peptide nucleic acid (PNA) clamps to reduce host DNA amplification. We tested PNA clamps on three plant species: Melaleuca quinquenervia (tree), Microlaena stipoides, and Themeda triandra (grasses). The effectiveness of PNA clamps was assessed through in silico analysis, axenic tissue culture, and metabarcoding techniques. In silico analysis confirmed the specificity of PNA clamps to the 16S rRNA gene V4 region of chloroplasts in the grass species. Axenic tissue culture experiments showed that applying PNA clamps at both 1 µM and 0.25 µM concentrations significantly reduced plant DNA amplification. Metabarcoding analyses further confirmed that PNA clamps effectively suppressed host DNA, enhancing microbial diversity estimates across all three species while preserving core microbial taxa. The efficacy of the clamps varied among host species, with T. triandra exhibiting the highest blocking efficacy, and chloroplast clamps outperforming mitochondrial ones. This study demonstrates that PNA clamps are a useful for improving seed endophyte metabarcoding datasets, although they require optimisation for some plant species. This knowledge will contribute to enhancing our understanding of seed microbiome diversity and its ecological implications. Full article

Microbial decomposition of persistent natural compounds such as phenolic lignin and polysaccharides in plant cell walls plays a crucial role in the global carbon cycle and underpins diverse biotechnological applications. Among microbial decomposers, fungi from the Ascomycota and Basidiomycota phyla have evolved specialized mechanisms for efficient lignocellulosic biomass degradation, employing extracellular enzymes and synergistic fungal consortia. Fungal coculture, defined as the controlled, axenic cultivation of multiple fungal species or strains in a single culture medium, is a promising strategy for industrial processes. This approach to biomass conversion offers potential for enhancing production of enzymes, biofuels, and other high-value bioproducts, while enabling investigation of ecological dynamics and metabolic pathways relevant to biorefinery operations. Lignocellulosic biomass conversion into fuels, energy, and biochemicals is central to the bioeconomy, integrating advanced biotechnology with sustainable resource use. Recent advancements in -omics technologies, including genomics, transcriptomics, and proteomics, have facilitated detailed analysis of fungal metabolism, uncovering novel secondary metabolites and enzymatic pathways activated under specific growth conditions. This review highlights the potential of fungal coculture systems to advance sustainable biomass conversion in alignment with circular bioeconomy goals. Full article

Urban expansion has led to two significant environmental challenges: the reduction in green spaces and the rise in urban temperatures, decreasing city livability. Green roofs have emerged as a sustainable solution to mitigate these issues, offering ecological and economic benefits while improving building energy efficiency. Some species of the genus Sedum, particularly Sedum sediforme and Sedum album, are ideal for such green infrastructure due to their non-aggressive and superficial root system, high drought tolerance, low nutrient needs, pest and disease resistance, and metabolic adaptability during dry periods. This study aims to optimize the large-scale production of two native ecotypes of S. sediforme and S. album from the Valencian Community through an efficient propagation system that enables uniform plant production in limited space. For this purpose, we have developed micropropagation systems that allow a rapid multiplication of these two species. A direct morphogenesis system was established using axenic plant shoots, and a protocol for adventitious organogenesis from leaves was also developed. These methods significantly enhance propagation speed, spatial efficiency, and plant uniformity. Notably, the metabolic plasticity of S. sediforme and S. album reduces abiotic stress during acclimatization, promoting efficient ex vitro establishment and functional integration into extensive green roof ecosystems. Full article

In insect–microbe symbiosis, understanding the diversity of associated bacteria is crucial. DNA-dependent sequence methods are widely used to assess microbial diversity in insects, but they cannot distinguish between live and dead microbes. In contrast, RNA-dependent sequencing can identify alive bacterial communities, making them more suitable for evaluating alive microbiota diversity. However, its practical reliability in insect–microbe symbiosis remains poorly validated. This study investigated larval gut bacteria diversity of Delia antiqua, a major pest of Liliaceae crops, by employing both DNA- and RNA-dependent 16S rRNA amplicon sequencing. The reliability of both sequencing methods was evaluated by comparing the effects of synthetic communities (SynComs, constructed according to DNA- or RNA-dependent sequencing) and bacterial communities from wild larvae on axenic larvae. Results revealed significant differences in bacterial community between DNA- and RNA-dependent sequence samples. Compared to bacterial communities from wild larvae, the SynCom constructed based on RNA-dependent sequencing exhibited inhibition effects on D. antiqua larvae survival and body weight, while DNA-dependent SynCom did not, suggesting that DNA-dependent methods were superior for assessing symbiotic microbiota in D. antiqua. This work will provide insights into microbial diversity detection in D. antiqua and offer a framework for other insect–microbe studies. Full article

The fungi belonging to the genus Pleurotus can be cultivated in different substrates and represent excellent producers of several extracellular enzymes. In this study, we analyzed eleven hydrolytic enzymes of the P. eryngii and P. ostreatus secretomes, which were collected at three different growth stages after 23 days (mycelial colonization of about 50% of the substrate), 34 days (100% colonization of the substrate) and 50 days (after the first flush). Mushrooms were axenically cultivated on the same substrate. The results demonstrate that proteases, lipases, amylases, α-glucosidase, cellulases (endoglucanase, β-cellobiohydrolase and β-glucosidase) and hemicellulase (xylosidase, glucuronidase, arabinosidase and mannosidase) activities were higher in the secretomes from P. eryngii than those from P. ostreatus. Time course analysis revealed for both species a similar enzymatic activity profile, in which in the early stages of mycelium development, both species use starch as the main carbon source. Protease and lipase activities increased and remained constant during the subsequent formation of fruiting bodies, whereas cellulase and hemicellulase activities decreased after the complete mycelial colonization of the substrate. The zymographic analysis suggested the presence in the secretomes of proteolytic activities belonging to different classes. In conclusion, both mushroom species released into the secretomes a broad spectrum of hydrolytic enzymes potentially useful in various biotechnological fields. Full article

Cyanobacteria are ubiquitous in freshwater environments, but their role in aquatic resistome remains unclear. In this work, we performed whole genome sequencing on 43 cyanobacterial strains isolated from Portuguese fresh/wastewaters. From 43 available non-axenic unicyanoabacterial cultures (containing only one cyanobacterial strain and their co-occurring bacteria), it was possible to recover 41 cyanobacterial genomes from the genomic assemblies using a genome binning software, 26 of which were classified as high-quality based on completeness, contamination, N50 and contig number thresholds. By using the comprehensive antibiotic resistance database (CARD) on the assembled samples, we detected four antibiotic resistance gene (ARG) variants, conferring resistance in pathogenic bacteria to tetracyclines, fluoroquinolones (adeF-type) and macrolides (ermF-type, mefC-type and mphG-type). Among these, adeF-type was the most prevalent gene, found across 11 cyanobacterial genomes from the Nostocales order. Planktothrix presented the highest variety of close ARG matches, with hits for the macrolide resistance genes ermF-type, mefC-type and mphG-type. An analysis of the genomic assemblies also revealed an additional 12 ARGs in bacteria from the phyla Firmicutes, Proteobacteria and Bacteroidetes, present in the cyanobacterial cultures, foreseeing the horizontal gene transfer of ARGs with cyanobacteria. Additionally, more than 200 partial ARGs were detected on each recovered cyanobacterial genome, allowing for future studies of antibiotic resistance genotype/phenotype in cyanobacteria. These findings highlight the importance of further efforts to understand the role of cyanobacteria on the aquatic resistome from a One Health perspective. Full article

Effective wastewater management is critical for mitigating environmental and health impacts in ecologically sensitive regions like the Peruvian Amazon, where rapid urbanization has led to increased discharge of nutrient-rich effluents into freshwater systems. Conventional treatment methods often fail to address nutrient imbalances while generating secondary pollutants. This study aims to evaluate the bioremediation potential of a non-axenic cyanobacterium, Synechococcus sp., isolated from the Amazon Basin, for municipal wastewater treatment within a circular bioeconomy framework. The strain was cultivated in different concentrations of municipal wastewater (25%, 50%, 75%, 100%) from Moronacocha Lake in the Peruvian Amazon to assess growth kinetics, ammonium removal efficiency, and biochemical composition. The cyanobacterium exhibited optimal performance in 25% wastewater, achieving the highest specific growth rate (22.8 × 10⁻² μ·day⁻¹) and biomass increase (393.2%), exceeding even the standard BG-11 medium. This treatment also demonstrated exceptional ammonium removal efficiency (95.4%) and enhanced phycocyanin production (33.6 μg/mg, 56% higher than the control). As wastewater concentration increased, both growth parameters and removal efficiency progressively declined. Biochemical analysis revealed that higher wastewater concentrations resulted in decreased protein content and increased lipid accumulation in the biomass. These findings demonstrate the dual potential of Synechococcus sp. for effective wastewater remediation and production of valuable biomass with modifiable biochemical characteristics, offering a sustainable approach for wastewater management in the Peruvian Amazon region. Full article

Wastewater (WW) treatment using biofilms harboring bacteria and microalgae is considered a promising polishing solution to improve current treatment technologies present in wastewater treatment plants (WWTPs), but their interaction in a sessile community remains to be understood. In this work, multi-species biofilms of Chlorella vulgaris, Chlorella sorokiniana, or Scenedesmus obliquus were selected as representative microalgae species of interest for WW bioremediation, and Rhodococcus fascians, Acinetobacter calcoaceticus, or Leucobacter sp. were selected as the bacteria for co-cultivation in a synthetic WW since they are normally found in WW treatment processes. The attached consortia were developed in specific carriers (K5 carriers) for 168 h, and their biofilm formation ability was evaluated in a profilometer and via scanning electron microscopy (SEM) imaging. From the selected microorganisms, C. sorokiniana was the microalga that adapted best to co-cultivation with R. fascians and A. calcoaceticus, developing a thicker biofilm in these two consortia (3.44 ± 0.5 and 4.51 ± 0.8 µm, respectively) in comparison to the respective axenic cultures (2.55 ± 0.7 µm). In contrast, Leucobacter sp. did not promote biofilm growth in association with C. vulgaris and C. sorokiniana, while S. obliquus was not disturbed by the presence of this bacterium. Some bacterial clusters were observed through SEM, especially in A. calcoaceticus cultures in the presence of microalgae. In some combinations (especially when C. vulgaris was co-cultivated with bacteria), the presence of bacteria was able to increase the number of microalga cells adhered to the K5 carrier. This study shows that biofilm development was distinctly dependent on the co-cultivated species, where synergy in biofilm formation was highly dependent on the microalgae and bacteria species. Moreover, profilometry appears to be a promising method for biofilm analyses. Full article

Drosophila melanogaster is broadly used to model host–pathogen interactions. Entomopathogenic nematodes are excellent research tools for dissecting the molecular and functional basis of parasitism and the host’s anti-parasitic response. In this work, we used discovery metabolomics to explore the differences in the metabolome composition of wild type D. melanogaster larvae that were infected with symbiotic nematodes (Steinernema carpocapsae carrying Xenorhabdus nematophila mutualistic bacteria) or axenic nematodes (S. carpocapsae lacking their bacterial partners). Benefiting from their high separation power, sensitivity, and compatibility with low amounts of the starting metabolome, we leveraged microanalytical capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to profile the small (<500 Da) polar portion of the metabolome among these experimental treatments. We detected and quantified 122 different small molecules, of which 50 were identified with high confidence. Supervised multivariate analysis revealed that the infection was paralleled with changes in amino acid biosynthesis (arginine, phenylalanine, tryptophan, and tyrosine), metabolism (alanine, arginine, aspartate, glutamate, glycine, proline, serine, and threonine), and classical signalling (aspartate, γ-aminobutyrate, glutamate, and pyridoxine). This study demonstrates the ability of high-sensitivity CE-ESI-MS to uncover metabolic perturbations during infection. The results from the metadata may facilitate the design of targeted studies to explore small biomolecules and their functions during host–pathogen interaction. Full article

Furfural is a relevant industrial product, but its presence in water and soil generates contamination and health risks. Moving bed biofilm reactors (MBBRs) are an increasingly used alternative to eliminate contaminants with the advantage of occupying small spaces, despite their high dependence on support and the microorganisms involved in the process. This work proposes furfural elimination through a laboratory-scale MBBR using Bacillus licheniformis GTQ1, Microbacterium sp. GISTAQ2, and Brevundimonas sp. GISTAQ1 isolated from an industrial effluent and agroforestry waste (rice husks, pine sawdust, and quebracho chips) as supports. The biofilm development was tested with both axenic and mixed cultures, confirming high coverage by Scanning Electron Microscope (SEM) images, especially in triple-mixed cultures. Biodegradation tests were carried out in the MBBR with 15 g rice husks or quebracho chips as supports and a 4000 mg L⁻¹ initial furfural concentration for 72 h. The mixed culture achieved almost a 100% furfural removal in three days with a rate of 3.97% per hour with rice husks and 2.61% per hour with quebracho chips. This laboratory-scale MBBR development is a promising first step ready for a scale-up for its implementation in industries to significantly reduce the environmental impact of the discharge of this type of effluent. Full article